Category: Biochemistry

Using Bio-Rad’s pGLO Bacterial Transformation Lab Kit (see Bio Rad’s link), our lab class made E. Coli glow in the dark.

See Bio-Rad’s Extensive Instructor and Student Documentation and Study Questions at this link [PDF]

Last edited: 08.24 .2019

Outline.

• Fatty Acid Synthesis
• Cholesterol Metabolism
• Fed and Fasting States

Fatty Acid Synthesis.

• Fatty acid (FA) synthesis has quite the different pathway from fatty acid catabolism/breakdown.
• FA synthesis is made from glucose mainly in the liver (mostly liver) and adipocytes and mammary glands during lactation.
• Glucose is converted to pyruvate in the cytosol. Pyruvate enter the mitochondrial matrix and is converted to acetyl CoA and oxaloacetate.
• Acetyl CoA + oxaloacetate + H2O –> citrate (in mitochondria)
• Citrate is moved out to the cytosol.
• In the cytosol, citrate is cleaved into acetyl CoA and oxaloacetate.
• The acetyl CoA (in cytosol now) transforms —acetyl CoA carboxylase–> malonyl CoA.
• Malonyl CoA is the activated form of acetyl CoA.
• Malonyl CoA (gets decarboxylated) is used to grow the FA chain.
• When glucose is abundant and in excess, glucose may be converted and used this way for FA synthesis.
• The fatty acid synthase complex is located in the cytosol (uses cytosolic acetyl CoA).
• Citrate-Malate-Pyruvate shuttle provides cytosolic acetate units and reducing equivalens for the FA synthesis process.
• 1 NADPH is made for each acetyl CoA that’s transferred from the mitochondria to the cytosol via malic enzyme.
• Other NADPH molecules are produced from the pentose phosphate pathway as well.

Characteristics	Synthesis	Degradation
Location	Cytosol	Mitochondrial Matrix
Activated Intermediates	Bound to ACP	Thioester of CoA
Intermediates	-SH of acyl carrier proteins	-SH of CoA
Activated Enzymes	FAS (multienzyme complex)	4 distinct enzymes
Substrte	Acetyl CoA	Fatty Acids
Direction	Starts at methyl end	Starts at carboxyl end
Cofactors	NADPH/NADP+	FADH2/NADH, FAD/NAD+
Major Sites	Liver	Muscle & Liver
Hormonal Regulation	High Insulin/Glucagon Ratio	Low Insulin/Glucagon Ratio
Activator	Citrate	Free FA
Inhibitor	FA CoA	Malonyl CoA

Acetyl CoA to Malonyl CoA.

• Acetyl CoA gets converted to Malonyl CoA via enzyme Acetyl CoA Carboxylase + coenzyme Biotin.
• This conversion is the most regulated step and it is the rate limiting step. It is reversible but this step is a commitment step to this pathway to make FAs.
• Note that not ALL our acetyl CoA is going down this path. Assume there’s plenty other acetyl CoA to make Acetyl ACP as well.
• 1 ATP is spent.
• One HCO3- (or CO2) is spent.

Acyl Carrier Protein (ACP).

• It is a complex with several binding sites for different activities.
• It is a dimer.
• The acyl carrier protein has a phosphopantetheine prosthetic group (can form thioester bonds) and a coenzyme A group.
• Prosthetic group: A non protein that combines with a protein/enzyme.
• The acetyl CoA and malonyl CoA get attached to ACP’s prosthetic group via the condensing enzyme acylmalonyl-ACP-condensing enzyme.
• The phosphopantetheine group (B5) is the “docking” location.
• The CoA part is where new materials are received.

Step 0: Activation/Loading.

Fatty Acid Synthesis Step 1. Condensation.

• Condensation
• We are taking the “omega end” (the last end part) of acetyl ACP which is the H3C-C=O (keeping that from the acetyl ACP) and we get rid of the -O-C=O part of the malonyl ACP.
• Loading.
• Move the 2-carbon unit from malonyl-ACP to acetyl-ACP. Forms a 2-carbon keto-aceyl-ACP.
• Omega carbon = last carbon in a chain.

Fatty Acid Synthesis Step 2. Reduction.

• Reduction
• Get rid of the double bond, carbonyl, left of the CH2 group.
• Convert keto-acyl-ACP to hydroxyl-ACP.
• Spends 1 NADPH.

Fatty Acid Synthesis Step 3. Dehydration.

• Dehydration
• Want to get rid of the hydroxyl OH group by making a double bond.
• Also, get rid of the OH group, water is leaving group.

Fatty Acid Synthesis Step 4. Reduction.

• Reduction
• Want to reduce to get rid of that double bond (trans H), left of the carbonyl.
• Spend 1 NADPH.
• With butyrl ACP, that is a 4-carbon chain with 2-carbons from the acetyl ACP.
• Hereafter, the cycle is repeated by adding malonyl ACP. This gives an addition of 2 more carbons to the chain.
• 1st time through the cycle, 2-carbons from acetyl ACP and 2-carbons from malonyl ACP = 4-carbon chain.
• 2nd time through the cycle, add 2 more carbons from malonyl to get a 6-carbon chain.
• 3rd time through the cycle, add 2 more carbons from malonyl to get a 8-carbon chain.
• 4th time through the cycle, add 2 more carbons from malonyl to get a 10-carbon chain.
• 5th time through the cycle, add 2 more carbons from malonyl to get a 12-carbon chain.
• 6th time through the cycle, add 2 more carbons from malonyl to get a 14-carbon chain.
• 7th time through the cycle, add 2 more carbons from malonyl to get a 16-carbon chain.
• STOP at 16-carbon chain which is Palmitate because fatty acid synthase cannot handle anything more than the 16-carbon chain.
• 7 cycles are needed to make the 16-carbon palmitate.
• 7 cycles >> 1 molecule of acetyl CoA and 6 molecules of malonyl CoA; 14 NADPH; 7 ATP.
• To make a complete palmitate:

How to Make a Long Fatty Acid? Keep repeating…

• Keep adding malonyl ACP (adds 2 carbons each time) until a 16-carbon chain is reached.
• Fatty acid synthase’s limit is 16 carbon chain.

Palmitate.

• At 16-carbon long FA chain, the enzyme thioesterase hydrolyzes the FA acyl group in order to make “free” palmitate.
• palmitoyl-ACP + H2O –thioesterase–> palmitate + ACP-SH
• Then palmitate can undergo elongation or unsaturation to make FAs.

Elongation and Unsaturation.

• Elongase is an enzyme in the smooth endoplasmic reticulum (ER) that adds 2-carbons onto a FA chain via malonyl CoA.
• Desaturase is an enzyme that adds cis double bonds up to position delta-9 in the ER in mammals. Humans can only use cis double bonds and not trans.

Summary of Key Points of FA Synthesis Steps.

• Step 0. Activation-Loading. Acetyl CoA and malonyl CoA need to be “loaded” on to the Acyl Carrier Protein (ACP). Enzymes: acetyl-CoA:ACP transacylase; malonyl-CoA:ACP transacylase. The original CoA’s are replaced by ACP designation.
• Step 1. Condensation. Acetyl-C0A:ACP and malonyl-CoA:ACP join to form a 4-carbon chain via fatty acid synthase. Enzyme: fatty acid synthase. A 2-carbon keto-acyl-ACP is formed. Released: CO3.
• Step 2. Reduction. Convert the carbonyl carbon adjacent to the omega carbon from a double bonded to O to a single bond OH group. Converts keto-acyl-ACP to hydroxyacyl-ACP. Enzyme: fatty acid synthase. Spends: NADPH + H+ to NADP+.
• Step 3. Dehydration. Need to remove the hydroxyl group at the carbon adjacent to the omega carbon. Forms a trans double bond between the second and third “to last” carbons in the chain. An enoyl is formed. Enzyme: fatty acid synthase. Released: H2O.
• Step 4. Reduction. Get rid of the double trans bond from step 3 via reduction to form a fully saturated 4-carbon chain. Enzyme: fatty acid synthase. Spends: NADPH + H+ to NADP+.

Release Palmitate.

• Fatty acid synthase can make up to a 16 carbon chain.
• Palmitate is a 16 carbon chain.
• When the cycle repeats and makes a 16-carbon long chain, thioesterase hydrolyzes it and frees up palmitate.
• Palmitate itself can then elongate or desaturate (convert double bonds to single bonds).

Elongation an Desaturation.

• After 16 carbons, elongase takes over. Elongase functions very much like FA synthase.

Regulation of Fatty Acid Synthesis.

• Acetyl CoA carboxylase (ACC) is controlled by: glucagon, epinephrine, insulin.
• ACC & glucagon. If glucagon levels are high, that means the body needs more blood sugar. It’s in a fasting state indicating that it is NOT the right time to be making fatty acids. Glucagon is an inhibitor.
• ACC & epinephrine. Increased levels of epinephrine inhibit ACC. Increased levels of epinephrine indicate that the body needs more blood sugar (fight or flight). That is NOT the right time to make FAs.
• ACC & insulin. Increased levels of insulin indicate that the blood has too much sugar floating around and insulin tells the cells to let blood sugar in (store or use). Insulin stimulates ACC which stimulates the production of FAs.
• Other regulation types: citrate, palmitoyl CoA, AMP.
• ACC is controlled via phosphorylation.
• Insulin –> stimulates FA –> causes dephosphorylation of ACC.
• Glucagon/epinephrine –> inhibits FA –> causes phosphorylation of ACC.
• Protein kinase (AMP-PK): activated by AMP; inhibited by ATP.
• ACC is inactivated when ATP is low.
• Citrate allosterically activates ACC.
• Citrate levels are high when acetyl CoA and ATP levels are high.
• Isocitrate dehydrogenase is inhibited by ATP.
• Carboxylase is allosterically inhibited by palmitoyl CoA.
• Global regulation: +insulin, -glucagon, -epinephrine.
• Local regulation: +citrate, -palmitoyl CoA, -AMP.
• Formation of malonyl CoA inhibits carnitine acyltransferase I

Global Regulation.

• If energy is low, then it is not the right time to make fatty acids.
• ACC is inactivated when ATP levels are low.
• Global regulations is achieved via reversible phosphorylation.
• Aceytyl CoA is inhibited by phosphorylation and stimulated by dephosphorylation.
• Insulin stimulates FA synthesis via dephoshorylation of ACC.
• Glucagon and epinephrine inhibit ACC via phosphorylation.
• Protein kinase is stimulated by AMP and inhibited by ATP.

Local Regulation.

• Citrate allosterically activates acetyl CoA.
• Citrate levels are high when acetyl CoA and ATP levels are in excess (isocitrate inhibited by ATP).
• Palmitoyl CoA allosterically inhibits carboxylase.

Fed.

• Increased insulin levels.
• Inhibit hydrolysis of triacylglycerides.
• Stimulates increase of malonyl CoA.
• Increased malonyl CoA inhibits carnitine acyltransferase I.
• FA stay in cytosol (FA oxidation enzymes are in mitochondria); so FA do not oxidize.

Fasting.

• Epinephrine and glucagon at increased levels.
• They stimulate adipose cell lipase.
• Levels of free FA increase.
• Inhibit ACC to decrease formation of malonyl CoA (causes more FA transported to mitochondria for beta-oxidation).

Cholesterol Metabolism.

• Cholesterol = steroid nucleus (sterane) that contains lipid and has a hydrocarbon tail.
• Cholesterol is a lipid.
• 4-ring steroid nucleus. It’s very hydrophobic.
• Ring A is a hexagon with OH at carbon-3 position.
• Ring B is a hexagon with a CH3 (methyl) at position carbon-10. The methyl’s carbon is #19.
• Ring C is a hexagon with a CH3 (methyl) at position carbon-12. The methyl’s carbon is #18.
• The hydrocarbon tail would be at carbon-17.
• A double bond from carbon-5 to carbon-6
• 27-carbon non-glyceride lipid.
• Amphipathic: polar OH at C3; non-polar nucleus and tail at C17.
• Made by most body cells: liver, intestines, cortex, brain, reproductive tissues.
• Plasma cholesterol is often esterified (cholesterol ester at C3).

Phytosterol.

• Plants don’t use cholesterol. Mammals only make cholesterol.
• The plant-equivalent of cholesterol is phyosterol.

Flow of Major Cholesterol Intermediates: Number of Carbons and Number of Acetyl CoA.

Stage 1. Acetyl CoA to Mevalonate (2-carbons to 6-carbons).

• 3 x acetyl CoA (2-carbon) –> mevalonate (6-carbon).
• Enzymes: thiolase, HMG-CoA synthase, HMG-CoA reductase.
• Cost: 2 NADPH.
• Leaving: 3x CoA-SH.
• Thiolase to cleave out the thiol and CoA groups. Also joining the first two acetyl CoA’s.
• CoA-SH is leaving group.
• B-Hydroxy-B-methylglutaryl-CoA Synthase (HMG-CoA synthase) to join the third acetyl CoA group and another CoA-SH leaves.
• HMG-CoA reductase uses 2 NADPH to get rid of the last CoA-SH group.
• B-Hydroxy-B-methylglutaryl-CoA is the rate-limiting step.

Stage 2. Mevalonate to Isopentenyl Pyrophosphate (IPP) (6-carbons, -1, 5-carbons).

• Enzymes: mevalonate-5-phosphotransferase, phosphomevalonate kinase, kinase, pyrophosphomevalonate decarboxylase, isopentenyl pyrophosphate isomerase.
• Cost: 3 ATP.
• Leaving: CO2, Pi.
• The CO2 is the -1 carbon from the mevalonate.

Stage 3A. IPP to Farnesyl Pyrophosphate.

• Need 6x mevalonate (5 carbon chain, needed 3 acetyl CoA) to make squalene (30 carbon chain, 18 acetyl CoA)
• Enzymes: cis-prenyl transferase.
• Cost:
• Leaving: 2 PPi.
• The end of stage 3A gives us a 15 carbon long chain (or 3 isoprenes) farnesyl pyrophosphate.

Stage 3B. Farnesyl Pyrophosphate to Squalene.

• Enzymes: squalene synthase.
• Cost: NADPH + H+.
• Leaving: 2 PPi.

Stage 4. Squalene to Lanosterol (both are 30 carbons long).

• Enzymes: squalene monooxygenase, cyclase.
• Cost: NADPH + H+, O2.
• Leaving: NADP+, H2O.
• This step is important to close the ring.

Stage 5. Lanosterol to Cholesterol (30 carbons to 27 carbons).

• Multistep.

Hydrolyzable	Non-Hydrolyzable
Esters	Hydrocarbons
Fats	Carotenoids
Waxes	Alcohols
Sterol Esters	Sterols
Phospholipids	Steroids
Phosphatidates	Acids
Phosphtids	Fatty Acids
Spingolipids	Eicosanoids
Glycolipids

Regulation of HMG-CoA Reductase.

• This is the rate-limiting enzyme to make cholesterol and other isoprenoids.
• It is a transmembrane (a type of integral membrane protein that spans the membrane) protein.
• It is anchored in the endoplasmic reticulum membrane.
• It has 8 domains.
• It is the target of many statin drugs (lipid lowering).
• Inihibits production of cholesterol in liver.
• Phosphorylated form: Inactive.
• DePhosphorylated form: Active.
• Insulin/thyroxine stimulates HMG-CoA reductase>>dephosphorylate HMG-CoA.
• Glucagon/cortisol inhibits HMG-CoA reductase>>phosphorylate HMG-CoA.
• If there’s high cellular cholesterol, the gene to make more HMG-CoA reductase enzyme is suppressed. This results in fewer and fewer copies of the HMG-CoA reductase enzyme and hence, the production of cholesterol slows down.

Statins, Muscle Pain, Ubiquinone (CoQ).

• It is unknown why statins may cause muscle pain in some people.
• CoQ supplementation may be helpful and CoQ is relatively “safe”.

Cholesterol Esterification.

• Cholesterol may “hitch a ride with” lipoproteins, but that limits cholesterol to the surface or lipid portion of those lipoproteins.
• Esterifying cholesterols allow them to become more incorporated or at least, not limited to reside “on the surface”.
• Esterification allows cholesterols more access to the interiors of lipoproteins or other structures that have both the hydrophobic and hydrophillic characteristics.
• Esterification also allows for greater carrying capacity of lipoproteins.
• Esterification converts cholesterol to an even more hydrophobic form.
• This is done in the liver via enzyme acyl-CoA-cholesterol acyl transferase (ACAT).

Bile Acids and Bile Salts.

• Bile is a mix of: water, electrolytes, organic materials, cholesterol, lipids, phospholipids, bilirubin and other waste products that get secreted into the bile and excreted.
• Bile helps us breakdown food.
• Bile helps us absorb fat soluble vitamins A, D, E, K.
• Adults can make 400-800 ml of bile/day.
• Liver cells (hepatocytes) make bile which run down the canaliculi ducts to the bile ducts. During this journey, bicarb is added into the mixture.
• Bile ends up in the gall bladder which helps to concentrate the mixture (during the body’s fasting state). Bile can be 5x more concentrated.
• To get rid of excess cholesterol, the bile is the way to go. The bile helps make cholesterol less hydrophobic via bile acids and lipids (e.g. lecithin). Cholesterol gets trapped in all the muck and can excrete it.
• Bile acids/salts can emulsify fats and take on a micelle form.
• Cholesterol can be converted into bile cholic (2nd most abundant) and chenodeoxycholic (most abundant) acids.
• Cholic and chenodeoxycholic acids can be conjugated to glycine or taurine. This would make the “active forms”.
• Primary bile acids made in liver via cholesterol made in the liver: cholic and chenodeoxycholic acids.
• These primary bile acids travel into the small intestine where bacteria reacts with the primary acids. They undergo dehydroxylation (a heating reaction where OH is released as water). The result are the secondary bile acids.
• Secondary bile acids: Deoxy Cholic Acid (Deoxy CA or DCA) and Litho Chenodeoxy Acid (Litho CA or LCA)
• To make bile acids…the rate determining step is:
  Cholesterol –7alpha-hydrolase–>7alpha-hydroxycholesterol.
• 7alpha-hydroxycholesterol is the precursor to bile acids.
• The glycine or taurine can be found attached onto the 24th carbon of the hydrocarbon tail.
• Bile salts glycocholic acid and taurocholic acid.
• Bile salts help digestion, eliminate waste products, help absorb fat-soluble vitamins and other components that the body needs.

Vitamin D, Calcitriol.

• Vitamin D3 (cholecalciferol) is made in the skin in the presence of sunlight.
• 25-hydroxycholecalciferol is made from cholecalciferol
• In the liver, Cholecalciferol –25-hydroxylase–> 25-hydroxycholecalciferol
• In the kidney with enzyme 1-alpha-hydroxylase helps convert 25-hydroxycholecalciferol into the active form of vitamin D, 1,25-dihydroxycholecalciferol.
• Sources of natural cholecalciferol (vit D3): fish and meat.
• Sources of ergocalciferol (vit D2): plants and fungi.

5 Types of Steroid Hormones Derived from Cholesterol.

• Mineralocortictoids. Made in adrenal cortex. Eg. Aldosterone C21 (conservation of Na+).
• Glucocorticoids. Made in almost every mammalian cell. Eg. cortisone C21.
• Androgens. Male testes. Eg. testosterone C19.
• Estrogens. Ovaries. Eg. Estradiol
• Progesterone. Ovaries, placenta, adrenal gland.

Lipoproteins (transport cholesterol).

• Lipoproteins are proteins that can help transport lipids.
• Types of lipids that may be transported via lipoprotein: fatty acids, triacylglycerols, phospholipids, free cholesterol, cholesterol ester, fat-soluble vitamins.
• 5 main types of lipoproteins:
  1.Chylomicron. Transport dietary lipids from intestines to other places in the body.
  2. VLDL, very low density lipoprotein. Carry cholesterol to tissues.
  3. IDL, intermediate density lipoprotein. Functions like LDL, carrying cholesterol and fats.
  4. LDL, low density lipoprotein. Can buildup in vessels.
  5. HDL, high density lipoprotein. Carry cholesterol back to liver.
• Density and size are inversely proportional (denser, smaller vs. less dense, fluffier and bigger).
• Liver: makes VLDL and HDL.
• Intestine: chylomicron, HDL.
  

Apolipoproteins (bind lipids to proteins to make lipoprotein).

• Apo A1. HDL.
• Apo B48. Chylomicron.
• Apo B100. VLDL, IDL, LDL.
• Apo C2. VLDL, chylomicron.
• Apo E. IDL, chylomicron remnants.

Hormones.

• Hormones are chemical messengers (from the endocrine system) that help regulate the body and maintain homeostasis.
• Some hormones travel all over the body via bloodstream.
• Some hormones are more localized.

Fed & Fasting States.

• Hormones are chemical messengers. Released from the endocrine glands, they get secreted into blood.
• Pancreas > Islet of Langerhans (beta cells) makes insulin.
• Pancreas > Alpha cells make glucagon.
• Pancreas > Delta cells make somatostatin.
• Note somatostatin: It’s made in more than one place. In the pancreas, somatostatin can inhibit insulin and glucagon. Somatostatin that’s produced in the pancreas affects/regulates the pancreatic hormones.

Insulin.

• Insulin dominates the FED state.
• Stimulates: glucose oxidation; glycogen synthesis; fat synthesis; protein synthesis.
• Insulin is a polypeptide derived from the prohormone proinsulin.
• The C-peptide is cleaved from the insulin precursor.
• Insulin has two chains connected via disulfide bridges.
• Insulin stimulates tissues (esp. liver, muscle, adipose) to increase the uptake of glucose and amino acids. If the glucose/amino acids isn’t needed for immediate use, then package it into a storage form.
• Insulin stimulates glycogen formation (for stored energy).
• Insulin increases FA synthesis in liver.
• Stimulates potassium uptake.
• Inhibits glucagon production.

Regulators of Insulin.

• +Blood glucose. When blood glucose is abundant, insulin is activated to tell cells to use/store the blood glucose.
• +Amino acids.
• +Neural input.
• +Gut hormones.
• -Epinephrine (adrenergic).

Regulators of Glucagon.

• -Glucose.
• -Insulin.
• -Amino acids.
• +Cortisol.
• +Neural (stress).
• +Epinephrine.

Other Regulators.

• Glucocorticoids (cortisol): stimulate gluconeogenesis and lipolysis and increase protein breakdown.
• Epinephrine/norepinephrine: stimulate glycogenolysis and lipolysis (exercise).
• Growth Hormone: stimulates glycogenolysis and lipolysis.

Thought Exercise: What Happens Right After a Meal vs. What Happens a Few Hours After a Meal??

Digestion.

• Digestion is the process of converting and breaking down food into simpler components that the body can absorb (and deal with) and use.
• The digestive/GI tract is where digestion-absorption takes place.
• The route is: mouth, esophagus, stomach, small intestine, large intestine.
• Denaturing: is unfolding of proteins or altering the structure in 3D space without breaking bonds.
• Breaking down: is snipping or cutting protein via break bonds.

Digestive Path.

• Salivary glands. Start salivating to help lubricate foodstuffs.
• Mouth. Amylase helps start the breakdown of carbs. Mechanical and chemical breakdown starts.
• Pharynx. Swallows foods/liquids.
• Esophagus. Transports food.
• Stomach. Churns foods. Pepsin helps break down protein. Stomach HCl helps breakdown food and kill germs. Mucosal lining helps protect the stomach and our tissues from the harsh acidity.
• Liver. Makes bile. Filters nutrients. Stores vitamins and iron. Destroys wastes and toxic materials.
• Gall Bladder. Stores and concentrates bile.
• Pancreas. Makes hormones. Bile ducts pass through to empty bile in duodenum of the small intestine. Hormones insulin and glucagon. Add bicarb to the bile and help neutralize stomach acid. Trypsin and chymotrypsin help digest proteins. Amylase helps digest polysaccharides. Lipase digests lipids.
• Small Intestine. Absorb most of our stuff.
• Large Intestine. Absorb most of our water.
• Rectum.
• Anus.
• Pancreatic amylase: helps break down dietary carbohydrates.
• Brush border disaccharidases: enzymes that help break down disaccharides in the small intestinal wall.
• Disaccharides : maltose (glucose x2); sucrose (glucose + fructose); lactose (glucose + galactose).

Resources.

• FA elongation & denaturation, https://youtu.be/eG08eClNoXA
• Fatty acid synthesis (Moof), https://youtu.be/w1cAMmXhJGs
• Fatty acid synthesis, part 1, https://www.khanacademy.org/test-prep/mcat/biomolecules/fat-and-protein-metabolism/v/fatty-acid-synthesis-part-i
• Fatty acid synthesis, https://www.khanacademy.org/test-prep/mcat/biomolecules/fat-and-protein-metabolism/v/fatty-acid-synthesis-part-ii
• Fatty acid synthesis, https://bio.libretexts.org/Bookshelves/Cell_and_Molecular_Biology/Book%3A_Cells_-_Molecules_and_Mechanisms_(Wong)/6%3A_Metabolism_II_%E2%80%93_Anabolic_Reactions/6.6%3A_Fatty_Acid_Synthesis
• Fatty acid synthesis, https://www.diapedia.org/metabolism-and-hormones/5105592814/fatty-acid-oxidation-and-synthesis
• Cholesterol, https://www.cdc.gov/cholesterol/index.htm
• Cholesterol, http://watcut.uwaterloo.ca/webnotes/Metabolism/Cholesterol.html
• Cholesterol, https://bio.libretexts.org/Bookshelves/Biochemistry/Book%3A_Biochemistry_Free_and_Easy_(Ahern_and_Rajagopal)/06%3A_Metabolism_I/6.08%3A_Cholesterol_Metabolism
• Cholesterol, https://youtu.be/3utYm3ouzaU
• Cholesterol, https://youtu.be/nurZ2nNovp8
• Cholesterol from the Biochem channel, https://youtu.be/seRkOCEfWpo
• HMG-CoA reductase, https://www.healio.com/cardiology/learn-the-heart/cardiology-review/topic-reviews/hmg-coa-reductase-inhibitors
• Cholesterol esterification, https://www.sigmaaldrich.com/technical-documents/articles/biofiles/cholesterol-esterification.html
• Bile, http://www.vivo.colostate.edu/hbooks/pathphys/digestion/liver/bile.html
• Cholic acid, https://en.wikipedia.org/wiki/Cholic_acid
• Chenodeoxy acid, https://en.wikipedia.org/wiki/Chenodeoxycholic_acid
• 7-alpha-hydroxycholesterol, https://themedicalbiochemistrypage.org/bileacids.php
• Vitamin D, http://www.vivo.colostate.edu/hbooks/pathphys/endocrine/otherendo/vitamind.html
• Lipoproteins, https://www.mabtech.com/knowledge-center/applied-research/apolipoproteins
• Insulin, https://www.yourhormones.info/hormones/insulin/
• Digestion, https://opentextbc.ca/anatomyandphysiology/chapter/23-7-chemical-digestion-and-absorption-a-closer-look/

References.

Bean, J. (2019). Cholesterol metabolism.

Bean, J. (2019). Fatty acid synthesis.

Bean, J. (2019). Fed and fasting states.

Ferrier, D. (2017). Biochemistry (7th ed.). Philidelphia, PA: Lippincott Illustrated Reviews.

Lieberman, M., & Peet, A. (2017). Marks’ basic medical biochemistry: A clinical approach(5th ed.). Philadelphia, PA: LWW.

Last edited: 08.24 .2019

Outline.

• Glycogen Metabolism
• Fatty Acid Metabolism
• Ketone Body Metabolism
• Amino Acid & Nitrogen Metabolism

Glycogen.

• Glycogen is stored in the cytosolic granules of the muscle and liver cells.
• Glycogen is important to the body particularly for the CNS because the CNS is an obligate user of glucose.
• IN THE LIVER, glycogen is broken down or stored in order to maintain the correct balance of blood sugar. There’s about 100g or about 10% glycogen in fresh weight well fed adult liver.
• IN THE LIVER, the enzyme that transforms glucose into glucose 6-phosphate is called glucokinase.
• IN THE MUSCLES, glycogen is used for energy especially for anaerobic high intensity bouts. There’s about 400g glycogen or 1-2% of fresh weight muscle.
• IN THE MUSCLES, the enzyme that transforms glucose into glucose 6-phosphate is hexokinase.
• Glycogen is a branched-chain polymer made up of glucose monomers linked up via alpha-1,4-glycosidic bonds in a straight chain, and alpha-1,6-glycosidic bonds at branches.
• There are more alpha-1,4-glycosidic bonds than the alpha-1,6 bonds.
• After about 8-14 glucose residues, a branch occurs.
• Nonreducing ends. The 1,4-glycosidic bonds dominate the structure and their “ends” have a free hydroxl group at carbon 4. These are called the nonreducing ends.
• Reducing end. The rightmost oriented glucose has a free anomeric carbon and is called the reducing end. This reducing end is usually attached to a protein (glycogenin).
• Reducing sugars: sugars that can freely open and close their ring-form.
• Is the anomeric carbon free? If something is bonded to the sugar at the anomeric carbon (carbon 1) this “blocks” the sugar from freely opening and closing its ring form. Thus the sugar is not reducible.
• When studying glycogen, a protein is often bound to the glycogen at the carbon 1 position. This improves stability as the sugar cannot freely “shift” between open- and closed-ring forms.
• Glycogenin: is an enzyme/protein that serves as the “head” or “anchor” where glycogen chains and branches can attach. Glycogenin is attached to the first glucose in a glycogen chain. Glycogenin initiates glycogen synthesis. Afterwards, glycogen synthase takes over.
• Futile cycling: aka substrate cycling; substrate gets converted into product via one pathway and then the product gets converted back to the substrate in another pathway.

Glycogenesis (Glycogen synthesis).

• Glycogenesis occurs in the cytosol.
• Glycogen is made from alpha-D-glucose.
• ATP and UTP (uridine triphosphate) are used for energy in this process.
• 3 enzymatic steps convert G6P into glycogen.
• Glycogen synthase is the major regulatory step. It makes alpha-1,4 bonds.
• UTP/UDP are carriers.
• Mutase is a special form of isomerase. Mutases take a functional group at one position and moves it to another position.

Uridine-5′-triphosphate ( C₉H₁₅N₂O₁₅P₃ ).

Step 1.

• In the liver, glucose-6-phosphatase may cleave a glucose monomer of glucose 6-phosphate so that a glucose molecule may enter the bloodstream and be used by cells for energy.
• glucose 6-phosphate + H2O –glucose-6-phosphatase–> glucose + Pi

Step 2.

• UDP-glucose is the activated form.
• UDP-glucose is the form which is used directly to form glycogen.
• Glucose 1-phosphate has one phosphate group.
• Take UTP, free two phosphates from it as PPi.
• Then attach UMP (monophosphate) to glucose 1-phosphate to get UDP-glucose (which now has 2 phosphate groups, one from previously and one from the UTP).
• Cleavage of the phosphate bonds give enough energy to attach monomers to the glycogen chain.

Step 3.

Glycogen Synthesis Enzymes.

• Glycogenin. Gets the process started and then glycogen synthase takes over. Glycogenin: is an enzyme/protein that serves as the “head” or “anchor” where glycogen chains and branches can attach. Glycogenin is attached to the first glucose in a glycogen chain. Glycogenin initiates glycogen synthesis. Afterwards, glycogen synthase takes over.
• Glycogen synthase. Adds a glucose monomer to the chain (elongation process). Creates alpha-1,4-linkages only. Adds one glucose unit to an existing chain of at least 8 molecules (elongation).
• 1,4-Alpha-Glucan Branching enzyme. Takes 6-8 residues at a time and forms a new branching point. Creates alpha-1,6-linkages only. Branching is important in that it creates a lot more terminals and potential sites for phosphorylation and synthesis (make it easier to multitask by increasing more sites/terminals). Also increases solubility by making more “pockets” for water molecules to get into.
• Putting a phosphate group onto something can alter it’s function and activate or deactivate it.
• Rarely do we say “turn on” or “turn off”, because it’s not that black and white.
• Glycogen phosphorylase. It is a dimer with quaternary structure. It has one copy “above” and one copy”below”. Specifically targets alpha-1,4-linkages. It cleaves off a glucose 1-phosphate. Once a glycogen chain gets down to 4 units of glucose monomer, glycogen phosphorylase cannot work. That’s where the debranching enzyme comes in.

Glycogenolysis.

• Catabolic/breakdown of glycogen to glucose units so that the glucose can enter the bloodstream and be utilized or so that glucose can be utilized in the muscle.
• This process uses Pi to cleave a glucose from glycogen (it’s not a hydrolysis reaction). It takes energy to cleave that glucose out.
• This cleavage using Pi is called phosphorylation.
• Glycogen phosphorylase is the enzyme that catalyzes this phosphorylation.

Glycogen Debranching Enzyme.

• Another way to catabolize glycogen.
• Glycogen phosphorylase (previously studied) was able to cleave a terminal glucose unit off of glycogen. Note that this unit is in the form glucose 1-phosphate.
• Glycogen phosphorylase CANNOT act on any branches that are less than 5 glucose monomers long.
• Glycogen debranching enzyme is a hydrolase (use water to cleave) NOT phosphorylase (using phosphate to cleave).
• Glucose is the result of cleavage and NOT glucose 1-phosphate.
• This enzyme has both a 4:4 transferase activity and a glucosidase activity.
• Once a branch has been pruned such that there are only 4 glucose units on it, the debranching enzyme (the 4:4 transferase role) will prune the 3 terminal units of glucose from that branch and attach them to a longer branch/chain.
• Then the debranching enzyme (glucosidase role) will prune off that lone glucose unit, making it available in the cell/bloodstream. Note that it is glucose and NOT glucose 1-phosphate or glucose 6-phosphate. It’s a straightup glucose.
• Note that the glucosidase role of the debranching enzyme can only act to prune off a glucose unit that is 1 unit long. For example, if there is a branch of 2 glucose units, the debranching enzyme cannot act on it.
• Note that the glucosidase role is a hydrolase. It uses water to cleave the glucose unit and set it free.

Regulation of Glycogen Metabolism.

• Glycogen synthase and glycogen phosphorylase help regulate glycogen metabolism.
• Both are allosteric regulators and respond to hormonal control (via phosphorylation and dephosphorylation).
• Glycogen synthase is activated by glucose 6-phosphate (indicating glucose levels are high).
• Glycogen phosphorylase is inhibited by high levels of glucose 6-phosphate and activated with high levels of AMP (indicating we need more energy).

Insulin.

• Insulin is a hormone that responds to high blood glucose levels.
• When blood glucose levels are high, insulin inhibits glycogen phosphorylase (don’t free up any more glucose because we don’t need it).
• Insulin activates glycogen synthase.
• Insulin is associated with the fed state.

Glucagon and Epinephrine.

• These are active when there is low blood glucose.
• These hormones inhibit glycogen synthase (we don’t want to store energy, we need energy to use now).
• These hormones activate glycogen phosphorylase.
• Associated with fasting state and/or exercise states.

Fatty Acid Metabolism (Oxidation).

• What are fatty acids (FAs)? They are a carboxylic acid head with a long alipathic (non-aromatic) hydrocarbon tail.
• What are fatty acids good for? Storage of energy; building blocks for other structures like phospholipid and glycolipids; used in eicosanoids structure and secondary messengers.
• Triacylglycerol = triglyceride = glycerol + 3 FA chains.
• Triacylglycerol is the simplest form of lipid.
• FAs are very reduced and have lots of potential for oxidation (great source of potential energy).
• FA oxidation produces NADH and FADH2 which can then be used in ETC to make lots of ATP.
• Fats are efficient energy storage.
• Carbohydrates = 4 kcal/g
• Fats = 9 kcal/g
• Advantages of fats over polysaccharides: FA has more energy per carbon because FAs are more reduced; FAs carry less water because they are nonpolar.
• Short-term energy, “quick” delivery: glucose and glycogen.
• Long-term energy, slower delivery: fats.
• 1/3 of our energy needs are from triacylglycerols.
• The CNS and red blood cells cannot use FA’s because they don’t have the machinery to process and utilize FAs.

Fatty Acid Breakdown.

• Bile salts. Made in the liver, trickle through bile ducts, and stored in the gallbladder until they are released into the duodenum. The gall bladder helps to concentrate the bile. Act as emulsifiers and keep fat from making large clumps of itself. As emulsifiers, help to increase the surface area of fat “droplets” so that enzymes can have an easier time to reach and act on the lipids. Without bile salts, enzymes may not be as efficient acting on large clumps of fats. Emulsifiers also help “fat droplets” move through the body better. Large fat clumps could potentially cause fatal blockages. Bile salts help to “solubilize” lipids/triacylglycerol.

• Lipases. Lipases are made in the pancreas and they help to hydrolyze ester bonds (RCOOR) of triacylglycerols.
• FAs and two monoacylglycerols can then cross the plasma membrane of epithelial cells.

Triglyceride Digestion & Absorption.

• What kinds of lipids are in our diet? Triglycerides (triacylglycerols), fat-soluble vitamins, sterols (e.g. cholesterol), and “neutral fats”.
• What needs to happen in order for lipids to serve our needs? Because lipids are hydrophobic and generally insoluble in water, lipids physically need to be broken down into teeny tiny particles held in a suspension so that they can travel in the body which is largely an aqueous environment. Then enzymes need to act on the lipid particles in order to convert them to a form that the body can use/absorb.
• Digestion starts at the mouth.
• Mouth: lingual lipase initiates the breakdown of lipids/fats.
• Gall bladder – Liver: bile salts (amphipathic meaning they have both hydrophilic and hydrophobic qualities) are made in the liver and secreted/concentrated in the gall bladder. These bile salts are then released in the duodenum.
• Pancreas: secretes HCO3-, lipase colipase (a protein coenzyme that aids the pancreatic lipase). Pancreatic lipase is largely responsible for the bulk of breaking down lipids into tiny particles. Pancreatic lipase is also water soluble.
• Down in the stomach, the gastric lipase helps to break triacylglycerols into diacylglycerols…then break those down into fatty acid chains. The stomach churning and movement down into and through the intestines help keep the lipids emulsified (along with the bile salts, pancreatic lipase and colipase).
• The bile salts help to surround the fats to form micelles–fatty acid core with a more water-soluble “shell” or exterior. Bile salts are like detergents.
• Intestinal cells (enterocytes), are able to absorb the FAs and monoacylglycerols via FA protein transporter (to cross the cell membrane). The monoacylglycerols and fatty acids reassemble to form triacylglycerols. They are then attached to a protein carrier (lipoprotein).
• Inside the cell, the FAs and monoacylglycerols goto the endoplasmic reticulum so that they can be used to build triglycerides (triacylglycerols).
• From the ER to the Golgi Complex (of the intestinal cell, enterocyte) in assembly-line fashion, the triglycerides are packaged with cholesterol and lipoproteins into what’s called a chylomicrons.
• This combination of lipoproteins (1-2%), cholesterol (1-3%), phospholipid (6-12%), and core of triacylglycerols (85-92%) result in what is called a chylomicron (like a “super-sized” micelle). Chylomicrons exit the enterocyte via exocytosis and can pass into the lymphatic system and out into the bloodstream. Once in the bloodstream, the chylomicrons can disassemble to be used.
• Hormones can signal for lipolysis in adipose tissues: epinephrine, glucagon, cortisol promote lipolysis.
• Insulin inhibits lipolysis.
• FAs are bound to serum albumin for transport around the body.
• The lipases break down chylomicrons into glycerol and FAs.
• Glycerol can get recycled to D-glyceraldehyde 3-phosphate which is an important intermediate for both glycolysis and gluconeogenesis.

Fatty Acid Activation.

• To oxidize FAs, first attach them to CoA and spend 2 ATP.

Getting the Fatty Acid into the Matrix.

• The fatty acyl CoA is shuttled via carnitine from the cytosol through the outer mitochondrial membrane (leaky) to the inner mitochondrial membrane.
• Carnitine palmitoyltransferase I (CPTI) enzyme is located on the outer mitochondrial membrane. It packages the fatty acyl CoA with carnitine.
• The fatty acyl CoA-carnitine is transported via carnitine translocase (enzyme) into the matrix.
• Carnitine palmitoyltransferase II (CPTII) located on the inner mitochondrial membrane then strips off the carnitine. The carnitine goes back out to the cytosol to be recycled. Then the fatty acyl CoA now present in the matrix can undergo beta-oxidation.

Beta-Oxidation.

• Beta-oxidation is so named because the activity is at the beta-carbon site.
• This is the catabolic pathway of fatty acids.

• Step 1. Oxidation. Makes 1 FADH2.
• Step 2. Hydration.
• Step 3. Oxidation. Makes NADH + H+
• Step 4. Thiolysis tomake one acyl CoA and one acetyl CoA

Calculating Energy Exchange of Fatty Acid Oxidation. What do you really get?

• Given n number of carbons.
• You have n/2 pairs of carbons.
• You cut the n long chain with (n/2)-1 many “snips”.
• You have (n/2)-1 many FADH2.
• You have (n/2)-1 many NADH and (n/2)-1 many H+
• You have n/2 many acetyl CoA.
• Each acetyl CoA will go through the TCA cycle to produce: 3 NADH, 1 FADH2, 1 GTP.
• You will go through n/2 rounds of the TCA cycle to produce: n/2 * 3 NADH; n/2 * 1 FADH2; n/2 * 1 GTP.
• FADH2 makes 1.5 ATP
• NADH makes 2.5 ATP
• 1 GTP makes 1 ATP
• As a shortcut you can think of each acetyl CoA that goes through the TCA makes 10 ATP.

What do you do with an odd-numbered FA chain?

Don’t panic. Odd numbered FA’s make propionyl-CoA. The last fragment of 3 carbons is called propionyl-CoA. HCO3- (bicarb) and ATP are added (coenzyme biotin). Coenzyme B12 is also active to make 4-carbon succinyl-CoA.

Let’s work an odd-numbered problem.

• Say you have a 17 carbon FA.
• (17/2) – 1.5 = 7 acetyl CoA.
• You can do the math for the 7 acetyl CoAs.
• REMEMBER to subtract the “cost” of 2 ATPs.
• But what’s left over?
• Propionyl undergoes carboxylations with biotin and B12 to produce succinyl-CoA.
• Add 5 ATP for the propionyl.

https://youtu.be/_MiJLxjh-OE

Fatty Acid Oxidation: Animals vs. Plants.

• Animals can’t use FAs to make glucose. Acetyl CoA can’t be converted to oxaloacetate.
• Plants have enzymes that let them convert acetyl CoA to oxaloacetate.

Ketone Body Metabolism.

• When lipid and carbohydrate metabolism are fairly balanced, most of the acetyl CoA from the FA beta-oxidation goes through the TCA cycle.
• Remember that the first step in the TCA cycle is oxaloacetate + water + acetyl CoA –citrate synthase–> citrate.
• BUT what if oxaloacetate supply is low? What do we do with the extra acetyl CoA?
• Oxaloacetate supply may be low if carbohydrates are scarce (starvation) or improperly used (misappropriated) such as diabetes.
• Oxaloacetate can be made from pyruvate via pyruvate carboxylase.
• Acetyl CoA drives ketone body production. When acetyl CoA levels persistantly rise, this triggers ketogenesis.

What are ketone bodies?

• Ketone bodies are 3 molecules produced in the mitochondrial matrix of hepatocytes (liver cells).
• These 3 molecules are: acetone, acetoacetate, and beta-hydroxybutyrate.
• The “making of” these 3 molecules is called ketogenesis. This occurs naturally in small amounts.

• When the body system is out of whack and acetone, acetoacetate, and/or D-beta-hydroxybutyrate are accumulated in eXcess, we call this state ketosis.
• When lots more ketone bodies accumulate in eXcess and the body’s pH is drastically lowered to acidic levels, we call this ketoacidosis.

Synthesis of Ketone Bodies.

• B-3-hydroxybutyrate is the dominant product and is useful for a source of CoA.
• Step 1. Two acetyl CoA get joined into 1 acetoacetyl CoA. This is a condensation reaction. CoA is a leaving group (and one CoA stays).
• Step 2. Acetoacetyl CoA is joined to another acetyl CoA in a condensation reaction with water. One CoA is a leaving group.
• Step 3. One acetyl CoA is cleaved, removing the thiol. We’re left with a four-carbon molecule that B-hydroxybutarate dehydrogenase acts on.
• Step 4. End product is B-3-hydroxybutyrate.
• All this is going on in the mitochondrial matrix of hepatocytes (liver cells).
• So acetoacetate is spontaneously converted sometimes to acetone. Acetoacetate can also spend 1 NADH to form B-3-hydroxybutyrate.
• We can breathe acetone out (fruity breath).
• Acetoacetate and B-3-hydroxybutyrate can be peed out.

Ketone Body Oxidation (we made them, now we break them).

Amino Acid & Nitrogen Metabolism.

• Proteins are broken down into amino acids which gets absorbed in the small intestine via intestinal epithelial cells.
• Amino acids make proteins and other nitrogen-containing compounds.
• The nitrogens that aren’t used for building things get converted to urea and other nitrogenous waste products which get excreted and leave the body.
• Amino acids can make glucose.
• AA’s can be substrated for FA synthesis.
• Amino acids can also be used as a fuel source like the BCAA (branched chain amino acids) that many people use as supplements in muscle-building activities.
• Other nitrogen-containing compounds are: ATP, nucleotides, hormones, porphyrin rings.
• Key idea: how can we interconvert things, how many ways are there to interconvert things?

How to move Nitrogen around: Transamination.

• Transamination. N-exchange.
• Catalyst: Transaminases/amino-transferases.
• Req: coenzyme PLP aka P5P (vitamin B6).
• Take the amine from one molecule and switch it with another group from another molecule. Turns amino acid to alpha-keto-acids and vice versa.
• All transamination reactions are reversible.
• Reactant-aminoacid1 becomes Product-alphaketoglutarate2; Reactant-alphaketoglutarate1 becomes Product-aminoacid2.
• Alanine <–> Pyruvate
• Aspartate <–> Oxaloacetate
• Alpha-Keto-Glutarate <–> Glutamate
• Asp + alphaketoglutarate <–asp aminotransferase, AST/GOT, PLP–> OA + Glu
• Ala + alphaketoglutarate <–Ala aminotransferase, ALT/GPT, PLP–> Pyr + Glu
• Leu + alphaketoglutarate <–Leu aminotransferase, LET/GOT, PLP–> OIC(oxoisocaproate) + Glu

How to move Nitrogen around: Amination.

• N-addition: +NH3
• Condensation reaction.
• Add nitrogen. For example, add +NH3.
• Aspartate + NH3 –> Asparagine
• Alpha-keto-glutarate + NH3 –> Glutamate
• Glutamate + NH3 –> Glutamine

How to move Nitrogen around: Deamination.

• N-removal.
• Remove the nitrogen.

Fate of AA Carbons and Nitrogen.

• Carbons are used for energy.
• Nitrogen goes to the Urea cycle.
• One N in urea comes from NH4+ and the other N comes from Aspartate

Glucogenic & Ketogenic AA Degradation.

• Threonine > Glycine > [Alanine, Serine, or Cysteine] > Pyruvate.
• Tryptophan > [Alanine, Serine, or Cysteine] > Pyruvate.
• Pyruvate > Alanine.
• [Aspartate or Asparagine] > Oxaloacetate.
• [Aspartate, Tyrosine, or Phenylanine] > Fumarate.
• [Valine Threonine, Isoleucine, or Methionine] > Propionyl CoA.
• [Arginine, Histidine, Glutamine, or Proline] > Glutamate > Alpha-Keto-Glutarate.
• Leucine > HMG CoA
• [Phenylanine or Tyrosine] > Acetoacetate (ketone bodies)
• [Threonine, Lysine, Isoleucine, or Tryptophan] > Acetyl CoA

Urea (ornithine) Cycle.

• Happens only in the liver–part in liver cell’s mitochondria and part in liver cell’s cyctoplasm.
• One nitrogen comes from NH4+
• The OTHER nitrogen comes from Aspartate
• UREA is excreted (2x N).

Resources.

• Glycogen, http://watcut.uwaterloo.ca/webnotes/Metabolism/Glycogen.html
• Glycogen, http://www2.csudh.edu/nsturm/CHE452/04_Glycogen.htm
• Glycogen, https://academic.oup.com/nutritionreviews/article/76/4/243/4851715
• Glycogen, http://www.wou.edu/~guralnl/450Glycogen%20metabolism.pdf
• Glycogen, http://www.uh.edu/dtu/01-Glycogen-07.htm
• Glycogen, https://youtu.be/d-HgDkWK3P0
• Glycogen, https://youtu.be/P_5ubq6MikQ
• Glycogen I (OSU), https://youtu.be/Cuuy97TcBs0
• Glycogen II (OSU), https://youtu.be/GrxW9GXf6QU
• Glycogen, https://themedicalbiochemistrypage.org/glycogen.php#catabolism
• Glycogen, https://themedicalbiochemistrypage.org/glycogen.php#synthesis
• Glycogen, https://themedicalbiochemistrypage.org/glycogen.php#catabreg
• Glycogen, https://themedicalbiochemistrypage.org/glycogen.php#synthreg
• Glycogen, https://themedicalbiochemistrypage.org/glycogen.php#clinical
• Glycogen, https://themedicalbiochemistrypage.org/glycogen.php#storage
• Glycogen Debranching Enzyme, https://youtu.be/USCDwSmXpPU
• Fatty Acid, https://www.khanacademy.org/test-prep/mcat/biomolecules/fat-and-protein-metabolism/v/overview-of-fatty-acid-oxidation
• Fatty Acid, http://oregonstate.edu/instruct/bb451/451material/Keynotes/30FattyAcidMetabolism.pdf
• Fatty Acid, https://opentextbc.ca/anatomyandphysiology/chapter/24-4-lipid-metabolism/
• Fatty Acid, https://youtu.be/nBFSz63T1c0
• Fatty Acid, https://youtu.be/ooOx3G0JFi8
• Fatty Acid, https://youtu.be/__jS-pjzb5k
• Fatty Acid, https://youtu.be/ZufZvbhPpws
• Ketone, https://www.khanacademy.org/test-prep/mcat/biomolecules/fat-and-protein-metabolism/v/how-does-the-body-adapt-to-starvation
• Ketone, https://youtu.be/BlR9DkUAUkI
• Ketone, https://youtu.be/AK9Q2ClI8y4
• Ketone, https://youtu.be/IC9NmcaKX-w
• Ketone, https://www.rose-hulman.edu/~brandt/Chem330/Ketone_bodies.pdf
• Ketone, https://physoc.onlinelibrary.wiley.com/doi/full/10.1113/JP273185
• Ketone, https://www.dpag.ox.ac.uk/files/news/stubbs-b-et-al-front-physiol-oct-2017.pdf
• Ketone, https://www.researchgate.net/publication/313464452_Multi-dimensional_Roles_of_Ketone_Bodies_in_Fuel_Metabolism_Signaling_and_Therapeutics
• Amino acid, https://www.khanacademy.org/test-prep/mcat/biomolecules/fat-and-protein-metabolism/v/overview-of-amino-acid-metabolism
• Amino acid, http://watcut.uwaterloo.ca/webnotes/Metabolism/AminoAcids.html
• Amino acid, https://youtu.be/0M-B2dOfcUo
• Amino acid, https://youtu.be/EyHMLGKV4Wk
• Amino acid, https://www.youtube.com/channel/UCbYmF43dpGHz8gi2ugiXr0Q/search?query=amino+acid
• Nitrogen, https://media.oregonstate.edu/media/t/0_fbnugu4r
• Nitrogen, https://youtu.be/XmLzMCQ37iI
• Nitrogen, https://youtu.be/iNcglLxJ7mM
• Nitrogen, https://youtu.be/Y5xwfKdNS5s
• Nitrogen, https://youtu.be/XZEJ5PNvjkk
• Bile salts, http://www.vivo.colostate.edu/hbooks/pathphys/digestion/liver/bile.html
• Lipid digestion, https://med.libretexts.org/Bookshelves/Nutrition/Book%3A_An_Introduction_to_Nutrition_(Zimmerman)/05%3A_Lipids/5.4%3A_Digestion_and_Absorption_of_Lipids
• Carnitine Shuttle, https://med.libretexts.org/Bookshelves/Nutrition/Book%3A_Intermediate_Nutrition_(Lindshield)/6%3A_Macronutrient_and_Alcohol_Metabolism/6.3%3A_Lipid_Metabolism/6.32%3A_Fatty_Acid_Oxidation_(Beta-oxidation)
• Carnitine Shuttle, https://youtu.be/__jS-pjzb5k
• Carnitine Shuttle, https://youtu.be/ZufZvbhPpws
• The Odd Number Carbon, https://biochemistryquestions.wordpress.com/2008/12/14/beta-oxidation-of-fatty-acids-with-an-odd-number-of-carbons-i/
• Calculate the Even Number Carbon, https://youtu.be/oMnCckEvtBY
• Oxidation FA odd-number, https://youtu.be/_MiJLxjh-OE

References.

Bean, J. (2019). Amino acid nitrogen metabolism.

Bean, J. (2019). Fatty acid metaboslim.

Bean, J. (2019). Glycogen metaboslim.

Bean, J. (2019). Ketone body metabolism.

Ferrier, D. (2017). Biochemistry (7th ed.). Philidelphia, PA: Lippincott Illustrated Reviews.

Lieberman, M., & Peet, A. (2017). Marks’ basic medical biochemistry: A clinical approach(5th ed.). Philadelphia, PA: LWW.

Ferrier, D. (2017). Biochemistry (7th ed.). Philidelphia, PA: Lippincott Illustrated Reviews.

Lieberman, M., & Peet, A. (2017). Marks’ basic medical biochemistry: A clinical approach(5th ed.). Philadelphia, PA: LWW.

Last edited: 08.14.2019

Outline.

• Tricarboxylic Acid Cycle (TCA Cycle).
• Electron Transport Chain (ETC) and Oxidative Phosphorylation.
• Glycolysis and Gluconeogenesis.

TCA Cycle.

Net Equation.
3NAD+ + FAD + FDP + Pi + Acetyl-CoA + 2H2O >>> 3NADH + FAD(2H) + GTP + CoASH + 2CO2

• Metabolism: sum total of all chemical reactions in a cell.
• Anabolism: building up; synthesis (especially regarding energy and building materials).
• Catabolism: breaking down (especially for energy).
• Tricarboxylic Acid Cycle (TCA) = Citric Acid Cycle = Krebs Cycle
• The TCA cycle is responsible for more than 2/3 of ATP production from fuel oxidation.
• The conversion of pyruvate into acetyl-CoA takes place inside the mitochondria.
• TCA occurs in the matrix of mitochondria (eukaryotes).
  
• Biochemical pathways are different routes that reactants, intermediates, products and etc. can take to lead to different outcomes in order to produce what is needed by the cell/organism. This builds on the concept of basic building blocks make more complex molecules that is needed, and then breaking down the complex molecules back to basic units in ordered to be recycled. Concept of “shuttling”. Intermediates may be recycled-reused and/or may be important to the biosynthesis of other biomolecules.
  
• One of the important goals for the oxidation of FAs, glucose, AAs, acetates and ketones is to convert these materials into acetyl coenzyme A (Acetyl CoA) which is the form that is needed in order to enter the TCA cycle.
  
• Goals is to conserve energy from all the oxidation reactions.
• Electrons from intermediates get transferred to NAD+ and FAD.
• 8 electrons given up from the acetyl group end up in 3 molecules of NADH and 1 FADH2.
• This is important because the carriers NADH and FADH2 give the electrons to oxygen as the final electron acceptor in the electron transport chain (ETC).
• Good nutrition plays an important role. This is what the TCA needs: lots of vitamin and minerals; niacin (NAD+); riboflavin (FAD) and flavinmononucleotide; pantothenic acid (coenZ A); thiamin; Mg 2+, Ca 2+, Fe 2+, phosphate.
  
• Sources of Acetyl-CoA include (but not limited to): beta-oxidation of fatty acids such as palmitate; breakdown of ketone bodies beta-hydroxybutyrate and acetoacetate; acetate (from diet or ethanol oxidation); glucose/carbohydrates oxidized to pyruvate; alanine and serine can also be oxidized to pyruvate.
  
• Regulatory steps tend NOT to be reversible (with the exception of step 8, malate <–> oxaloacetate).

Quick Redox Review.

• LEO, Lose Electrons Oxidation. Gain oxygen or lose hydrogen.
• GER, Gain Electrons Reduction. Lose oxygen or gain hydrogen.
• *The more multiple bonds, the greater degree of oxidation.
• In biochem, it’s not as “obvious” to see how the electrons flow and what is oxidized and what is reduced.
• Sometimes it helps to look at the number of bonds and how the bonds change.
• If A is losing electrons/bonds, then it is getting oxidized (it is being oxidized).
  A’s role is the reducing agent.
• If B is gaining electrons/bonds, then it is being reduced.
  B’s role is the oxidizing agent.

Acetyl CoA.

• Examining Acetyl CoA, 2 main structural parts are easily recognizable: phosphopantetheine chain and nucleotide.
• The phosphopantetheine chain has -SH thiol group (can form thioesters with acyl groups) at the end of the pantothenic acid.
• The nucleotide is adenosine 3′, 5′ -biphosphate.
• The CoA-S-(C=O)-R helps to activate fatty acid or acetyl group.

Pyruvate Dehydrogenase Complex (PDHC/PDC).

• From the alpha-ketoacid dehydrogenase complex family.
  
• 3 catalytic subunits:
  1. Pyruvate decarboxylase subunits (bind TPP E1)
  2. Transacetylase subunits bind lipoate (E2)
  3. Dihydrolipoyl dehydrogenase subunits that bind FAD (E3)
  
• PDHC has 2 regulatory enzymes: pyruvate dehydrogenase kinase (PDH kinase) and pyruvate dehydrogenase phosphatase (PDH phosphatase).

STEP 1. Citrate Synthase Rxn.

• Acetyl-CoA + oxaloacetate + H2O >>citrate synthase>> Citrate (6 carbons) + CoA-SH
• Reactants: acetyl-CoA, oxaloacetate, water.
• Enzyme: citrate synthase.
• Products: citrate.
• Leaving: CoASH.
• Irreversible reaction.
• Regulation: -citrate.
• “Synthases” generally work by catalyzing the condensation reaction of 2 organic molecules to form a C-C bond when no high-energy phosphate bond energy is available.
• “Synthetase” (note the spelling looks a lot like “synthase”) are enzymes that DO need the high-energy phosphate bond energy to do their work.
• *Oxaloacetate is regenerated w/each turn of this cycle.
• The condensation of Acetyl-CoA and oxaloacetate (OAA) is irreversible .
• Acetyl-CoA can come from: palmitate, acetoacetate, glucose, pyruvate, alanine, and ethanol (PPAAGE). However, the major source is oxidative decarboxylation of pyruvate via pyruvate dehydrogenase complex (PDHC).

STEP 2. Isomerization of Citrate.

• Citrate (a tricarboxylate acid) is rearranged to isocitrate.
• Reactants: citrate.
• Enzyme aconitase (Fe-S protein), isocitrate dehydrogenase.
• Intermediate: cis-aconitate.
• Product: isocitrate.
• Reversible reaction.
• Aconitase catalyzes the migration of -OH group to a neighboring carbon so that it can be oxidized to form part of a keto-group.
• Aconitase is inhibited by a pesticide/plant toxin called fluoroacetate. Fluoroacetate gets converted to fluorocitrate which is an inhibitor.
• Isocitrate dehydrogenase catalyzes the oxidation of the -OH and cleaves the carboxyl, releaseing CO2.

STEP 3. Isocitrate Dehydrogenase Rxn.

• Oxidative decarboxylations: these are oxidations reactions (Lose Electrons Oxidation) where a carboxylate is removed and CO2 is produced.
• Isocitrate undergoes oxidative decarboxylation (releasing CO2) to form alpha-ketoglutarate.
• Reactants: Isocitrate, NAD+.
• Enzyme: isocitrate dehydrogenase.
• Products: alpha-ketoglutarate, CO2, NADH + H+.
• Irreversible reaction.
• Regulation: -NADH, +ADP, +Ca+2.
• One NAD+ is reduced to NADH.
• This is the first NADH produced in the TCA cycle.
• Isocitrate + NAD+ >>isocitrate dehydrogenase>> Oxalosuccinate + NADH + H+
• This is a rate-limiting step as isocitrate dehydrogenase is allosterically activated by ADP and Ca+2 and inhibited by ATP and NADH.

STEP 4. Oxidation of Alpha-Ketoglutarate.

• Alpha-ketoglutarate undergoes oxidative decarboxylation to form succinyl-CoA.
• Reactants: alpha-ketoglutarate, CoASH.
• Enzymes: alpha-ketoglutarate dehydrogenase complex, protein aggregate (contains conenzymes TPP or thiamine pyrophosphate, lipoic acid, FAD).
• Products: succinyl CoA, CO2, NADH + H+.
• Irreversible reaction.
• Regulation: -NADH, +Ca+2.
• CO2 is released from one of the carboxyl groups of alpha-ketoglutarate.
• Second NADH is released.

STEP 5. Formation of Succinate.

• Succinyl-CoA >> succinate thiokinase >> Succinate
• Reactants: succinyl CoA, GDP + Pi.
• Enzyme: succinate thiokinase (cleaves thioester bond and the bond energy is used to make GTP from GDP and Pi).
• Products: succinate, GTP, CoASH.
• Reversible reaction.
• Note that guanosine diphosphate (GDP) gets phosphorylated to GTP.
  GTP and ATP are interconvertible via nucleoside diphosphate kinase:
  GTP + ADP <–> GDP + ATP
• Substrate-level phosphorylation: forming a high-energy phosphate bond (with none having preexisted) from molecular O2 and NOT from oxidative phosphorylation.

STEP 6. Formation of Fumarate.

• Succinate >>succinate dehydrogenase>> Fumarate
• Reactants: succinate, FAD.
• Enzyme: succinate dehydrogenase.
• Products: fumarate, FADH2.
• Reversible reaction.
• The coenzyme FAD gets reduced to FADH2.
• Succinate dehydrogenase is the only enzyme in the TCA that is in the inner mitochondrial membrane.

STEP 7. Formation of Malate.

• Fumarate >>fumarate hydratase>> Malate
• Reactants: fumarate, water.
• Enzyme: fumarase.
• Product: Malate.
• Reversible reaction.
• This is a hydration reaction.

STEP 8. Formation of Oxaloacetate.

• Malate >>malate dehydrogenase>> Oxaloacetate
• Reactants: malate, NAD+.
• Enzyme: malate dehydrogenase.
• Products: oxaloacetate + NADH + H.
• Reversible reaction AND a regulatory step.
• Regulation: -NADH.
• This step makes the third NADH.

Summary of TCA Step 1-8.

TCA Regulation.

• The rate of TCA is regulated to correspond to the rate of ETC.
• ETC is regulated by the ratio of ATP:ADP and the rate of ATP usage.
• ATP Rate of Usage Feedback:
  1. phosphorylation state of ATP (ratio of ATP:ADP)
  2. reduction state of NAD+ (ratio of NADH:NAD+)
• In the cell and also in mitochondria, the pools of total adenine and the pools of total NAD are fairly/relatively constant.

Citrate Synthase Regulation.

• Has no allosteric regulators.
• Rate is controlled by concentration of oxaloacetate and the concentration of citrate (product inhibitor and competitor of oxaloacetate)
• Malate-oxaloacetate equilibrium favors malate. Oxaloacetate concentration is low inside mitochondria.
• As the NADH:NAD+ ratio increases, the oxaloacetate:malate ratio also increases.
• Activation of isocitrate dehydrogenase decreases the concentration of citrate, suppressing the inhibitory effect of citrate synthase.
• Liver NADH:NAD+ ratio helps determine if Acetyl-CoA enters the TCA or another pathway for ketone production.

Isocitrate Dehydrogenase Regulation.

• Isocitrate dehydrogenase is made up of 6 subunits.
• Is one of the rate-limiting steps in TCA.
• Allosterically activated by ADP.
• Also activated by Ca+2. The release of Ca+2 from the sarcoplasmic reticulum may provide additional activation.
• Inhibited by NADH.
• When ADP is absent, the subunits bind to each other and are converted to an active conformation.
• When ADP is present, all the subunits are in active conformation enabling isocitrate to bind more easily and readily.
• A small change in the concentration of ADP can significantly affect this step.

Alpha-Ketoglutarate Dehydrogenase Regulation.

• Alpha-ketoglutarate is a complex.
• It isn’t allosterically regulated.
• It is product-inhibited (negative feedback) by NADH and succinyl-CoA (and maybe GTP as well).
• Also activated by Ca+2. The release of Ca+2 from the sarcoplasmic reticulum may provide additional activation.

TCA Intermediates Regulation.

• TCA regulation ensures that the rate of NADH generation is sufficient to maintain ATP homeostasis, AND regulates the concentration of all the intermediates.

The Electron Transport Chain (ETC) & Oxidative Phosphorylation.

• Chemiosmotic Model of ATP Synthesis.
• ETC occurs in the inner mitochondrial membrane, the site of oxidative phosphorylation via ATP synthase.
• A series of oxidative reactions using oxygen as the final electron acceptor, release water and carbon dioxide.
• The intermediates of this oxidative series donate their electrons to coenzymes, adenine dinucleotide (NAD+), and flavin adenine dinucleotide (FAD). The reduced NADH and FADH2 have lots of energy stored in their bonds.
• As electrons are shuttled down this chain, they lose their free energy. This energy gets redirected to transport H+ (protons) from the mitochondrial matrix across the inner membrane of the mitochondria to cytosolic side thus causing an H+ gradient enabling the production of ATP from ADP + inorganic phosphate, Pi.
• This “proton pump” creates an electrochemical gradient. This is key.
• ATP synthase has a pore from the inner membrane to the headpiece sticking out in the matrix. Protons are pushed through this pore changing the conformation of the headpiece and this causes the simultaneous release of ATP at one site and the formation of ATP from ADP + Pi at another site.

Mitochondria.

• Inner mitochondrial membrane, rich in proteins, is impermeable to most small ions (including H+) and molecules like ADP, ATP, pyruvate, etc. The mitochondria need specialized carriers to transport goods inside. Cristae (convolutions) help to increase the surface area.
• The inner membrane has 4 separate complexes which are part of the ETC.
• ETC has 3 large protein complex I, III, & IV spanning the inner mitochondrial membrane.
• All members of the ETC, except CoQ (lipid-soluble quinone), are proteins.
• ATP that’s made is actively transported into the intermembrane space by adenine nucleotide translocase (ANT).
• Yields 30-32 ATP.
• Matrix maintains a low concentration of H+, high pH.
• Intermembrane space maintains a high concentration of H+ low pH.
• Proton pumps pump H+ from the matrix to the intermembrane space. Because this is against the concentration, it takes energy/work to maintain this potential gradient.

Electron Carriers.

• NAD+ (nicotinamide adenine dinucleotide). Related to vitamin B3, niacin. Indirectly produces 2.5-3 ATPs.
  NADH –> NAD+ + H + 2e-
• FAD (flavin adenine dinucleotide). Related to vitamin B2, riboflavin. Its electrons are at a lower energy state. Indirectly produces 1.5-2 ATPs.
• FMN (flavin mononucleotide). Vitamin B2, riboflavin. A prosthetic group, a non-protein molecule that is needed for the function of a protein.
• Other: Fe-S complexes, cytochromes, copper-based carriers.
• 2e- + H+ + 1/2O2 = water (this is the reduction of oxygen to water, or the oxidation of NADH to NAD+)
• Ubiquinone (Q) carries electrons from complex I and complex II to complex III. It is lipid-soluble and moves in the hydrophobic core of the membrane. Q carries pairs of electrons (2e-).
• Cytochrome C. Carries electrons from complex III to complex IV. Cytochrome C can carry only one e- at a time.
• Cytochrome B.
• Cytochrome A.
• Cytochrome A3.

Complex I and Q.

• NADH carries 2e- to complex I.
• Complex I is the entrypoint for NADH.
• Complex I is made of FMN (vitamin B2) and Fe-S.
• NADH dehydrogenase is the enzyme in complex I.
• Complex I pumps 4 H+ into the intermembrane space.
• Ubiquinone (Q) takes the 2e- and shuttles them to cytochrome C which shuttles them to complex III.

Complex II (succinate dehydrogenase).

• Complex II is the entry point for FADH2.
• FADH2 ‘s electrons are dropped off and Q shuttles them to complex III.
• Q, a mobile electron carrier like a “taxi”, is reduced to QH2.
• Complex II is bound to the membrane.
• Complex II is not a pump.

Complex III (cytochrome oxidoreductase).

• Complex III is composed of: Fe-S, cytochrome b, Reiske centers (2Fe-2S), cytochrome c1.
• Complex III pumps 4 H+ to the intermembrane space.
• The electrons get passed to complex IV via cytochrome c. Cytochrome c can carry only one e- at a time (not 2e-).
• Electrons get passed from cytochrome b to Fe-S to cytochrome c1.

Complex IV.

• Complex IV is made of cytochromes c, a, and a3.
• Complex IV has 2 heme groups per cytochromes a and a3. An oxygen molecule is bound very tightly between the iron and copper ions until the oxygen can be totally reduced at which point it picks up two H+ from the surrounds for the creation of water.
• 2H+ gets pumped out to the intermembrane spae.

Summary of ETC.

So thus far, the main purpose of ETC is two-fold: maintain the [H+] concentration gradient (high [H+] in the intermembrane space, and low [H+] in the matrix), and the shuttling of e- from a higher energy state to lower energy state where oxygen is the final electron acceptor and is expelled by binding it to two H+ to form water. Complexes I, III, and IV are proton pumps while complex II is like a proton funnel.

Chemiosmosis & ATP Synthase.

• Chemiosmosis is when the movement of ions down their electrochemical gradient is harnessed “to do work”. In this case, “work” is putting ADP and Pi together to form ATP.
• This process accounts for more than 80% of the ATP produced in the human body.
• ATP synthase is a proton channel (the only kind in the intermembrane space of mitochondria) allowing H+ from the intermembrane space to flow down their concentration gradient back into the matrix.
• ATP synthase is like a water-wheel or wind/water turbine. For every four H+ that flows through ATP synthase (from the intermembrane space to the matrix), one ADP + Pi bond can be “built” resulting in ATP.

Glycolysis.

• Glycolysis is the breakdown of glucose.
• This process occurs in the cytosol and doesn’t require oxygen (can work in the presence of oxygen or without oxygen).
• There are 2 stages. Stage 1 is the “spending” stage where ATP is spent. Stage 2 is the “money-making” stage where we make some “ATP money” back.
• In the larger picture, the real advantage of glycolysis is making that pyruvate, then converting the pyruvate into acetyl CoA (in the mitochondria) and entering the TCA cycle and ETC (where the most amount of ATP is made…like winning the lottery).

Stage 1. Step 1. Glucose to Glucose 6-phosphate.

• This step is a commitment. Once a phosphate groups is attached to glucose, glucose cannot leave the cell.

Stage 1. Step 2.

Stage 1. Step 3.

• This is the primary regulatory step for glycolysis.
• Fructose 1,6-bisphosphate is not a “real intermediate” (meaning it doesn’t act as an “intermediate”) but plays a more regulatory role in that its presence will either inhibit or promote an action.
• PFK1 is a rate-limiting enzyme via allosteric regulation.
• Inhibited by high concentrations of ATP.
• Inhibited by citrate concentrations.
• Promoted/stimulated by high concentrations of AMP (adenosine monophosphate).
• Promoted/stimulated by high concentrations of fructose 2,6-bisphosphate.
• If citrate appears in the cytosol, it can “leak out” and slow down the TCA cycle because an abundance of citrate indicates that the energy needs requirements are met–we have adequate energy/fuel. Citrate builds up in the mitochondrial matrix and “leaks” out into the cytosol.

Stage 1. Step 4.

• A 6-carbon molecule splits into two 3-carbon molecules which are isomers of each other. The only form that will continue on in the glycolysis process is glyceraldehyde 3-phosphate (GAP).
• The two 3-carbon isomers “flip” configuration back-and-forth via triose phosphate isomerase. This is noted in the next step 5.

Stage 1. Step 5.

• While these two isomers coexist with equal likelihood, the only form that will continue on in the glycolysis process is glyceraldehyde 3-phosphate.

Stage 2. Step 6.

• This is the only step where oxidation occurs.
• This step is a redox reaction.

Stage 2. Step 7.

• Substrate level formation of ATP.
• Note the resonance structure of 3-phosphoglycerate.

Stage 2. Step 8.

• Mutase phosphate on carbon 2.

Stage 2. Step 9.

Stage 2. Step 10.

• Substrate level ATP formation.
• Inhibited by high concentration of ATP, product-inhibition.
• Promoted/activated by fructose 1,6-bisphosphate.

Recap.

• In Stage 1, two ATP was “spent” or -2 ATP.
• In Stage 2, 4 ATP were created.
• Net ATP = 2.
• In Stage 2, 2 NADH were created.
• In Stage 2, 2 molecules of water were created.
• In Stage 2, 2 molecules of pyruvate were created.

Regulation of Glycolysis.

• Glycolysis is regulated at 5 enzymatic areas (and their corresponding step): hexokinase; phosphofructokinase; glyceraldehyde 3-phosphate dehydrogenase; phosphoglycerate kinase; and pyruvate kinase.
• Hexokinase (glucose to glucose 6-phosphate): inihibited by high levels of glucose 6-phosphate (product inhibition which is a form of negative feedback).
• Phosphofructokinase (fructose 6-phosphate to fructose 1,6-bisphosphate): most important as a rate-limiting step in mammalian glycolysis; this is the first committed step which means that if we make the product for this step, we are committed to the rest of the glycolytic pathway. Fructose 1,6-bisphosphate is the product. Inhibited by: high ATP concentrations; high citrate concentrations. Activated by: high AMP concentrations; high fructose 2,6-bisphosphate (formed in the FED state when glucose is abundant). Fed/absorptive state is up to 4 hours after a meal.
• Pyruvate kinase (phosphoenolpyruvate to pyruvate): inhibited by high ATP concentrations; activated by high fructose 1,6-bisphosphate concentratoins.

Alternate Paths for Pyruvate.

• Glycolysis makes pyruvate.
• From there, pyruvate can be used in many different pathways: alcohol fermentation (anaerobic microbial/bacterial process); lactate fermentation (anaerobic); aerobic respiration (conversion of pyruvate to acetyl CoA, TCA cycle, ETC).

Alcohol Fermentation.

• Results in 2 ATP.
• Anaerobic pathway.

Lactate Fermentation.

• Results in making 2 ATP.
• Anaerobic pathway.
• Occurs in muscle cells under anaerobic conditions.

Entrypoints of Other Sugars.

• Fructose (mobilized from adipose tissue) enters glycolysis at fructose 6-phosphate.
• Galactose enters glycolysis at glucose 6-phosphate.

Gluconeogenesis.

• Gluconeogenesis is the process by which pyruvate is converted to glucose; glucose is generated from non-carbohydrate sources (lactate, amino acids, fatty acids).
• If there’s an increase in glycolytic activity, gluconeogenesis is suppressed.
• If there’s a decrease in glycolytic activity, gluconeogenesis is promoted/activated.
• Gluconeogenesis is not “reverse glycolysis” because glycolysis contains 3 irreversible steps. SO, we need to find a “workaround” for those 3 irreversible steps in glycolysis.
• Gluconeogenesis is a highly aerobic pathway!

Roadblock 1. How can we get to phosphoenol pyruvate from pyruvate?

• Side note: lactate can be converted to pyruvate.
• Let’s start with pyruvate. We want to convert pyruvate to phosphoenol pyruvate so that we can reuse and follow most of the reversible steps in glycolysis (don’t reinvent the wheel when you don’t need to).
• Pyruvate can be converted to oxaloacetate (OAA) via enzyme pyruvate carboxylase and using 1 ATP.
• As a side note: amino acids can be converted to OAA and enter the pathway here.
• Then we can use GTP and enzyme PEPcarboxylase to convert OAA to phosphoenol pyruvate (at which point we can join the reversible steps in glycolysis).

Roadblock 2. How can we get from fructose 1,6-bisphosphate to fructose 6-phosphate?

• We can use enzyme fructose 1,6-bisphosphotase to work-around this roadblock (irreversible reaction in glycolysis) to form fructose 6-phosphate. From fructose 6-phosphate, we can use the reversible steps of glycolysis. Again, don’t reinvent the wheel!

Roadblock 3. How can we get from glucose 6-phosphate back to glucose?

• No problem…we just wanna get back to good ole regular glucose!!
• We use this slick enzyme glucose 6-phosphotase to convert glucose 6-phosphate back to glucose.

Cori Cycle (aka Lactic Acid Cycle).

• In the muscle, glucose can make pyruvate which can be converted to lactate which can enter the bloodstream, which can enter the liver to be converted to pyruvate and back to glucose.
• Glycolysis happens in the muscles.
• Gluconeogenesis happens in the liver.
• When oxygen levels are low, anaerobic glycolysis happens.
• We need to regenerate NAD+.
• Side note: the heart muscle can also use lactate as an energy source.

Resources.

• Acetyl CoA, https://library.med.utah.edu/NetBiochem/FattyAcids/2_4.html
• Acetyl CoA, https://www.humpath.com/spip.php?article3611
• Major metabolic pathways, http://homepage.ufp.pt/pedros/bq/integration.htm
• Major metabolic pathways, http://opencourse.ncyu.edu.tw/ncyu/file.php/23/Central_metabolic_pathways_II.pdf
• Major metabolic pathways, https://www.sciencedirect.com/science/article/pii/S1097276510006672
• TCA, https://www.khanacademy.org/science/biology/cellular-respiration-and-fermentation/pyruvate-oxidation-and-the-citric-acid-cycle/a/the-citric-acid-cycle
• TCA, http://www.bioinfo.org.cn/book/biochemistry/chapt15/sim2.htm
• TCA, http://equilibrator.weizmann.ac.il/static/classic_rxns/classic_reactions/tca.html
• ETC, https://www.khanacademy.org/science/biology/cellular-respiration-and-fermentation/oxidative-phosphorylation/a/oxidative-phosphorylation-etc
• ETC, https://courses.lumenlearning.com/wm-biology1/chapter/reading-electron-transport-chain/
• ETC, https://www.unm.edu/~lkravitz/Exercise%20Phys/ETCstory.html
• ETC, https://www.cureffi.org/2013/11/08/biochemistry-08-the-citric-acid-cycle-and-the-electron-transport-chain/
• PDC, https://www.khanacademy.org/test-prep/mcat/biomolecules/krebs-citric-acid-cycle-and-oxidative-phosphorylation/v/regulation-of-pyruvate-dehydrogenase
• Pyruvate dehydrogenase, http://watcut.uwaterloo.ca/webnotes/Metabolism/TCAcycle.html
• ETC, http://watcut.uwaterloo.ca/webnotes/Metabolism/RespiratoryChain.html
• Gluconeogenesis, http://watcut.uwaterloo.ca/webnotes/Metabolism/Gluconeogenesis.html
• Glycolysis, http://watcut.uwaterloo.ca/webnotes/Metabolism/Glycolysis.html
• ETC, https://www.khanacademy.org/test-prep/mcat/biomolecules/krebs-citric-acid-cycle-and-oxidative-phosphorylation/v/electron-transport-chain
• Glycolysis, https://www.khanacademy.org/science/biology/cellular-respiration-and-fermentation/glycolysis/v/glycolysis-overview
• Glycolysis, https://www.khanacademy.org/science/biology/cellular-respiration-and-fermentation/glycolysis/a/glycolysis
• Glycolysis, http://watcut.uwaterloo.ca/webnotes/Metabolism/Glycolysis.html
• Glycolysis, https://courses.lumenlearning.com/boundless-biology/chapter/glycolysis/
• Pyruvate, https://courses.lumenlearning.com/wm-biology1/chapter/reading-pyruvate-oxidation/
• Pyruvate, https://www.chem.purdue.edu/courses/chm333/images/Fermentation%20Pyruvate.pdf
• Metabolic states, https://opentextbc.ca/anatomyandphysiology/chapter/24-5-metabolic-states-of-the-body/
• Metabolic states, https://courses.lumenlearning.com/suny-ap2/chapter/metabolic-states-of-the-body/
• Gluconeogenesis, https://www.khanacademy.org/test-prep/mcat/biomolecules/carbohydrate-metabolism/v/gluconeogenesis

References.

Bean, J. (2019). ETC & oxidative phosphorylation.

Bean, J. (2019). Glycolysis and gluconeogenesis.

Bean, J. (2019). TCA cycle.

Ferrier, D. (2017). Biochemistry (7th ed.). Philidelphia, PA: Lippincott Illustrated Reviews.

Lieberman, M., & Peet, A. (2017). Marks’ basic medical biochemistry: A clinical approach(5th ed.). Philadelphia, PA: LWW.

Last edited: 08.08.2019

See Appendix A Amino acid abbreviations; Appendix B Glucogenic, Ketogenic, and Glucoketogenic Amino acids; Appendix C 20 Amino Acids to Know Drawn at Physiological pH 7.4.

Review of Macromolecules and Functional Groups.

• What is a monomer? A monomer is a base unit of something. A base unit is something that cannot be broken down further without losing is character, traits, physical/chemical properties, behavior, etc. A monomer of a chocolate bar (a polymer) is one square of chocolate. Several squares of chocolate (monomers) make up that one large chocolate bar (polymer).
• What is a polymer? A polymer is a larger compound built up from base units of monomers.
• Examples of polymers and monomers.

Condensation (dehydration) synthesis.

Hydrolysis.

Carbohydrates.

A. Structure.

A sugar monomer is a single sugar unit called a monosaccharide. Two monosaccharides make up a disaccharide via glycosidic linkage. Sucrose = glucose + fructose. Maltose = glucose + glucose.

Glycosidic linkages are condensation/dehydration reactions in which H2O is a product (is produced in the reaction). The formula for a typical sugar is CnH2nOn.

Sugars may be aldoses (aldehyde group at top) or ketoses (ketone group at top). On a Fischer projection, L-sugars have the hydroxyl group on the left of the last chiral carbon (carbon farthest away from the aldehyde or ketone group). D-sugars have the hydroxyl group to the right of the last chiral carbon. The human body almost exclusively uses D-sugars.

Sugars can have the same molecular formula but still be different due to stereochemistry. Galactose and glucose can have the same molecular formula. They are different sugars because they are arranged differently. It’s like having 5 red legos and 5 blue legos; you can put them together in many different combinations.

B.Function.

Examples of polysaccharides: starch (plant energy storage), cellulose (fiber-like used for plant cell walls), glycogen (animals’s energy storage in muscles and liver), chitin (exoskeletons and fungal cell walls). Example function of carbohydrates and used for energy storage and structural support.

Proteins.

A. Structure.

Amino acids are monomers for proteins which are 1+ polypeptide chains (polymer). 2 AA’s are joined via peptide bonding. Polypeptide chains can get longer and longer and you can put multiples of them together to form complex molecules. Once AA’s become part of a chain they are called “residues”. AA’s are made up of: an amine group, a carboxylic acid group, a hydrogen at the alpha-carbon, and a R-sidechain.

At physiological pH of 7.4, the amino group is protonated (+NH3) and the carboxylic acid group is deprotonated (COO-). Because the molecule has a dual charge, it’s called a zwitterion. The charged amino group at the leftmost position of the chain is called the N-terminus; the carboxylate ion at the rightmost position of the chain is called the C-terminus. AA’s are joined together to form a chain via peptide bonding.

Remember that if the pH < pKa, then that group will be protonated (e.g. +NH3, COOH). If pH > pKa, then that group will be deprotonated (e.g. NH2, and COO-). Physiological pH is 7.4; pKa is ~9.0 for amines; pKa is ~4.75 for carboxylic acid. Histidine’s pKa is very close but a little on the basic side at physiological pH.

Amino acids have stereochemistry (they have mirror images).

The “L” designation in L-amino acids denotes that the zwitterion is oriented with the protonated amine group at the left and the carboxylate ion on the right. The human body uses L-amino acids with the exception of glycine which is achiral. This is also important because the L stereochemistry is what our body uses in ligand-receptor binding (e.g. enzymes).

The alpha-carbon is the carbon directly bonded to the carboxylic acid group. The alpha amine is the amine group directly bonded to the alpha carbon.

Peptide bonding is when the carboxylate end of one AA covalently bonds with the charged amino end of another AA giving off a water molecule. Peptide bonding is a special form of dehydration synthesis (condensation reaction) between two amino acids. A more general term for peptide bonding is amide bonding. Bonding sequence matters. If amino A is bonded to amino B and then to amino C, the charged amino group of A is retained. In the order A-B-C, the carboxylate ion of amino C is retained. Bonding order matters. A-B-C is not the same as B-C-A.

Proteins have primary, secondary, tertiary, and quaternary structures.

Primary structure is characterized by an unique sequence of amino acids in a polypeptide. Structure and function go hand in hand. Any tiny change in structure could impact the function of the protein.

Secondary structure is characterized by repeated folding of the polypeptide BACKBONE. The keyword is backbone. Hydrogen bonds (H-bonds) stabilize peptide bonds. The alpha helix and B-pleated sheets (parallel or antiparallel). Antiparallel configuration is more stable as steric and angle strain.

http://www.chem.ucla.edu/~harding/notes/strain_01.pdf

https://chem.libretexts.org/Bookshelves/Organic_Chemistry/Supplemental_Modules_(Organic_Chemistry)/Alkanes/Properties_of_Alkanes/Cycloalkanes/Ring_Strain_and_the_Structure_of_Cycloalkanes

Tertiary structure is characterized by lots more globular folding and interactions between R-sidechains/groups (H bonds, ionic bonds, sulfhydral bonds, disulfide bridges, salt bridges). This is where the polar/nonpolar, acidic/basic, etc. properties of amino residues becomes an important consideration.

Quaternary structure is characterized by 1+ separate tertiary structures assembling together to form a more complex molecule. Often the “core” or pockets created may hold inorganic ions and some of the gaps created may be ligand-receptor sites. The act of binding and releasing can cause conformational changes altering the function of the protein.

B. Function.

Amino acids may be classified as essential (ones the human body cannot make and must come from exogenous sources). Nonessential amino acids are ones the human body can make.

Amino acids may be classified based on the polar/nonpolar characteristics of the sidechain: polar, nonpolar; acidic/basic/uncharged or neutral.

Amino acids may be classified based on aromatic or aliphatic (structural comparison).

Amino acids may be classified based on what they can make: glucogenic (make glucose); ketogenic (make ketones); glucoketogenic (make both glucose and ketones).

Proteins can have all sorts of functions: acting as enzymes, structural support, antibodies, cell signalling, transport, and movement.

See Appendix A Amino acid abbreviations; Appendix B Glucogenic, Ketogenic, and Glucoketogenic Amino acids; Appendix C 20 Amino Acids to Know Drawn at Physiological pH 7.4.

Lipids.

A. Structure.

A functional unit of lipids is a triglyceride which is a glycerol (trihydric alcohol) and fatty acids (acyl groups which can be saturated or unsaturated long hydrocarbon with a carboxylic acid head. The carboxylate head bonds with the hydroxyl (of the glycerol) via ester linkages. The fatty acids that get attached to the glycerol do not have to be the same. Triglycerides are amphipathic (the key to this structure is its hydrophilic and hydrophobic duality).

Phospholipid = glycerol + 2 FA chains + phosphate + head-group. The key to this structure is its hydrophilic and hydrophobic duality. This is important to cell membranes, micelles, etc.

Waxes = 2 long hydrocarbon tails connected via ester bond.

Steroids = 4 carbon rings with no fatty acid tails.

http://www.biologie.ens.fr/~mthomas/L3/intro_biologie/2-sucres-lipides-acides-nucleiques.pdf

**Circle the ester bond(s) and circle the glycerol component. BE CAREFUL that the ester bonds will overlap with the glycerol oxygen.

B. Function.

Some functions include (but not limited to): creating a barrier; insulation; protection; energy storage; hormones, carriers (e.g. fat soluble vitamins) and signalling.

Nucleic Acids.

A. Structure

Nucleotides are the monomers for nucleic acids. A nucleotide = pentose sugar (e.g. deoxyribose or ribose) + phosphate (mono, di, or tri) + nitrogenous base. “Deoxyribose” just means that the second carbon in the ring does not have a hydroxyl group (i.e. carbon 2 is bonded to two hydrogens). A nucleoSide = pentose sugar (e.g. deoxyribose or ribose) + nitrogenous base = nucleoTide without the phosphates.

The pentose sugar is bonded to the nitrogenous base via N-glycosidic bond. The phosphates are bonded to the pentose sugar via phosphoester bonding.

https://chem.libretexts.org/Courses/Sacramento_City_College/SCC%3A_Chem_309_-_General%2C_Organic_and_Biochemistry_(Bennett)/Text/13%3A_Functional_Group_Reactions/13.10%3A_Phosphoester_Formation

The nitrogen bases used are guanine, adenosine, cytosine, thymine, and uracil (DNA pairs are C-G and A-T, RNA pairs are C-G and A-U). Adenine and guanine are purines (5 ring connected to a 6 ring); cytosine, thymine, and uracil are pyrimidines. Base pairs between C and G are stronger due to an extra H-bond.

How to tell them apart?

Cytosine has an amine group bonded to one of the carbons on the ring. Adenosine has a carbonyl group with the carbonyl carbon as part of the ring. Uracil has two carbonyl groups, each with the carbonyl carbon as part of the ring.

Adenine has an amine group hanging off the 6-ring structure; guanine has a carbonyl group with the carbonyl carbon as part of the ring structure.

The nucleotide monomers are connected via covalent bonding carbon 5 of one sugar to the carbon 3 of the other sugar (phosphate group sandwiched inbetween). We call this the 5′ (five prime) position and 3′ (three prime) position. It is a condensation reaction forming a phosphodiester bond between the sugars and phosphate groups (alternating sugar-phosphate-sugar-phosphate etc.) with water given off as a product.

Differences between DNA and RNA.

1. Sugar. DNA has deoxyribose (missing -OH on 2nd carbon); RNA has ribose.
2. Base pairs. DNA’s base pairs are C-G, A-T. RNA’s base pairs are C-G, A-U.
3. # of strands. DNA has two strands in a double helix configuration (H-bonds help to stabilize). RNA is single-stranded.

B. Function.

Nucleic acids are in genetic material like DNA and RNA.

The nitrogen bases used are guanine, adenosine, cytosine, thymine, and uracil (DNA pairs are C-G and A-T, RNA pairs are C-G and A-U). Adenine and guanine are purines (5 ring connected to a 6 ring); cytosine, thymine, and uracil are pyrimidines. Base pairs between C and G are stronger due to an extra H-bond.

How to tell them apart?

Cytosine has an amine group bonded to one of the carbons on the ring. Adenosine has a carbonyl group with the carbonyl carbon as part of the ring. Uracil has two carbonyl groups, each with the carbonyl carbon as part of the ring.

Adenine has an amine group hanging off the 6-ring structure; guanine has a carbonyl group with the carbonyl carbon as part of the ring structure.

The nucleotide monomers are connected via covalent bonding carbon 5 of one sugar to the carbon 3 of the other sugar (phosphate group sandwiched inbetween). We call this the 5′ (five prime) position and 3′ (three prime) position. It is a condensation reaction forming a phosphodiester bond between the sugars and phosphate groups (alternating sugar-phosphate-sugar-phosphate etc.) with water given off as a product.

Differences between DNA and RNA.

1. Sugar. DNA has deoxyribose (missing -OH on 2nd carbon); RNA has ribose.
2. Base pairs. DNA’s base pairs are C-G, A-T. RNA’s base pairs are C-G, A-U.
3. # of strands. DNA has two strands in a double helix configuration (H-bonds help to stabilize). RNA is single-stranded.

B. Function.

Nucleic acids are in genetic material like DNA and RNA.

Central Dogma.

As far as our understanding today, scientists believe that genes determine the sequence of mRNA which in turn determines the sequence of proteins.

https://www.khanacademy.org/science/biology/gene-expression-central-dogma/central-dogma-transcription/a/the-genetic-code-discovery-and-properties

Basic Steps: From Gene to the Primary Structure of Protein.

Step 1. Transcription.

In the nucleus, RNA polymerase unzips DNA and reads DNA in order to make and spit out pre-mRNA (transcribed from the DNA) using nucleoside triphosphates as building materials. Emphasis on triphosphates because the process requires lots of energy which is available in those phosphate bonds (e.g. each time phosphate gets cleaved).

Step 2. Editing the pre-mRNA.

In the nucleus, the pre-mRNA needs to be edited. A methylated structure called a “cap” is placed on pre-mRNA’s 5′ end. A poly-adenosine monophosphate tail is added at the 3′ end. Introns are spliced out leaving only the exons.

https://www.cell.com/trends/biochemical-sciences/fulltext/S0968-0004(19)30002-7

Step 3. Translation.

The “edited/fixed-up” mRNA is transported out of the nucleus and into the cytoplasm at the rough endoplasmic reticulum. The mRNA gets sandwiched between the large and small ribosomal subunits. The ribosome is the assembly site. The tRNA is the shuttle that pairs its anticodon to the codon on the mRNA and brings in the correct amino acid for assembly. As the ribosome moves along reading the mRNA, the protein strand that’s being formed (queue-fashioned) starts to stick out of the ribosome. This step is called “translation” because it’s like decoding a secret message on the mRNA to make the protein that you want.

https://www.khanacademy.org/science/biology/gene-expression-central-dogma/translation-polypeptides/v/translation-mrna-to-protein

See Appendix A Amino acid abbreviations; Appendix B Glucogenic, Ketogenic, and Glucoketogenic Amino acids; Appendix C 20 Amino Acids to Know Drawn at Physiological pH 7.4.

Appendix A. Amino acid abbreviations.

• Ala, Alanine, A
• Arg, Arginine, R (aRRRRRRRg arginine)
• Asn, Asparagine, N
• Asp, Aspartic acid, D (aspar-dic)
• Cys, Cysteine, C
• Glu, Glutamic acid, E (glue-EE the glutamic acid)
• Gln, Glutamine, Q (GQ guy got hot glutes)
• Gly, Glycine, G (Trivia: the achiral amino with an H sidechain)
• His, Histidine, H (Trivia: the pKa is so damn close to physiological 7.4)
• Ile, Isoleucine, I (iso-i or iso-eye)
• Leu, Leucine, L (leu-leu, loo-loo)
• Lys, Lysine, K
• Met, Methionine, M (methionine the meth head)
• Phe, Phenylalanine, F (fee-fye-fo-fum, says the giant in Jack in the Beanstalk)
• Pro, Proline, P (it get’s a p for just being weird and attaching to both the alpha carbon and amine, looks like an urinal…pee pee pee)
• Ser, Serine, S (reminds me of sarin gas)
• Thr, Threonine, T (three-o-nineTy time for pinty glug glug)
• Trp, Tryptophan, W (only cuz teacher said it looks like a W, the turkey amino)
• Tyr, Tyrosine, Y (looks Y-ish)
• Val, Valine, V (Val-in-tine’s day, V-day)

Appendix B. Glucogenic, Ketogenic, and Glucoketogenic Amino acids.

Ketogenic (LK): lysine, leucine.

Ketoglucogenic (ITFWY): isoleucine, threonine, phenylalanine, tryptophan, tyrosine.

Glucogenic: alanine, arginine, asparagine, aspartate, cysteine, glutamate, glutamine, glycine, histidine, methionine, proline, serine, valine.

Appendix C. 20 Amino Acids to Know Drawn at Physiological pH 7.4

Glycine

The only achiral amino acid because the sidechain is just a hydrogen.

Alanine

The shortest sidechain, methyl group.

Valine

Looks like a short “V”.

Isoleucine

Isomer of leucine.

Leucine

Isomer of isoleucine.

Resources.

References.

Bean, J. (2019). Amino.

Bean, J. (2019). Macromolecules.

Bean, J. (2019). Nucleotides and nucleic acids.

Bean, J. (2019). Protein structure.

Ferrier, D. (2017). Biochemistry (7th ed.). Philidelphia, PA: Lippincott Illustrated Reviews.

Bean, J. (2019). Nucleotides and nucleic acids.

Bean, J. (2019). Protein structure.

Ferrier, D. (2017). Biochemistry (7th ed.). Philidelphia, PA: Lippincott Illustrated Reviews.

Lieberman, M., & Peet, A. (2017). Marks’ basic medical biochemistry: A clinical approach(5th ed.). Philadelphia, PA: LWW.

[Study guide covering chapter 7, PDF]

Last edited: 08.05.2019

Characteristics of 3D Structure.

• 3D conformation and the type of amino acid side chains determine the character and function of the protein.
• Globular proteins usu. water soluble.
• Fibrous proteins are linear, arranged about a single axis, have repeating units.
• Transmembrane proteins that have 1+ regions aligned so they cross the lipid membrane.
• Primary structure. Linear AA residues joined by peptide bonds to form polypeptide chain.
• Secondary structure. Recurring structures that form in short localized regions. Beta pleats, parallel, antiparallel.
• Tertiary structure. 3d globular structure.
• Quatenary structure. 2+ protein structures.

Requirements for 3D Structure.

• Creating a binding site that’s specific for just one molecule or a group of molecules w/similar structural properities/characteristics. Binding sites on a protein define that protein’s role.
• Has appropriate rigidity and flexibility allow the protein to fulfill its role.
• Structural flexibility and mobility so that the protein can fold appropriately.
• The external surface of the protein must be appropriate for its environment and aid in the protein’s functionality.
• Conformations must be stable.
• When the protein is degraded/damaged, it must be able to be broken down and disposed of or recycled.

3D Structure of Peptide Bond.

• When AA are joined to form a polypeptide chain, the peptide bond takes on a “trans” configuration to minimize steric strain between different alpha carbons and side chains.
• The backbone is a resonance structure and has very restricted “bends”. The carboxyl and amide groups are planar.
• Torsion angles, are limited allowed rotation about the alpha carbon and alpha amino group around the bond between the alpha carbon and carbonyl group. Limited by steric constraints and favor the max distance between substituents.

Secondary Structure.

• Recurring localized structures in polypeptide chains are secondary structures.
• Alpha helix: secondary structure of proteins; membrane spanning; rigid, stable conformation; maximize H-bond. Peptide backbone formed by H-bonds betw. carbonyl oxygen and amide hydrogen located 4 residues down. Ea. peptide bond is connected to the other peptide bond +4 and -4 amino residues away. Proline is known as the “helix breaker” because it cannot fit in the alpha helix due to proline’s dual attachment points to a backbone.
• Beta sheets: maximize H-bond betw. peptide backbones; maintain allowed torsional angles; paralllel or antiparallel “weaving”; the carbonyl oxygen of one peptide bond is H-bonded to the amide hydrogen of a peptide bond on adjacent strand. Antiparallel is like hairpin turns or the chain folded backwards on itself. Sheets have hydrophilic and hydrophobic sides.
• Nonrepetitive secondary structures: bends, loops, and turns that don’t have the same organization as alpha helixes and beta sheets.
• Patterns of secondary structure.

Tertiary Structure.

• Secondary elements folded into 3d structure.
• 3d is dynamic and flexible.
• Fluctuating movement of the side chains and domains. Don’t require “unfolding”.
• Maintains appropriate surface resides respective of its environs.
• Flexibility is a key feature.
• Forces: ionic bonds, H-bonds, disulfide deformations, London forces, hydrophobia-hydrophilia.
• Tertiary structural domains: physically indep. regions that fold independently. Domains are fairly obvious to visual inspection.
• Domain: formed from continuous AA sequence in a polypeptide chain that’s folded into 3d indep. of rest of the protein. 2 domains are connected via a loop or similar simple structure.
• Folds in globular proteins: relatively large patterns of 3d structure recognizable in many proteins. Each”fold” hosts a certain kind of activity.
• Actin fold: G-actin.
• Nucleotide binding fold.
• Globular proteins solubility in aqueous environment. Most are soluble in the cell. The core is mostly hydrophobic and is densely packed to maximize London forces.
• Charged/polar parts of the polypeptide chains generally on the surface or close to it.
• Charged side chains bind to inorganic ions to minimize repulsion betwen like groups.
• Some charged aminos in the interior usu. serve as binding sites.
• Polar uncharged: serine, threonine, asparagine, glutamine, tyrosine, tryptophan.
• Transmembrane proteins have characteristics that allow them to cross membrane structures. They usu. have hydrophobic and hydrophilic components. Transmembrane proteins usu. have many post-translational modifications providing additional chemical groups to interact as needed. Receptors need both flexibility and “rigidity”.
• Binding domain.

Quaternary Structure.

• Homo-
• Hetero-
• Protomer: an unit made from two identical units.
• Oligomer: multi subunit proteinmade of identical G-actin subunits.
• Multimer: a complex w/lots of subunits of 1+ types. “Melting/melding pot”.
• The larger a complex protein, the more difficult it is to fold and unfold etc. Requires much larger effort. It also means that it’s more stable, too. It has fewer “options”. It can also operate in more of a coordinated effort due to less subunits “going rogue”.

Quantication of Ligand Binding.

• Ka. Association constant. Binding affinity of a protein for a ligand. The Keq for the binding reaction of P protein and L ligand. Used for comparing proteins made by diff. alleles.
• Kd. Dissociation constant for the ligand-protein binding. Is the reciprocal of Ka.

Structure-Function in Myoglobin and Hemoglobin.

• Myoglobin and hemoglobin are oxygen-binding proteins similar in structure.
• Myoglobin: globular; simple polypeptide that has one oxygen binding site; 8 alpha helices connected via short coils called globin fold; no beta pleated sheets (unusual); helicies create hydrophobic oxygen binding pocket w/tightly bound heme w/an iron atom Fe2+.
• Hemoglobin: tetramer made up of different subunits (2 alpha and 2 beta polypeptide chains called alpha-beta-protomers). Maximizies oxygen carrying capacity. Planar porphyrin ring of 4 pyrrole rings linked by methenyl bridges lying w/nitrogen atoms in the center, binding Fe(II) in the center. Negatively charged areas interact w/arginine and histidine side chains from hemoglobin; hydrophobic methyl and vinyl groups interact w/hydrophobic amino acid side chains as well that help position the heme group. There are 16 diff. interactions betw. myoglobin amino acids and diff. groups in porphyrin ring.
• When PO2 (partial pressure of oxygen) is high (in lungs), myoglobin and hemoglobin are oxygen-saturated.
• When PO2 is lower (e.g. in tissues), hemoglobin can’t bind to oxygen as well as myoglobin can.
• Myoglobin is in heart and skeletal muscle and is able to bind to (and store) O2 released by hemoglobin.
• Cytochrome oxidase, a heme-containing enZ in ETC( electron transport chain), has higher affinity than myoglobin.
• Prosthetic groups: organic ligands tightly bound to proteins. They are a part of the protein and don’t dissociate until the protein is degraded.
• Holoprotein: a proteinw/prosthetic groups.
• Apoprotein: a protein w/out its prosthetic group.
• In the binding pocket of myoglobin and hemoglobin, oxygen binds to the Fe2+. The Fe2+ can chelate (bind to) 6 different ligands (4 ligands are co-planar, 2 ligand positions are pependicular). 4 ligand positons taken by N, 1 of the perpendicular positions is taken by N on histadine called proximal histidine, the other position taken by O2.
• When O2 binds, conformational changes occur >> Tertiary structure changes from T-state (tense state) with low O2 affinity >> to R (relaxed) state w/high O2 affinity. Binding rate for the first O2 is low but with subsequent O2’s the binding rate is higher. This is known as positive cooperativity. The first O2 is difficult but subsequent binding of O2’s gets easier.
• HbO2 –> Hb + O2
• Agents that affect O2 binding: hydrogen ions; 2,3-bisphosphoglycerate; covalent binding of CO2.
• 2,3-bisphosphoglycerate (2,3-BPG). Formed in red blood cells. 2,3-BPG binds to hemoglobin increasing energy requirements for the conformational changes that facilitate O2 binding. Lowers hemoglobin affinity for O2. RBC can use 2,3-BPG to modulate affinity to O2 binding as needed by changing the rates of synthesis/degradation of 2,3-BPG.
• Proton binding, (Bohr Effect). When hemoglobin binds protons, it has less affinity for oxygen. pH decreases in tissues (proton concentration is higher) as metabolic CO2 is converted to carbonic acid via carbonic anhydrase in RBC’s. Dissociated protons react w/AAs >> conformational changes >> promote release of O2. In the lungs, this process is reversed. Lungs have high O2 concentration >> O2 binds to hemoglobin causing release of protons >> pH of blood rises >> carbonic anhydrase cleaves carbonic acid to H2O and CO2 >> CO2 exhaled.
• Carbon dioxide. Most of it is from tissue metabolism. CO2 is carried to lungs as bicarbonate. Some of the CO2 is covalently bonded to hemoglobin.
• https://www.khanacademy.org/science/health-and-medicine/advanced-hematologic-system/hematologic-system-introduction/v/bohr-effect-vs-haldane-effect
• https://www.khanacademy.org/science/health-and-medicine/advanced-hematologic-system/hematologic-system-introduction/v/hemoglobin-moves-o2-and-co2
• http://www.pathwaymedicine.org/bohr-effect
• https://youtu.be/FtA4Xy-lMSY

Structure-Function Relationships in Immunoglobin.

• Immunoglobins/antibodies bind to antigens (ligands) on the invaders, like marking them for inactivation/destruction. Marking the invaders as “not self”.
• Immunoglobins have the same structure: ea. antibody molecule has 2 identical polypeptide chains (L light chains) and 2 identical large polypeptide (H heavy) chains. L and H chains are joined via disulfide bonds.
• 5 major classes of immunoglobins.
• IgG gamma (most abundant); 220 aminos in light chain; 440 aminos in heavy chain. L and H chains have immunoglobulin fold (collapsed number of B sheets called Beta-Barrel). Have attached oligosaccharides that help mark “not self”.
• V = variable regions. VL (variable light chain) and VH (variable heavy chain) interact to make one antigen-binding site at each branch of the Y-shaped molecule. V regions have different AA compositions.
• C = constant regions. Form the fragment, crystallizable part of the antibody important for the antigen-antibody complex.
• Antigens bind tightly w/almost no tendency to dissociate. Kd is betw. 10^-7 to 10^-11 M.

Protein Folding.

• Peptide bonds may be rigid, but other bonds in the molecule can allow some flexibility.
• Native conformation: every molecule of the same protein has the same stable “native” conformational state.
• Primary structure (sequence of AA side chains) determines folding and assembly of subunits.
• Denaturation can affect protein structure. However, under certain conditions, the denaturation may be reversed.
• Not all proteins fold into their native state by themselves. Sometimes the folding and refolding occurs as the protein searches for its most stable state.
• Kinetic barriers are the higher energy states and conformation the protein may pass through.
• These kinetic barriers may be overcome by heat-shock (chaperonins) which use energy from ATP hydrolysis to help in the folding process.
• Cis-trans isomerase.
• Protein disulfide isomerase.
• Collagen: fibrous protein; made mostly by fibroblasts (cells in interstitial connective tissue), muscle cells, and epithelial cells. Type 1 collagen most abundant in mammals and major component in connective tissue. Found in ECM, loose conn. tissue, bone, tendons, skin, blood vessels, and cornea. 33% glycine, 21% proline, and hydroxyproline.
• Hydroxyproline is an AA made by posttranslational mods of peptidyl proline residues.
• Procollagen 1 is the precursor of collagen 1; triple helix of 3 pro-alpha polypeptide chains twisted around ea. other (ropelike). Interchain H-bonds. Every 3rd residue is a Glycine.
• Collagen 1 polymerizes >> collagen fibrils, great tensile strength.
• Vit C functions as a cofactor of prolyl hydroxylase and lysyl hydroxylase. These hydroxylase aid in H-bond formation >> strength and stability. Vit C deficiency causes the melting point to drop from 42 deg C to 24 deg C.
• Aldehyde residues, allysine, make covalent crosslinkages betw. collagen and other structures >> improve stability and structural support.
• Allysine on one collagen molecule + lysine of another molecule >> Schiff base (N=C double bond).
• Aldol condensation may occur betw. 2 allysine to form lysinonorleucine.
• Protein denaturation via nonenzymatic modification of proteins.
• Protein denaturation via temp, pH, and solvent.
• Protein denaturration via misfolding and prions.

Resources.

• http://leah4sci.com/mcat/mcat-biochemistry/hemoglobin-and-oxygen-dissociation-curves-on-the-mcat/
• https://www.youtube.com/watch?v=57P2Gsdq7is
• https://youtu.be/dxCd1yaMi-8

References.

Lieberman, M., & Peet, A. (2017). Marks’ basic medical biochemistry: A clinical approach(5th ed.). Philadelphia, PA: LWW.

[Study guide covering chapter 6, PDF]

Last edited: 08.05.2019

Structure.

• Phosphate group.
• Nitrogen/amino group: attached to the alpha carbon of the carboxylate.
• Carboxylate group.
• R-side chain.
• Concept of zwitterion.
• Amine group pH ~9.5
• Physiological pH ~7.5
• Alpha carbon is chiral except in glycine.
• Glycine, the carbon is achiral (has two H).
• Mammalian AA are L configuration (as opposed to D).
• Alpha aminos are AA attached to alpha carbon.
• Primary structure: sequence of AAs.
• AA joined via peptide bonds betw. COO- of one AA and the NH3+ of the other.
• Backbone >> carbolxylate, alpha carbon, and amino group.
• AA are the side chains.
• Binding sites.
• Ligand-receptor pairs.
• N-terminal (where the amino group is).
• C-terminal (where the carboxylate is).

Classification of Amino Acids.

• pKa
• Hydropathic index: a scale to describe hydrophobicity of the side chain.
• Glycine is a special case where its R-group is just H. Has the least steric hinderance. Can be found in the “nooks and crannies”.
• Nonpolar: alanine, valine, leucine, isoleucine; aliphatic; very hydrophobic; typically form the hydrophobic cores.
• Proline is another special case as it’s attached to the backbone twice. It has both an alpha carbon and alpha amino group. Proline is an imino acid. Rigid and forms kinks. Restricted conformations.
• Aromatic acids: 6-member ring of C and H; conjugated double bonds. H on the ring doesn’t participate in H-bonding. The substituents on the ring influence side chains characteristics & determine what action takes place (e.g. hydrophilic or hydrophobic).
• Aliphatic, polar, uncharged AA: has amide group (asparagine, glutamine); or hydroxyl group (serine, threonine); form H-bonds with water. Typically found on surfaces.
• AA with sulfur: cysteine and methionine. Can form disulfide bridges via oxidation of the sulfhydral groups, but it doesn’t always do that. The action/function depends on it’s surrounding environment and “what else” there is.
• Acidic AA: carboxylic acid groups; e.g. aspartate and glutamate. Their negative charge can form ionic bonds with cations.
• Basic AA: have side chains containing N (+) and tends towards basicity; e.g. histidine, lysine, arginine. Their positive charges can form ionic bonds with anions. Lysine and arginine can form bonds to anionic compounds w/protein binding sites (i.e. those protein binding sites can become incorporated into a new compound as well as become altered). Those ligand-receptor sites can become “grandfathered in”.
• Acidic/basic characteristics can allow AAs to participate in H-bonds and salt bridge formations.
• Carbon positions can be described using the Greek letters: alpha, beta, gamma, delta, epsilon).
• If pH < pKa then it favors the protonated form (-COOH, -NH3+). If pH>pKa, then it favors the deprotonated form (-COO- and -NH2).
• Imidazole ring. C3N2H4 (compound).
• In proteins, only the side chains, N-terminal, and C-terminal are dissociateable. Other C, N, H that form part of the backbone do not participate in acidic/basic characteristics.
• Electrophoresis: separate proteins via charge differences. Helps to identify proteins and components.

Variations in Protein Structure.

• Protein structure and characteristics can vary between different individuals and different ages. This variant nature is called variant regions.
• Hypervariable describes a situation where variation is tolerable within reason.
• Invariant regions, in contrast, do NOT vary between individuals, species, etc. Variation is NOT tolerated.
• Polymorphisms: when allele variations occur with great frequency.
• Homologous proteins: these belong to the same ancestral proteins.
• Paralogs: proteins that have similar structure and function that have evolved from the same gene after gene duplication.
• Divergent evolution: when one gene performs it’s expected function yet a copy mutates into a different function or have different characteristics.
• Superfamily: large family of homologous proteins.
• Isoforms: 2+ functionally similar proteins with similar structure (but not identical) or AA sequence; isoforms of a protein have the same function.
• Isozymes: 2+ enZ w/similar functions but differ in structure; isozymes catalyze the same reactions.
• Developmental variation: structures and functions differ at different developmental stages.
• Tissue-specific isoforms: proteins that function “the same” but they vary in structure/characteristics from tissue group to another tissue group.
• Tissue-specific isozymes: enZ that function “the same” but they vary in structure/function from tissue group to tissue group.
• Species variation: proteins and enzymes (and their structure and function) can vary from species to species.

Modified Amino Acids.

• Post-translational modification. Post-protein synthesis, some AA residues in the primary sequence may be modified. These changes may or may not serve/enhance function or characteristics. Usu. occur after protein has already folded into its specific conformation.
• Glycosylation: the addition of carbohydrates to a molecule.
• Fatty acylation: the addition of lipid group(s) to a molecule. These types of changes (N- or O-) can enhance barrier/surface protection (e.g. N-linked oligosaccharides). O-linked oligosaccharides can enhance secretions.
• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4975971/
• https://www.nature.com/articles/nrm.2015.11
• Prenylation: the addition of farnesyl or geranylgeranyl groups via thioether linkate to specific cysteine residues of membrane proteins.
• Regulatory modifications. Phosphorylation, acetylation, and adenosine diphosphate (ADP)-ribosylation of some AAs can alter bonding characteristics of the AA.
• Other amino acid posttranslational modifications: can alter the activity of the protein.
• Selenocysteine: found in a few enZ and is required to activate those enZ.

Resources.

References.

Lieberman, M., & Peet, A. (2017). Marks’ basic medical biochemistry: A clinical approach(5th ed.). Philadelphia, PA: LWW.

[Study guide covering chapter 5, PDF]

Last edited: 08.04.2019

Important functional groups: those with oxygen, nitrogen, phosphorus, sulfur.

• Acyl group: the part of the structure that provides the –C=O carbonyl group in an ester/amide linkage. “-yl” ending.
• Aliphatic: open chains (non-ring).
• Aromatics/Benzene rings. “Phenyl” if ring is a substituent.

Carbohydrates (sugars).

• Formula: CnH2nOn. Glucose is C6H12O6.
• Classified by: carbonyl group (aldose/ketose); # of carbons; positions of the -OH group on the anomeric carbon (D=right/L=left OH position, stereoisomers, epimers); any additional substituents; number of saccharides; how the components are linked (e.g. glycosidic bonds).
• For n asym centers >> 2^n stereoisomers generally speaking.
• Humans use D-sugars. “D for Delightful sugars!”
• Epimer: a pair of stereoisomers that differ only in one position of the OH at a chiral carbon.
• Epimerases: enzymes that make epimer conversions.
• Glycoproteins: proteins + sugars.
• Proteoglycans: proteins that are heavily glycosylated. Many long unbranched polysaccharide chains attached to a protein core. VIP to extracellular matrix, aqueous humor, cells that make mucous secretions, & cartilage.
• Glycosaminoglycans: polypeptide chains with repeating disaccharide units w/oxidized acid sugars, sulfated sugars, and N-acetylated amino sugars. Structure looks like a bottle brush.
• https://themedicalbiochemistrypage.org/glycans.php
• https://www.mdpi.com/1424-8247/11/1/27/pdf
• Glycosylation: a reaction where a carbohydrate is attached to a hydroxyl or other functional group.
• Glycolipids: lipids + sugars.
• In solution, OH on anomeric carbon spontaneously changes (mutarotation) from alpha to beta and back to change from open to ring forms such as chair/boat etc. Chair/boat etc are usually more stable so there’s a greater chance that a compound will be in those configurations.
• If the anomeric carbon forms a bond with another molecule, those mutarotations cannot happen due to the bond which limits configurational possibilities.
• Common substituted groups: phosphate, amino, sulfate or N-acetyl.
• Most free monosaccharides in the body are phosphorylated at the terminal carbons preventing transport out of cell.
• Galactosamine & glucosamine are examples of an amino group replacing one of the OH groups. Usu. the amino group gets acetylated forming an N-acetylated sugar.
• Acetylation: adding an acetyl functional group to a compound.
• Acyl group. http://www.chem.ucla.edu/~harding/IGOC/A/acetyl_group.html
• https://www.oit.edu/docs/default-source/library-documents/library-publishing/che102-intro-organic-chemistry/chapter-1-7.pdf
• Sugars can get oxidized at the aldehyde carbon to form “-onic acid” or “-onate”.
• Uronic (“-uronic acid”) acid forms when the the terminal OH group gets oxidized.
• Polyol sugar: a sugar where the aldehyde gets reduced where all the carbon atoms have OH. Eg. Sorbitol.
• Deoxy sugar: a sugar that has reduced such that 1+ carbons contains only H’s. Carbon 2 of deoxyribose.
• The OH of the anomeric carbon can react with an OH (O glycosidic bonds found in sugar-sugar, sugar-hydroxyl bonds) or NH (N glycosidic bonds found in nucleosides and nucleotides) group to form an alpha/beta glycosidic bond.
• Alpha glycosidic bond. The Greek alpha looks like a fish which is DOWN in the sea.
• Beta glycosidic bond. The Greek beta looks like a bird UP in the air.
• Disaccharide: 2 monosaccharides joined by O-glycosidic bond.
• Oligosaccharide: 3-12 linked monosaccharides via N or O glycosidic bonds.
• Polysaccharides: thousands of monosaccharides joined to make chains and/or branches.

Lipids.

• Hydrophobic.
• Usu. straight chains, methyl group at one end (w-carbon) and carboxyl at the other end.
• Most FA in humans have even number of carbons betw. 16-20.
• Most common FA in cells are stearic and palmitic FAs.
• *Special notation for FA’s pg. 69-70.
• http://rogersal.people.cofc.edu/Lipids.pdf
• https://www.cs.mcgill.ca/~rwest/wikispeedia/wpcd/wp/f/Fatty_acid.htm
• https://courses.lumenlearning.com/suny-nutrition/chapter/2-33-fatty-acid-naming-food-sources/
• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3646453/
• https://www.uio.no/studier/emner/matnat/farmasi/nedlagte-emner/FRM2041/v06/undervisningsmateriale/fatty_acids.pdf
• FA also classified by distance from w-carbon to the double bond.
• Fatty acids. Esterified to glycerol >> triacylglycerols (triglycerides) or phosphoacylglycerols (phosphoglycerols).
• Tri acyl glyderols: 3 acyl FA groups attached to glycerol; fat stores in the body.
• Sphingolipids: FA + sphingosine (serine + palmitate FA). No glycerol backbone.
• Ceramides (type of amides) = sphingosine + FA attached at the amino group.
• More sphingolipids formed from attaching substituents onto the OH of the ceramide.
• Cerebrosides + gangliosides = sugars glycosidically bonded to OH of ceramides.
• Sphingomyelin = phosphorylcholine + ceramide; vip part of cell membranes and myelin sheath.
• Glycolipids: lipids + sugar hydroxyl group.
• Polyunsaturated FA: building blocks of eicosanoids.
• Eicosanoids: signalling molecules via enZ or non-enZ oxidation of arachidonic acid or other polyunsat. FA. Hormone-like compounds. Polyunsat FA with 20 carbons (eicosa) and have 3-4-or-5 double bonds (e.g. prostaglandins, thromboxanes, leukotrienes).
• Naturally occurring FA typically cis.
• There are also trans.
• Cholesterol: formed from isoprene units.
• Bile salts.
• Steroid hormones.
• Isoprenyl unit: combined in long chains to make structures such as side chains of Coenzyme Q in humans and Vit A in plants.
• Geranyl groups = 10 carbons & polymers of isoprenyl units.
• Farnesyl groups = 15 carbons + isoprenyl units.
• Geranyl and farnesyl groups often get attached to proteins so that proteins can interact w/other cellular structures.
• Acylglycerols: glycerol with 1+ FAs (acyls via ester linkages). Mono- di- and tri-acylglycerols contain 1, 2, and 3 FA esterified to glycerol. Triacylglycerols don’t usu. have the same FA at all 3 positions (usu. mixed).
• Phosphoacylglycerols: FA at positions 1 and 2; phosphate group (or substituent attached to phosphate group) at position 3. If it’s only the phosphate group and NO other substituents at position 3, then it’s a phosphatidic acid.
• Phosphatidylcholine (lecithin) found in membranes. Has polar and nonpolar duality.
• Lysolipid = phosphoacylglycerols – fatty acyl group
• Steroids: 4-ring steroid nucleus; cholesterol precursor; diff species made by modifying ring or C20-side chains.
• Cholesterol hydrophobic can convert to hydrophilic bile salt (eg. cholic acid). Branched 5-carbon units w/1 double bond (isoprenyl unit)
• Bile salts are on micelles survaces in the intestinal lumen.

Nitrogen Compounds.

• Amino groups.
• Heterocyclic ring.
• Amino acids: carboxyl group; amino group; 1+carbons.
• Purines: has heterocyclic N ring.
• Pyrimidines: has heterocyclic N ring.
• Pyridines: has heterocyclic N ring.
• Nucleosides: N ring + sugar (usu. ribose or deoxyribose) via N-glycosidic bond.
• Nucleotide: nucleoside + phosphate.
• In proteins, amino acids (AA) are L-alpha-AA.
• While it is rare that beta or gamma AAs get formed, only L-alpha-AAs get incorporated into proteins.
• D-aminos are used by bacterial in their cell walls.
• N as a component in heterocyclic rings or N-bases.
• Common rings: purines, pyrimidines, pyridines.
• “-ines” denotes nitrogen in the structure. Uracil (pyrimidine) is an exception to the naming.
• Tautomers: in N rings, hydrogen can shift positions with the double bonds and this is called tautomers or tautomerization.
• https://chem.libretexts.org/Bookshelves/Organic_Chemistry/Book%3A_Organic_Chemistry_with_a_Biological_Emphasis_(Soderberg)/13%3A_Reactions_with_stabilized_carbanion_intermediates_I/13.1%3A_Tautomers
• http://www.chem.ox.ac.uk/vrchemistry/nor/notes/tautomers.htm
• https://www.khanacademy.org/science/organic-chemistry/ochem-alpha-carbon-chemistry/formation-of-enolate-anions/v/keto-enol-tautomerization

Free Radicals.

• Compounds w/single electron in outer shell.
• Extremely reactive and unstable.
• Usually formed as intermediates.
• Usu. negative effects.

Oxidation/Reduction.

• Carbon-carbon or carbon-oxygen bonds said to be oxidized or reduced depending on # of electrons around the carbon.
• LEO: lose electrons (lose H atoms) oxidation.
• GER: gain (gain H or lose O) electrons reduction.
• More oxidized from alcohol to aldehyde/ketone to carboxyl.

Acid/Base.

• Cations are catfabulous (+)! Anions (onions) make you cry (-).
• Common anionic groups: carboxylate; phosphates (P); sulfates.
• Common cationic groups: N, amines.

Bond Polarity & Partial Charges.

• Carboxylate.
• Phosphate.
• Sulfate.
• Ester = carboxylic acid + alcohol – water
• Thioester = acid + sulfhydryl
• Amide = acid + amine
• Phosphoester = phosphoric acid + alcohol
• Anhydride = acid1 + acid2

Resources.

• https://www.researchgate.net/publication/268224079_A_New_and_Simple_Method_for_Drawing_of_the_Monosaccharide_Fischer_Projection_Based_on_New_Monosaccharide’s_Barcodes
• Isoprene unit. https://chem.libretexts.org/Ancillary_Materials/Reference/Organic_Chemistry_Glossary/Isoprene_Rule
• Drawing sugars. http://www.chtf.stuba.sk/~szolcsanyi/education/files/Chemia%20heterocyklickych%20zlucenin/Prednaska%206/Odporucane%20studijne%20materialy/Drawing%20sugar%20structures.pdf
• Aldose configurations. https://chem.libretexts.org/Bookshelves/Organic_Chemistry/Map%3A_Organic_Chemistry_(McMurry)/Chapter_25%3A_Biomolecules%3A_Carbohydrates/25.04_Configurations_of_Aldoses
• https://chem.libretexts.org/Bookshelves/Organic_Chemistry/Map%3A_Organic_Chemistry_(McMurry)/Chapter_25%3A_Biomolecules%3A_Carbohydrates/25.01_Classification_of_Carbohydrates
• https://chem.libretexts.org/Bookshelves/Organic_Chemistry/Map%3A_Organic_Chemistry_(McMurry)/Chapter_25%3A_Biomolecules%3A_Carbohydrates/25.02_Depicting_Carbohydrate_Stereochemistry%3A_Fischer_Projections
• https://chem.libretexts.org/Bookshelves/Organic_Chemistry/Map%3A_Organic_Chemistry_(McMurry)/Chapter_25%3A_Biomolecules%3A_Carbohydrates/25.03_D%2C_L_Sugars
• https://chem.libretexts.org/Bookshelves/Organic_Chemistry/Map%3A_Organic_Chemistry_(McMurry)/Chapter_25%3A_Biomolecules%3A_Carbohydrates/25.05_Cyclic_Structures_of_Monosaccharides%3A_Anomers
• https://chem.libretexts.org/Bookshelves/Organic_Chemistry/Map%3A_Organic_Chemistry_(McMurry)/Chapter_25%3A_Biomolecules%3A_Carbohydrates/25.06_Reactions_of_Monosaccharides
• https://chem.libretexts.org/Bookshelves/Organic_Chemistry/Map%3A_Organic_Chemistry_(McMurry)/Chapter_25%3A_Biomolecules%3A_Carbohydrates/25.08_Disaccharides
• https://chem.libretexts.org/Bookshelves/Organic_Chemistry/Map%3A_Organic_Chemistry_(McMurry)/Chapter_25%3A_Biomolecules%3A_Carbohydrates/25.09_Polysaccharides_and_Their_Synthesis
• https://chem.libretexts.org/Bookshelves/Organic_Chemistry/Map%3A_Organic_Chemistry_(McMurry)/Chapter_25%3A_Biomolecules%3A_Carbohydrates/25.10_Other_Important_Carbohydrates
• https://chem.libretexts.org/Bookshelves/Organic_Chemistry/Map%3A_Organic_Chemistry_(McMurry)/Chapter_26%3A_Biomolecules%3A_Amino_Acids%2C_Peptides%2C_and_Proteins

References.

Lieberman, M., & Peet, A. (2017). Marks’ basic medical biochemistry: A clinical approach(5th ed.). Philadelphia, PA: LWW.