biochem: mark's medical biochem chapter 27: digestion and transport of carbohydrates

this chapter covers several aspects of carbohydrate digestion. first it goes over some basics about carbohydrates and digestion in general, then talks about the specific enzymes at work in the brush border of the small intestine, then a brief section about lactose intolerance, and finally, a section about the transport of glucose from the intestine into the blood.

the normal american diet is made of 40-50% carbohydrates, and of this, 50-60% is made of the starch molecules (10,000 to 1 million glucosyl units long) amylose and amylopectin. amylose consists of glucose molecules bonded with alpha 1-4 bonds, while amylopectin is the same, but with alpha 1-6 bonds (and therefore branches) as well. digestion of these starches begins in the mouth with alpha amylase, which is an example of an endoglucosidase-- an enzyme which cleaves alpha 1-4 bonds at random intervals. this initial digestion leaves chunks of polysaccharides called alpha-dextrins. stomach acid deactivates the amylase, and carbohydrate digestion continues in the small intestine.

in the small intestine, carbohydrate digestion takes place mainly on the "brush border" of the intestinal mucosa, which have enzymes that are embedded in the intestinal membrane that poke into the intestinal lumen. the four major "glucosidases" are glucoamylase, trehalase, beta-glucosidase, and sucrase-isomaltase. each of these enzymes has a specific structure and catalytic sites that are specific to certain types of carbohydrates. glucoamylase breaks down alpha 1-4 bonds from the non reducing end ("tail end") of the sugar, until isomaltase remains-- isomaltase is basically just two sugar units branched together in an alpha 1-6 bond. the action of glucoamylase on polysaccharides seems similar to the action of glycogen phosphorylase in the breakdown of glycogen in that both are removing glucose residues one at a time from the tail end, and both can not remove the last glucose unit (or in glucose phosphorylase's case, the last 4).

sucrase-isomaltase is another brush border glucosidase with two catalytic sites. one is specific to isomaltose and maltose (and thus can break down the isomaltose from glucoamylase's activity) and one is specific to sucrose and maltose. beta glucosidase also has two catalytic sites. one is specific to breaking the beta 1-4 bond between glucose and galactose in lactose, and the other is specific to cleaving beta 1-4 bonds in glycolipids. the last glucosidase in the brush border is trehalase, which has only one catalytic site which is specific to trehalose, a sugar found only in some insects, mushrooms, and algae. these four enzymes work in tandem to break down the different types of carbohydrates that are dumped into the duodenum. the relative concentration of these enzymes change depending on the location in the gut. for example, pancreatic alpha amylase is secreted mainly in the duodenum, sucrase-maltase and beta-glucosidase is mainly in the jejunum, and glucoamylases is most common in the ileum.

a couple other notes about carbohydrate digestion. lactose intolerance occurs with either a lactase deficiency or intestinal damage. most adults only have 10% of the lactase activity that they had as children. lactose intolerance can ultimately lead to malabsorption of nutrients: lactose is undigested in the gut, and is instead metabolized by the bacterial flora in the colon, producing gases and lactic acid. the lactic acid can increase the intestinal lumen's osmolarity and cause water to distend the abdomen, which increases peristalsis and potentially causes diarrhea and malabsorption of other nutrients.


the last section of the chapter focused on the membrane channels that transport glucose across the intestinal lining into the blood. there are two types of such transport proteins, regular facilitated glucose channels and Na+ facilitated channels. Na+ facilitated channels use an ATPase Na+/K+ pump to establish a low concentration of Na+ inside the intestinal cell. the concentration gradient that is formed from the higher Na+ concentration in the intestinal lumen is then coupled with glucose transport in these membrane proteins. facilitative glucose transporters (also called "GLUT" transporters) do not require ATP and simply allow glucose to flow down its concentration gradient from the intestinal lumen, into the epithelium, and out into the serosa side ("blood side").

a few interesting notes relating to these glucose transporting membrane proteins. the liver's GLUT transporters have a higher than usual Km (the concentration at which the substrate is half-saturated with enzyme-- generally representing the enzyme's affinity for substrate and in this case the GLUT for glucose) for glucose, because it will only accept glucose into its cells (to be converted into glycogen for storage) when the blood glucose concentration is very high, like after a high carb meal. also, in muscle and fat cells, insulin stimulates glucose absorption by means of recruiting intracellular vesicles of glucose transport proteins to the cell membrane, where they can facilitate glucose transport into the cell.

questions
1. what percentage of a normal american diet consists of carbohydrates?
2. what percentage of the carbohydrate calories consists of amylose and amylopectin?
3. how many glucosyl units do amylose and amylopectin have?
4. what types of bonds do amylose and amylopectin have?
5. what are the major natural sweeteners found in fruit, honey, and vegetables?
6. what is the major dietary carb found from animal sources?
7. how much liquid do the salivary glands secrete per day?
8. what is an "endoglucosidase" and what is an example of one?
9. what is salivary amylase inactivated by?
10. how much digestive enzyme is secreted by the pancreas per day?
11. what are in the pancreatic secretions?
12. what are oligosaccharides?

13. what are the glucosidases found in the brush border of the small intestine?
14. describe the digestion of lactose and sucrose in the small intestine.
15. describe the structure and activity of glucoamylase?
16. what are alpha-dextrins vs. limit-dextrins?
17. describe the structure and activity of the sucrase-isomaltase complex.
18. what percentage of maltase activity can be attributed to the sucrose-isomaltose complex?
19. describe the structure and activity of trehalase.
20. describe the structure and activity of the beta-glucosidase complex
21. pancreatic alpha-amylase activity is highest in...
22. sucrase-isomaltase activity is highest in...
23. beta-glucosidase activity is highest in...
24. glucoamylase activity is highest in...

25. what type of carbohydrates enter the colon?
26. what are the fatty acids that result from bacterial starch digestion in the colon?
27. what are the gases that result from bacterial starch digestion in the colon?

28. lactose intolerance can be caused by...
29. what are normal lactase levels of an adult as compared to a child?
30. what happens when lactose is ingested by a lactose intolerant person?

31. what does the glycemic index represent?
32. which sugars have the highest glycemic index?
33. what are the two types of glucose transport proteins?
34. describe the mechanism of the Na+ dependent glucose transporter.
35. what are facilitative glucose transporters?
36. compare the digestion of glucose with that of galactose and fructose.
37. in body tissues, why is glucose transport across membranes not the rate limiting step of glucose metabolism?
38. how does the high Km of glucose transport proteins in the liver relate to the liver's blood glucose regulation?
39. how is insulin related to glucose transport proteins in the liver?


answers
1. 40-45%
2. 50-60%
3. 10,000 to 1 million
4. amylose has alpha 1-4 bonds between glucosyl residues. amylopectin has alpha 1-4 bonds between glucosyl units as well as alpha 1-6 bonds between branches.
5. fructose, sucrose, glucose.
6. lactose, which is made of glucose and galactose.
7. ~1 liter a day
8. an enzyme that breaks internal alpha 1-4 bonds in a polysaccharide at random intervals, such as amylase.
9. acidity of the stomach
10. ~1.5 liters a day
11. trypsinogen, chymotrypsinogen, carboxypeptidase (for digestion of proteins), alpha-amylase (for carbohydrates), lipase (fat), and bicarbonate (neutralizing gastric acidity)
12. 4-9 glucosyl units long, contain one or more alpha-1,6 branches.

13. beta-glucoamylase, sucrase-isomaltase, beta-glycosidase, trehalase.
14. converted to monosaccharides by glucosidases attached to the brush border lining.
15. it has two domains that have different substrate specificity, and acts as an exoglucosidase by breaking alpha 1-4 bonds on the non reducing ("tail end") of the saccharides, releasing glucose units until only isomaltose remains.
16. alpha dextrins are the pieces of polysaccharides that result from salivary alpha-amylase's endoglucosidase activity. limit dextrins are oligosaccharides that have been formed from the further breakdown of polysaccharides by pancreatic alpha-amylase.
17. made of two subunits: sucrose-maltose subunit cleaves alpha 1-4 bonds in sucrose, maltose. isomaltose-maltose unit cleaves alpha 1-6 bonds in isomaltose, and also breaks down maltose.
18. 80%
19. trehalose is a smaller dissaccharidase that only has one catalytic site with specificity for trehalose, which is a relatively rare source of carbohydrate found in some insects, algae, and mushrooms.
20. has two catalytic sites: glucosyl-ceramidase site, which cleaves beta bonds in glycolipids, and lactase site, which breaks beta 1-4 bonds between glucose and galactose in lactose.
21. duodenum
22. jejunum
23. jejunum
24. ileum

25. any undigested starches: starches high in amylose, poorly hydrated starches (like in dried beans), dietary fiber.
26. acetic acid, propionic acid, butyric acid. 2,3,4 carbon.
27. hydrogen gas, CO2, methane

28. low lactase levels or intestinal injury
29. 10% of the level of a child
30. lactose is undigested by the lactase in the small intestine and is therefore metabolized by the colonic bacteria, which produces lactic acid, methane, and H2. the increased lactic acid increases osmolarity of the intestinal lumen, causing more water to be dumped into the lumen, causing excess peristalsis, causing malabsorption of other nutrients.

31. how quickly blood glucose levels rise after consumption of a food.
32. glucose and maltose.
33. sodium dependent glucose transporters and facilitative glucose transporters
34. a Na+ / K+ ATPase pump pumps Na+ out of the intestinal epithelium cells so that there is a low Na+ concentration within. the Na+ dependent transporter channels then use the resulting Na+ concentration gradient to power movement of glucose within the cell.
35. these are glucose channels that exist on both the luminal and serosal side of the intestinal epithelium that allow glucose to move down its concentration gradient without expenditure of energy.
36. galactose passes through the intestinal membrane in a similar way to glucose- via both Na+ facilitated channels and facilitative glucose transporters. fructose passes through by facilitated diffusion only.
37. because the transport proteins have a high affinity for glucose (a low Km) or are present in high numbers.
38. the liver will only transport glucose into its cells (and therefore convert glucose to glycogen for storage) when the blood glucose level is high, such as right after a high carb meal.
39. binding of insulin recruits GLUT proteins from intracellular vescicles onto the membrane.

biochem: mark's medical biochem chapter 29- fructose, galactose, pentose pathway

this chapter looked at the metabolic pathways for two other common dietary carbohydrates, fructose and galactose, as well as the pentose pathway, which produces NADPH and ribose 5-phosphate (used in nucleotide synthesis). fructose and galactose metabolism are similar in that they are basically phosphorylated and then converted into intermediates of the glycolytic pathway.

fructose metabolism:
1. fructose is phosphorylated to fructose 1-phosphate via fructokinase
2. fructose 1-phosphate is cleaved to DHAP and glyceraldehyde via aldolase B
3. glyceraldehyde phosphorylated to G3P via triose kinase
4. G3P and DHAP can be used in the glycolytic pathway

galactose metabolism:
1. galactose is phosphorylated to galactose 1-phosphate via galactokinase
2. galactose 1-phosphate plus UDP-glucose yields glucose 1-phosphate plus UDP-galactose
3. glucose 1-phosphate is converted to glucose 6-phosphate, which can be used in the glycolytic pathway.

production of fructose from glucose was also looked at in the "polyol" pathway:
1. glucose is reduced to sorbitol (polyol) via aldolase reductase, using NADPH as the reducing equivalents.
2. sorbitol is oxidized to fructose via sorbital dehydrogenase, producing NADH.

this conversion of glucose to fructose is used mainly in the seminiferous tubules, where spermatozoans use fructose instead of glucose in order to maintain the integrity of their plasma membrane (apparently glucose causes "acrosomal damage")



the pentose pathway is introduced as an alternate use of glucose 6-phosphate: instead of oxidizing for ATP, glucose 6-phosphate can taken through the pentose pathway to create NADPH (provides reducing equivalents, aids in catabolic reactions and helps protect against reactive oxygen species) and ribose 5-phosphate (used in nucleotide synthesis). the pentose pathway has two parts, an oxidative and non oxidative pathway. the oxidative pathway takes glucose 6-phosphate and creates 2 NADPH and ribulose 5-phosphate:

1. glucose 6-phosphate is oxidized to 6-phosphoglucanolactone via glucose 6 phosphate dehydrogenase, producing NADPH
2. 6-phosphoglucanolactone is hydrolyzed to 6-phosphoglucanolactate via glucanolactase
3. 6-phosphoglucanolactate undergoes oxidative decarboxylation to ribulose 5-phosphate, releasing CO2 and producing another NADPH

the non-oxidative portion of the pentose pathway takes three molecules of 5-ribulose phosphate, the product of the oxidative portion, and rearranges the molecules using transferases to eventually form 2 molecules of fructose 6-phosphate and 1 molecule of G3P. the reactions are as follows:

1. out of 3 molecules of 5-ribulose phosphate, 2 are converted to xylulose 5-phosphate via epimerases
2. the 3rd molecule of 5-ribulose phosphate is converted to ribose 5-phosphate (which can be used for nucleotide synthesis if necessary)
3. xylulose 5-P and ribose 5-P are rearranged to G3P and sedoheptulose 7-P via transketolase
4. sedoheptulose 7-P and G3P are rearranged to fructose 6-P and erythrose 4-P via transaldolase
5. erythrose 4-P and xylulose 5-P are rearranged to fructose 6-P and G3P
thus, 3 molecules of ribulose 5-phosphate are converted to 2 molecules of fructose 6-phosphate and one molecule of G3P.

we then look at how the pentose pathway can be used to accommodate and balance cellular needs for ATP, ribose 5-phosphate, and NADPH. several scenarios are considered-- when only NADPH is needed, only the oxidative portion of the pathway is activated, producing two molecules of NADPH per glucose molecule, and the nonoxidative pathway converts ribulose 5-phosphate to glucose 6-phosphate (presumably by isomerization of fructose 6-phosphate) which is then put back into the oxidative portion to create even more NADPH. when NADPH and ribose 5-phosphate are both needed, the oxidative pathway is activated to produce NADPH, and ribose 5-phosphate is produced from the non-oxidative pathway via isomerase. when only ribose 5-phosphate is needed, this implies that NADPH levels are relatively high-- which actually inhibits the first enzyme in the oxidative pathway. thus the oxidative pathway is inhibited, but the nonoxidative pathway produces ribose 5-phosphate from ribulose 5-phosphate. finally, in the case when NADPH and pyruvate are needed, both the oxidative and non oxidative portions are stimulated.


questions
1. describe the mechanism for the metabolism of fructose.
2. where does metabolism of fructose occur?
3. describe the mechanism for the polyol pathway.
4. where is the polyol pathway mainly used and why?
5. what is the galactose metabolism pathway?
6. what does the pentose pathway produce and why are the products useful?
7. why is NADP+ used instead of NAD+?
8. describe the mechanism for the oxidative branch of the pentose pathway.
9. describe the mechanism for the non-oxidative branch of the pentose pathway.
10. describe how the glycolytic intermediates can be used to produce ribose 5-phosphate for nucleotide synthesis.

11. what are some uses of NADPH in the body?
12. the entry of glucose 6-phosphate into the pentose phosphate pathway is regulated by...

describe how the pentose phosphate pathway is used to respond to cellular needs for:
13. NADPH
14. NADPH and ribose 5-phosphate
15. ribose 5-P only
16. NADPH and pyruvate

answers
1. fructose is phosphorylated by fructokinase to fructose 1-phosphate. fructose 1-phosphate is cleaved by aldosase B into DHAP and glyceraldehyde. glyceraldehyde is phosphorylated by triose kinase into G3P. G3P can then be converted to 1,3 bisphosphoglycerate in the glycolytic pathway or combined with DHAP to form fructose 1,6 bisphosphate in the gluconeogenic / glycolytic pathway.
2. mainly in the liver, but also in the mucosa of the small intestine.
3. glucose is reduced to sorbitol (polyol) by aldolase reductase, forming NADPH. sorbitol is oxidized to fructose via sorbitol dehydrogenase.
4. glucose conversion into fructose is mainly used in the seminal vescicles, which store the spermatozoans. spermatozoans use fructose rather than glucose to avoid plasma membrane damage while in the male reproductive system.
5. galactose is phosphorylate by galactokinase to galactose 1-phosphate. galactose 1-phosphate is converted to glucose 1-phosphate via galactose 1-phosphate uridylyltransferase. glucose 1-phosphate is then isomerized to glucose 6 phosphate for use in other metabolic pathways.
6. an alternative pathway to the first few steps of glycolysis that produces ribose 5-phosphate, which is used in nucleotide synthesis, and NADPH, which is used in reductive detoxification.
7. NADPH is used in reactions that have need for reducing equivalents because the ratio of NADPH to NADP+ is much higher than that of NADH to NAD (because NADH is immediately used in the electron transport chain)
8. glucose 6-phosphate is oxidized to 6-phosphogluconolactone via glucose 6-phosphate dehydrogenase (producing NADPH). 6-phosphogluconolactone is hydrolyzed to 6-phosphogluconolactate via gluconolactase. 6-phosphate gluconolactate undergoes oxidative decarboxylation to ribulose 5 phosphate, releasing CO2 and forming NADPH.
9. 3 molecules of ribulose 5-phosphate are converted via isomerases, epimerases, transketolases, and transaldolases into two molecules of fructose 6-phosphate and one molecule of G3P, which can be used in glycolysis.
10. ribose 5-phosphate can be synthesized using the non-oxidative portion of the pentose pathway because the reactions are reversible. 2 molecules of fructose 6-phosphate and 1 molecule of G3P can ultimately produce 3 molecules of ribose 5-phosphate.

11. NADPH is mainly produced from the oxidative portion of the pentose pathway and is used to provide reducing equivalents and is involved in protecting the body against reactive oxygen species. it is also involved in anabolic processes such as cholesterol synthesis, fatty acid synthesis and chain elongation.
12. the concentration of NADPH in the cell. high NADPH inhibits glucose 6-phosphate dehydrogenase.

13. the oxidative portion of the pentose phosphate pathway is utilized to produced 2 moles of NADPH per molecule of glucose. the non oxidative portion of the pentose pathway is used to convert ribulose 5-phosphate back to glucose 6-phosphate, where it can be used to generated more NADPH.
14. the oxidative portion creates NADPH and ribulose 5-phosphate. isomerases convert ribulose 5 phosphate into ribose 5-phosphate.
15. high levels of NADPH will inhibit the oxidative portion; just the non-oxidative portion will create ribose 5-phosphate from fructose 6-phosphate and G3P.
16. the oxidative portion will create NADPH and the nonoxidative will create fructose 6-phosphate and G3P, which can be used in glycolysis to produce pyruvate.

biochem: mark's medical biochem chapter 28- glycogen

this chapter is about the synthesis and breakdown of glycogen in the liver and skeletal muscles. glycogen is a huge branched molecule which serves as a storage form for glucose. its role in the liver is to aid in regulating blood sugar levels by degradation of glycogen into free glucose, which can be used to replenish flagging blood glucose levels. in skeletal muscle, glycogen is broken down expressly for the purpose of supplying glucose 6-phosphate for anaerobic glycolysis when ATP demand is high.

glycogen itself is a large, highly branched polysaccharide made of repeating glucosyl units with alpha 1-4 bonds (elongating a given branch) and alpha 1-6 bonds (creating a new branch point). glycogen's synthesis mechanism is as follows:

1. glucose is phosphorylated to glucose 6-phosphate via glucokinase.
2. glucose 6-phosphate is isomerized to glucose 1-phosphate via phosphoglucomutase.
3. glucose 1-phosphate is activated by UTP into UDP glucose.
4. UDP glucose units are repeatedly added to a glycogen primer via glycogen synthase.
5. when a given branch is ~11 units (or "residues") long, a 6-8 residue piece is transferred via a transferase to another glucosyl unit with a alpha 1-6 bond, creating new branches.

glycogen breakdown is not the reverse of the synthesis pathway:

1. glycogen residues are phosphorylated by glycogen phosphorylase, forming molecules of glucose 1-phosphate (which can be isomerized to glucose 6-phosphate for use in glycolysis, etc)
2. when any given branch is shortened to 4 residues long, the glycogen phosphorylase can not remove any more due to steric hindrance.
3. the "debrancher" enzyme then transfers the last 3 residues to another branch, where they can be acted on by glycogen phosphorylase.
4. the last residue is hydrolyzed by alpha 1,6 glucosidase into a glucose molecule.

the regulation of glycogen synthesis and degradation is somewhat involved and is different in the liver and muscle due to the different uses of glycogen as mentioned above. in the liver, the primary factor that regulates glycogen synthesis/breakdown activity is the glucagon/insulin ratio in the blood, which reflects the liver's need to maintain blood sugar levels. in skeletal muscle the primary factor that regulates glycogen synthesis/degradation is AMP levels, which indicate relative ATP usage -- reflecting skeletal muscle's use of glycogen as a direct backup energy source for contraction. during times of stress, epinephrine is released and stimulates glycogen degradation in both liver and skeletal muscle.

the stimulation of glycogen synthesis by rising glucagon, AMP levels, or epinephrine levels occurs via an enzymatic cascade which begins with the synthesis of cAMP via adenylate cyclate. cAMP then activates protein kinase A, which phosphorylates two enzymes, glycogen synthase and phosphorylase kinase. glycogen synthase, which (as described above) synthesizes glycogen from glucose 6-phosphate, is inactivated by this phosphorylation whereas phosphorylase kinase, which begins the degradation pathway, is activated by the phosphorylation. thus, activated protein kinase A's net effect is to simultaneously shut down the synthesis of new glycogen as well as initiate glycogen breakdown. in the breakdown pathway: phosphorylase kinase activates glycogen phosphorylase, which then removes glycosyl residues from glycogen as described above.

protein kinase A's counterparts are the "protein phosphorylases" that removes the phosphates from glycogen synthase (thereby activating synthesis) and phosphorylase kinase (thereby deactivating breakdown). this stimulation of synthesis of glycogen can happen after a high carbohydrate meal, when glucose levels are high and need to be converted into glycogen for storage and lowering blood glucose levels. during a high carb meal, glucagon levels fall, and insulin levels rise -- it is thought that compared to glucagon, the level of insulin is more actively involved in the regulation of glycogen synthesis and breakdown although the exact mechanisms are not well understood. however, higher insulin levels are known to activate these protein phosphorylases, which inhibit the breakdown pathway and stimulate the synthesis pathway.

questions
1. what is the structure of glycogen?
2. describe the usage of glycogen in skeletal muscle vs. in the liver.
3. describe the synthesis pathway of glycogen.
4. what are the two enzymes involved in the breakdown of glycogen?
5. what are the two functions of the "debrancher" enzyme?
6. describe the mechanism for the breakdown of glycogen.

7. describe the three factors that regulate glycogen synthesis and breakdown in the liver.
8. describe the three factors that regulate glycogen synthesis and breakdown in skeletal muscle.

9. compare glycogenolysis and gluconeogenesis as means of replenishing blood glucose levels.
10. describe the role of phosphorylation states in the regulation of glycogen synthesis / regulation in the liver.
11. what is synergistic phosphorylation and how does it relate to glycogen degradation?
12. what is hepatic PP-1 and what does it do?

13. why is insulin considered the primary hormone that regulates glycogen synthesis / breakdown?
14. describe how glucose levels affect glycogen synthesis / breakdown.
15. describe epinephrine's actions on beta-receptors in the liver.
16. describe epinephrine's actions on alpha-receptors in the liver.

17. why is glucose 6-phosphate produced from glycogenolysis in skeletal muscle "committed" to the glycolytic pathway?
18. how does glucagon affect the regulation of skeletal muscle glycogen synthesis/degradation?
19. how does AMP affect the regulation of skeletal muscle glycogen synthesis/degradation?
20. contrast the intracellular Ca2+ production in the liver and skeletal muscle.

answers
1. glucosyl units linked by alpha 1-4 glycosidic bonds with alpha 1,6 branches every 8-10 residues.
2. in skeletal muscle, when ATP demands are high or when glucose 6-phosphate is used up by anaerobic glycolysis, glucose 6-phosphate can be replenished via glycogen breakdown in order to ultimately enter the glycolytic pathway. in the liver, glycogen breakdown produces glucose 6-phosphate, which is then converted to via glucose 6-phosphatase to glucose, which is then released into the blood.
3. glucose is converted to glucose 6-phosphate by hexokinases (or glucokinases in the liver). glucose 6-phosphate is converted to glucose 1-phosphate by phosphoglucomutase. glucose 1-phosphate is activated by UTP and converted to UDP-glucose, which can then be attached via glycogen synthase to a glycogen primer. when the chain of glycosyl is 11 residues long, amylotransferase transfers the chain back onto another glycogen branch in an alpha 1-4 bond-- this process happens repeatedly and creates a highly branched structure.
4. glycogen phosphorylase and the "debrancher enzyme"
5. it acts as a transferase and an alpha 1-6 glucosidase.
6. glycogen phosphorylase continually removes glucosyl residues by phosphorylating the terminal glycosidic bond, creating glucose 1-phosphate. however, due to steric hindrance, it can not free glucosyl residues that are closer than 4 units away from a branch point. the transferase portion of the debrancher enzyme transfers the end three residues onto another chain, where it can be acted on by the glycogen phosphorylase enzyme. the alpha-1,6 glucosidase portion of the debrancher enzyme then hydrolyzes the final glucosyl residue on the branch to glucose.

7. the liver's glycogenolytic activity is regulated by glucagon, insulin, and epinephrine levels: when fasting, glucagon is high and insulin is low, which stimulates glycogenolysis and inhibits glycogen synthesis. during a high carbohydrate meal, insulin levels are high and glucagon levels are low -- stimulating glycogen synthesis and inhibiting glycogen breakdown. during exercise, epinephrine stimulates glycogen breakdown and inhibits glycogen synthesis as well.
8. epinephrine, AMP, and Ca2+. higher levels of all three molecules signal the need for greater energy production, which stimulates glycogen degradation and inhibits glycogen synthesis.

9. while both are employed by the liver to replenish blood glucose levels, glycogenolysis is both faster and supplies more glucose.
10. glucagon and insulin regulate glycogen synthesis / breakdown via a mechanism involving phosphorylating the glycogen synthase and glycogen phosphorylase enzymes between inactive and active states. for example, during fasting, high glucagon stimulates phosphorylation of glycogen phosphorylase to the active form, beginning glycogen degradation, while also phosphorylating glycogen synthase to an inactive form, inhibiting glycogen synthesis.
11. the phosphorylation of glycogen synthase into the inactive form is much more complex than that of glycogen phosphorylase into the active form, in that it has up to 10 different phosphorylation sites. "synergistic phosphorylation" is the process by which glycogen synthase is inactivated, where phosphorylation of one site (by protein kinase A) changes the conformation of the enzyme and facilitates phosphorylation at the remaining sites. (analogous to oxygen binding to hemoglobin)
12. hepatic PP-1 is a protein phosphatase that works in opposition to the protein kinase A in that it removes the phosphates from phosphorylase kinase and glycogen phosphorylase (thereby inhibiting glycogen breakdown), and glycogen synthase (thereby stimulates glycogen synthesis).
13. because its levels change to a greater degree in response to changing blood sugar levels than glucagon.
14. high glucose levels inhibit glycogen breakdown almost immediately (faster than the effect of glucagon's cAMP and protein kinase A pathway, which takes 10-15 minutes). glucose stimulates protein phosphatases to remove the phosphates from glycogen synthase b and phosphorylase a, the net effect being inhibition of glycogen degradation.
15. when epinephrine binds to beta receptors in the liver, it stimulates adenylate cyclase to produce cAMP and activate protein kinase A in a similar fashion as glucagon.
16. when epinephrine binds to alpha receptors, it activates the PIP-Ca2+ signal transduction system which increases intracellular levels of Ca2+.

17. because skeletal muscle has no glucose 6-phosphatase to facilitate the conversion to glucose.
18. glucagon has no effect on skeletal muscle regulation of glycogen synthesis/degradation and therefore glycogen levels in skeletal muscle do not vary much depending on food intake.
19. AMP represents the usage of ATP and activates the muscle isozyme of glycogen phosphorylase.

biochem: chart

courtesy of christina "smooshy" nelson

biochem: mark's medical biochemistry chapter 31- gluconeogenesis

gluconeogenesis is the process that occurs mainly in the liver in which glucose is produced from non-carbohydrate sources during times of fasting, exercise, or stress. it is essentially the opposite of glycolysis, in that instead of producing pyruvate from glucose, it synthesizes glucose from pyruvate. pyruvate itself is supplied by several different sources: lactate can be oxidized into pyruvate and is available from anaerobic glycolysis or by adipocytes in red blood cells. alanine can be transaminated into pyruvate and is produced from other amino acids released from the muscle. glycerol also serves as the precursor to an intermediate of gluconeogenesis, DHAP. the reactions of gluconeogenesis can be divided into three major sections:

conversion of pyruvate to phosphoenolpyruvate (PEP)
1. pyruvate is formed from alanine or lactate in the cytosol.
2. pyruvate diffuses into the mitochondria and is carboxylated to oxaloacetate via pyruvate carboxylase (recall that this is an anaplerotic reaction of the TCA cycle)
3. oxaloacetate is either transaminated to aspartate, or reduced to malate (using NADH as an electron source) and transported back out of the mitochondria.
4. oxaloacetate is reformed in the cytosol either by transamination of aspartate or oxidation of malate.
5. oxaloacetate is converted to phosphoenolpyruvate via phosphoenolpyruvate carboxykinase, using one GTP.

conversion of PEP to fructose 1,6 bisphosphate (reverse of glycolysis)
6. PEP is converted to 2-phosphoglycerate
7. 2-phosphoglycerate is converted to 3-phosphoglycerate
8. 3-phosphoglycerate is converted to 1,3-bisphosphoglycerate
9. 1,3-bisphosphoglycerate is converted to G3P.
10. for every two molecules of G3P that are formed, one isomerizes to DHAP
11. G3P condenses with DHAP to form fructose 1,6 bisphosphate.

conversion of fructose 1,6 bisphosphate to glucose (reverse of glycolysis)
12. fructose 1,6 bisphosphate is converted to fructose 6-phosphate via fructose 1,6 bisphosphatase.
13. fructose 6-phosphate is isomerized to glucose 6-phosphate via phosphoglucoisomerase.
14. glucose 6-phosphate is converted to glucose via glucose 6-phosphatase.

the reactions in bold are irreversible, endergonic reactions that use enzymes that are not used in the reverse glycolytic pathway. this is significant because the relative activity of these competing enzymes determines whether the reaction will proceed in the glycolytic or gluconeogenic pathway. the regulation of the first of these reactions, the conversion of oxaloacetate to PEP, is the most complex and is regulated by several enzymes in upstream reactions beginning with pyruvate production. the first, pyruvate dehydrogenase, is normally responsible for oxidizing pyruvate to acetyl CoA but is deactivated during gluconeogenesis, allowing pyruvate to instead be carboxylated into oxaloacetate. pyruvate carboxylase, the enzyme that catalyzes this reaction, is in turn activated by acetyl CoA, which is produced during the fatty acid oxidation which occurs during fasting or stress. these two reactions work in tandem during fasting conditions to ensure production of oxaloacetate from pyruvate rather than acetyl CoA.

the third enzyme which takes place in the regulation of the production of PEP is PEP carboxykinase, which converts oxaloacetate to PEP. in fasting conditions, glucagon and epinephrine stimulate cAMP to increase transcription of PEPCK enzymes, increasing the quantity of enzyme in the cell (called inducing). finally, the last enzyme involved is pyruvate kinase, which normally converts PEP back into pyruvate (recall the last step of glycolysis). high glucagon levels causes phosphorylation of the enzyme (using a mechanism involving cAMP and protein kinase A) and inactivates it-- thus allowing PEP to be used for gluconeogenesis instead of being uselessly cycled back to pyruvate. these four enzymes basically act as "switches" which first turn on the gluconeogenic pathway by allowing pyruvate to be converted to PEP.

the next places for enzymatic regulation of the gluconeogenic pathway are: the conversion of fructose 1,6-bisphosphate to fructose 6-phosphate, and the conversion of glucose 6-phosphate into glucose. both reactions are similar in that (as mentioned earlier) they use enzymes that are not the same as the reverse glycolytic reaction. in fasting conditions, the enzymes that catalyze the glycolytic reaction are deactivated, allowing the reaction to proceed in the gluconeogenic direction.

the book then talks about what happens in the liver and body tissues during, after, and long after a meal. during a high carbohydrate meal, blood glucose levels can rise from the normal 80-100 mg/dL to a high of 140 mg/dL. during this time insulin is secreted from the beta cells in the pancreas, and glucagon levels decrease. the net result is a storage of glucose in the liver as glycogen. within a few hours after eating, blood glucose and insulin levels fall back down, and glucagon levels start to rise- this initiates the process of glycogenolysis, which is the conversion of the stored glycogen in the liver back into glucose to maintain blood glucose levels. glucagon stimulates glycogenolysis and inhibits glycogen storage concurrently via production of cAMP, which stimulates protein kinase A to inactivate the enzyme related to glycogen synthesis as well as activate the glycogenolytic pathway. within 4 hours after a meal, as the liver's glycogen supply is decreasing (it takes about 30 hours to deplete the liver's supply of glycogen), gluconeogenesis is also stimulated by glucagon and falling blood sugar levels.

questions
1. what happens in the liver during fasting?
2. what is gluconeogenesis?
3. what are the three carbon sources for gluconeogenesis in humans?

4. describe the role of lactate as a gluconeogenic precursor.
5. describe the role of alanine as a gluconeogenic precursor.
6. describe the role of glycerol in gluconeogenesis.

7. describe the conversion of pyruvate to PEP.
8. what determines the path in which oxaloacetate will be converted and transported across the mitochondrial membrane?
9. describe the conversion of PEP to fructose 1,6 bisphosphate.
10. describe the conversion of fructose 1,6 bisphosphate to glucose.
11. describe the conversion of glycerol to DHAP.

12. what are other factors that can stimulate gluconeogenesis?
13. what are the three main reactions that are regulated in gluconeogenesis?
14. how does the fasting state deactivate pyruvate dehydrogenase?
15. how does the fasting state activate pyruvate carboxylase?
16. how is PEP carboxykinase regulated?
17. what is pyruvate kinase and how is it regulated?
18. describe the regulation of the reaction from fructose 1,6 bisphosphate to fructose 6-phosphate.
19. describe the regulation of the reaction from glucose 6-phosphate to glucose.

20. what is the energy consumption during gluconeogenesis and where does it happen?

21. what are normal blood glucose levels for fasting, right after a meal, 2 hours after a meal, and starvation?
22. describe the pancreas's actions after ingestion of a high glucose meal.
23. glycerol, glucagon, glycogen.
24. describe the stimulation of glycogenolysis in the liver.
25. describe what happens roughly 4 hours after a meal.
26. describe what happens during prolonged starvation.
27. how long does it take to deplete liver glycogen stores? (and therefore halt glycogenolysis)


answers

1. liver releases glucose into the blood via glycogenolysis and gluconeogenesis.
2. the process by which glucose is created in the liver from non carbohydrate sources.
3. lactate, glycerol, and amino acids- particularly alanine.

4. lactate is produced by anaerobic glycolysis through reduction of pyruvate or by adipocytes in the fed state or by red blood cells. lactate is oxidized into pyruvate, which is a precursor for gluconeogenesis.
5. alanine is produced in the muscle from other amino acids (whenever insulin is low or stress hormones are high) and from glucose. it is converted to pyruvate via alanine aminotransferase.
6. glycerol is released from adipose tissue whenever insulin levels are low or stress hormones are high. it is converted to DHAP, which is a gluconeogenetic intermediate (as well as a glycolytic one)

7. pyruvate is created from alanine or lactate in the cytosol, and then travels into the mitochondria, where it is carboxylated to oxaloacetate via pyruvate carboxylate (an anaplerotic reaction of the TCA cycle). oxaloacetate is then transaminated to aspartate or reduced to malate and transported back out into the cytosol, and reformed back into oxaloacetate (via oxidation or transamination). in the cytosol, oxaloacetate is decarboxylated by phosphoenolpyruvate carboxylkinase to form PEP.
8. the reduction of oxaloacetate into malate requires reducing equivalents; if the mitochondria has need for reducing equivalents for other reactions, it will use the other venue, the conversion to aspartate.
9. PEP is converted to fructose 1,6 bisphophate through a reversal of the glycolytic reactions. PEP is converted into 2-phosphoglycerate, to 3-phosphoglycerate, to 1,3 bisphosphoglycerate, and reduced to G3P. for every two molecules of G3P produced, one isomerizes to DHAP. G3P and DHAP condense to form fructose 1,6 bisphosphate.
10. fructose 1,6 bisphosphate has a phosphate removed by fructose 1,6bisphosphatase to form fructose 6 phosphate. fructose 6 phosphate is isomerized to glucose 6 phosphate by phosphoglucose isomerase. glucose 6 phosphate has a phosphate removed by glucose 6-phosphatase, producing glucose.
11. glycerol is converted to glycerol 3-phosphate via glycerol kinase, and then oxidized to DHAP.

12. prolonged exercise, stress, and a high protein diet.
13. OAA to PEP, fructose 1,6 bisphosphate to fructose 6 phosphate, glucose 6 phosphate to glucose. all three reactions use regulatory enzymes which are not involved in the reverse glycolytic pathway.

14. during the fasting state, fatty acids are released from adipose tissue and undergo beta oxidation, producing NADH, acetyl CoA, and ATP. the higher ATP / ADP ratio phosphorylates pyruvate dehydrogenase into the inactive form.
15. fatty acid oxidation produces acetyl CoA, which activates pyruvate carboxylase.
16. glucagon is released during fasting and EP is released during exercise/stress, both of which stimulate production of cAMP, which increases transcription of PEPCK genes.
17. pyruvate kinase is the enzyme that catalyzes the conversion of PEP back into pyruvate. when glucagon levels are high, pyruvate kinase is phosphorylated and inactive through a mechanism involving cAMP and protein kinase A.

18. this reaction occurs via the fructose 1,6 bisphosphotase enzyme, and normally would compete with the reverse reaction from glycolysis, fructose 6-phosphate to fructose 1,6 biphosphate via PFK-1. however, under conditions favoring gluconeogenesis, the enzymes that stimulate PFK-1 are inactive, allowing the reaction to head towards the production of glucose.
19. low insulin and glucose levels deactivate the enzyme for the glycolytic forward reaction and allow the glucose synthesis to occur.


20. for every mole of glucose that is produced, 6 moles of ATP and 2 moles of NADH are used. 2 moles of ATP at the conversion of pyruvate to oxaloacetate, 2 moles of ATP at the conversion of oxaloacetate to PEP, 2 moles of ATP at the conversion from 3-phosphoglycerate to 1,3 bisphosphoglycerate, and 2 moles of NADH at the reduction of 1,3 bisphospholycerate to G3P. (2 moles at each reaction because 2 molecules of pyruvate combine into one molecule of glucose)

21. fasting: 80-100mg/dL. right after a meal: up to 140mg/dL. 2 hours after a meal: back to 80-100mg/dL. starvation: not lower than 65mg/dL.
22. during a meal, the high glucose concentration in the blood stimulates the beta cells of the pancreas to increase insulin production. glucagon levels decrease in response to a high carbohydrate meal but increase in response to a high protein meal.
23. glycerol is released from adipose whenever levels of insulin are low and levels of glucagon is high-- and is converted into DHAP. glucagon is a hormone released by the alpha cells of the pancreas in response to decreasing blood glucose levels-- stimulating gluconeogenesis. glucagon also activates production of cAMP in liver cells, which activates protein kiase A, which inactivates glycogen synthase-- thus high glucagon levels inhibit glycogen production. glycogen is synthesized from glucose and stored in the liver.
24. high glucagon levels stimulate adenylate cyclate, which synthesizes cAMP. cAMP activates protein kinase A, which inactivates glycogen synthase, and activates phosphorylase kinase. phosphorylase activates phosphorylase b, which converts glycogen to glucose 1-P, which is then converted to glucose 6-P and then free glucose in the liver, which can then enter the blood.
25. in addition to supplementing blood glucose levels with glycogenolysis, gluconeogenesis is stimulated by the release of precursor material such as glycerol, alanine, and lactate from peripheral body tissues.
26. the body switches to fatty acid and ketone body oxidation and requires much less glucose.
27. ~30 hours

biochem: mark's medical biochem chapter 20- TCA cycle

this chapter is about the tricarboxylic acid cycle, which takes place inside the matrix of the mitochondria and takes a three carbon molecule, acetyl coenzyme A, and through a cycle of chemical changes produces 3 molecules of NADH2, one molecule of FADH2, one molecule of GTP, and two molecules of CO2. the reactions of the TCA cycle are as follows:

1. oxaloacetate and acetyl coenzyme A are condensed into citrate via citrate synthase.
2. citrate is isomerized to isocitrate via aconase.
3. isocitrate undergoes oxidative decarboxylation to
alpha-ketoglutarate via isocitrate dehydrogenase. NAD is reduced to NADH and CO2 is formed.
4. alpha-ketoglutarate undergoes another oxidative decarboxylation to succinyl CoA via alpha-ketoglutarate dehydrogenase. another NAD is reduced and CO2 is formed.
5. succinyl CoA undergoes substrate level phosphorylation via succinate thiokinase, forming succinate and GTP (from GDP).
6. succinate is oxidized to fumarate via succinate dehydrogenase. FADH2 is formed.
7. a H2O molecule is added to fumarate via fumarase, forming malate.
8. malate is oxidized back to oxaloacetate via malate dehydrogenase. NADH is formed.

the next sections cover some intricacies of the cycle. for example, the only reaction that uses FAD as an electron acceptor is the oxidation of succinate to fumarate via succinate dehydrogenase. the reason for this is that FAD can accept single electrons, forming a high energy, half reduced intermediate, whereas NAD can only accept pairs of electrons. this particular oxidation reaction involves formation of a C=C double bond, which apparently involves transferring one electron at a time- thus FAD is used instead of NAD. the enzyme that catalyzes this reaction, succinate dehydrogenase, has an FAD molecule near the active site, which accepts electrons and transfers them to an adjoining Fe-S complex, which then transfers the electrons to Coenzyme Q of the electron transport chain (recall that succinate dehydrogenase is actually "complex 2" of the electron transport chain).

another detail that is looked at: CoA contains a thioester bond, which is a C-S-C bond (as opposed to a regular C-O-C ester), which yields some special properties for molecules that contain it: first off, it is a relatively high energy bond which can provide a high and negative free energy change-- in the TCA cycle, this comes into play in the oxidation of succinyl CoA to succinate via succinate thiokinase, which provides enough energy to phosphorylate a GDP to GTP. secondly, the thioester does not participate in resonance stabilization as the regular ester does, activating the adjacent carbons to react with other molecules. this is seen in the citrate synthesis reaction, where the thioester bond on acetyl CoA activates a neighboring C, which then reacts with oxaloacetate to form citrate.

yet another detail: alpha-keto glutamate is oxidized with the help of alpha-ketoglutarate dehydrogenase, which is one of a family of three enzymes called alpha-ketoacid dehydrogenase complexes. these enzyme complexes have three subunits, labeled E1 through E3. E1 contains thiamine pyrophosphate, which cleaves a CO2 off of the alpha-keto acid, which is then oxidized and joined to a CoASH molecule by the lipoate on the E2 subunit. the electrons that lipoate received are then transferred FAD on E3, which then transfers them to NAD. the net result: these dehydrogenase complexes cleave a CO2, oxidize (hence "oxidative decarboxylation"), and combine the alpha-ketoacid with an CoA. this comes into play in the TCA cycle with the oxidative decarboxylation of alpha-ketoglutarate into succinyl CoA. later on the book talks about another enzyme in this same class, pyruvate dehydrogenase, which oxidizes pyruvate into acetyl CoA in a similar fashion.

the energetic efficiency of the TCA is touched upon: comparing the actual energetic output of the TCA cycle via the intermediates NADH and FADH2 versus what the energy output would be from combusting the intermediates in a bomb calorimeter yields an energy efficiency of ~90%. the reactions that have a large and negative free energy change: the synthesis of citrate, the oxidative decarboxylation of isocitrate as well as alpha-ketoglutarate. the reactions with a positive free change (non-spontaneous) are the oxidation of malate to oxaloacetate, and the isomerization of citrate into isocitrate. for the malate reaction, this means that the equilibrium favors malate, leaving a low concentration of oxaloacetate.

the low concentration of oxaloacetate facilitates regulation of the first reaction of the TCA cycle, citrate synthesis from oxaloacetate and Acetyl CoA. the TCA cycle is regulated in many different places, generally inhibited by the products that it creates and stimulated by buildup of reactants so as to maintain homeostasis and adequate ATP synthesis. in this fashion, citrate synthase is inhibited by the product it creates, citrate and stimulated by the reactant it uses, oxaloacetate. oxaloacetate production is itself stimulated by the NAD/NADH ratio, which in turn reflects the ATP usage: demonstrating that all activity in the cycle is ultimately correlated back to ATP usage. another example of enzymatic regulation is alpha-ketoglutarate dehydrogenase being activated by ADP (reflecting the need for more oxidative activity), inhibited by NADH (reflecting the lack of oxidative activity downstream -- therefore signalling the reaction to stop), and also activated by Ca2+ (which is released intracellularly during rapid muscle contraction, reflecting the need for greater oxidative activity).



after regulation we then take a look at the synthesis and usage of the precursors and intermediates of the TCA cycle, beginning with acetyl coA and pyruvate. this is an important reaction, as it converts the product of aerobic glycolysis, pyruvate, into the first compound of the TCA cycle, acetyl CoA. as was mentioned earlier, pyruvate is converted to Acetyl CoA by one of the three enzymes in the alpha-ketoacid dehydrogenase complex family, called pyruvate dehydrogenase complex (PDC). PDC is intricately regulated both by the reactant/product system described earlier (inhibited by NADH and acetyl CoA, two products of the pyruvate oxidation reaction), as well as a system of two enzymes which deactivate/activate it by phosphorylation/removing a phosphate, named PDC kinase and PDC phosphatase. PDC kinase is inhibited by high ADP levels and high pyruvate levels- in the first case the high ATP usage signals PDC kinase to stop its inhibitory activity of PDC, allowing pyruvate to be oxidized. in the second case the high pyruvate levels signals an excess of reactant, which produces the same effect. PDC phosphatase removes the phosphate from PDC, allowing it to catalyze the oxidation of pyruvate. it is stimulated by Ca2+, which can signal rapid muscle contraction and reflect the need for greater oxidative activity- as in with alpha-ketoglutarate dehydrogenase.

the intermediates of the TCA cycle are used for other purposes, especially so in the liver, where the TCA is called an "open cycle" for this reason. oxaloacetate and alpha-ketoglutarate are both used for amino acid synthesis, citrate is used for fatty acid synthesis, succinyl CoA is used for heme synthesis, and malate is used for gluconeogenesis. additionally, in skeletal muscle, alpha-ketoglutarate is converted into glutamine, and in the brain, converted into the neurotransmitter GABA. to replenish the intermediates used in these reactions (called "efflux" of intermediates), "anaplerotic" reactions take place. the major anaplerotic reactions are: pyruvate carboxylase adding an activated CO2 to pyruvate to form and replenish oxaloacetate. glutamate being converted back into alpha-ketoglutarate via transaminases or glutamate dehydrogenases. valine or isoleucine being converted into succinyl CoA. other amino acids being converted to fumarate and oxaloacetate.

questions

1. what are the byproducts of the TCA cycle?
2. which reactions produce NADH as a byproduct?
3. which reaction produces FADH2 as a byproduct?
4. why is FAD used as an electron acceptor in the oxidation of succinate as opposed to NADH?
5. describe the role of succinate dehydrogenase in the oxidation of succinate.
6. what is the relationship of the NAD/NADH ratio to oxidative enzymes?

7. what is unique about the thioester bond in CoA and how is it used in the TCA cycle?
8. what are the three components to alpha-ketoacid dehydrogenase complexes?

9. what is the energetic efficiency of the TCA cycle?
10. which reactions of the TCA cycle have a large and negative change in free energy?
11. which reactions of the TCA cycle have a positive change in free energy?

12. describe the regulation of the first step in the TCA cycle.
13. describe the regulation of isocitrate dehydrogenase.
14. describe the regulation of alpha-ketoglutarate dehydrogenase.

15. what does the pyruvate dehydrogenase complex do?
16. describe the actions of PDC kinase on PDC and what it is influenced by.
17. describe the actions of PDC phosphatase on PDC and what it is influenced by.

18. why is the TCA cycle in the liver called an "open cycle"?
19. what are examples of efflux of TCA intermediates in the brain and skeletal muscle?
20. what are anaplerotic reactions?
21. describe the actions of the pyruvate carboxylase enzyme. what is it activated by?
22. what are the other major anaplerotic reactions that replenish TCA intermediates?

answers

1. 3 molecules of NADH, 1 molecule of FADH2, 2 molecules of CO2.
2. oxidative decarboxylation of isocitrate to alpha-ketoglutarate, oxidative decarboxylation of alpha-ketoglutarate to succinyl CoA, oxidation of malate to oxaloacetate.
3. oxidation of succinate to fumarate via succinate dehydrogenase.
4. FAD can accept single electrons, forming a half-reduced intermediate. the oxidation of succinate involves the formation of a double bond, which involves transfer of single electrons, making FAD better suited as an electron acceptor.
5. succinate dehydrogenase contains a covalently bound FAD molecule near its active site, which can accept electrons and transfer them directly to an Fe-S complex, which then donates them directly to Coenzyme Q (of the electron transport chain)
6. free NAD+ molecules bind to dehydrogenases and are reduced to NADH, which can then bind to and inhibit the oxidation activity of other dehydrogenases. thus the NAD/NADH regulates the activity of oxidative enzymes to coordinate the rate of fuel oxidation with ATP production.

7. the thioester bond is unique because it is a high energy bond (-13kJ/mol), and it differs from regular oxygen esters in that it does not share its electrons, and therefore does not allow for resonance stabilization, thereby allowing neighboring carbons to be activated for different reactions. this is demonstrated in citrate's synthesis, where the alpha-carbon in CoA is activated by the thioester, allowing it to undergo a condensation reaction with oxaloacetate. the release of energy from the breakage of the thioester bond in CoA is harnessed in the reaction of succinyl CoA to succinate, where an enzyme-bound phosphate is transferred to GDP to create GTP.
8. TPP's, lipoases, and FAD. thiamine pyrophosphates cleave the C-C bonds next to the carboxyl group, which is how the CO2 is freed in the decarboxylation reactions. lipoases are responsible for transferring the acyl portion of the alpha-ketoacid to CoASH, as well as accepting and donating electrons to FAD, which then transfers them to NAD.

9. ~90%
10. synthesis of citrate, oxidation of isocitrate and alphaketoglutarate.
11. oxidation of malate, isomerization of citrate.

12. the synthesis of citrate from oxaloacetate and acetyl coA is regulated by the concentration of oxaloacetate, (availability of reactant), as well as concentration of citrate (which can competitively inhibit the reaction). the concentration of oxaloacetate is regulated by the NAD/NADH ratio, which is a reflection of the level of oxidative phosphorylation, and the concentration of citrate is regulated by the level of activity of the isocitrate dehydrogenase enzyme.
13. the presence of ADP causes isocitrate dehydrogenase to activate all subunits on the enzyme, allowing the reaction to proceed more rapidly (lowering the michaelis constant). NADH inhibits isocitrate activity.
14. this enzyme is inhibited by NADH, activated by Ca2+ and ADP.

15. the pyruvate dehydrogenase complex (PDC) is an enzyme that catalyzes the oxidation of pyruvate into acetyl coA, connecting glycolysis with the TCA cycle.
16. PDC kinase phosphorylates and deactivates PDC. PDC kinase is inhibited by ADP, pyruvate and activated by Acetyl CoA and NADH: when ATP is used up, the ADP concentration rises, inhibiting PDC kinase activity, leaving PDC in the active form, allowing it to oxidize pyruvate to acetyl CoA to be used in the TCA cycle and ultimately ATP production. (thus high ADP levels stimulate more ATP production). presence of pyruvate has the same inhibitory effect on PDC kinase, allowing PDC to oxidize pyruvate. (thus high pyruvate levels allow PDC to oxidize pyruvate). Acetyl CoA and NADH are products of the PDC reaction which activates PDC kinase, which phosphorylates PDC and inhibits its activity (thus accumulation of the products from the oxidation of pyruvate inhibits the oxidation of pyruvate)
17. PDC phosphatase, the enzyme that activates PDC, is activated by Ca2+ -- as in when Ca2+ is released from sarcoplasmic reticulum during smooth muscle contraction (thus rapid muscle contraction stimulates pyruvate oxiation).

18. because of the high efflux of intermediates that are used for amino acid synthesis (oxaloacetate, alpha-ketoglutarate), fatty acid synthesis (citrate), heme synthesis (succinyl CoA), and gluconeogenesis (malate).
19. in the brain, alpha-ketoglutarate is used to form GABA, a neurotransmitter. in skeletal muscle, it is converted to glutamine, which is transported in the blood to other tissues.
20. reactions that replenish the intermediates of the TCA cycle via carbohydrates or amino acids.
21. pyruvate carboxylase is an enzyme that catalyzes the anaplerotic reaction which replenishes oxaloacetate. it does so by addition of an activated CO2 to pyruvate to form oxaloacetate, expending one ATP in the process. it is activated by Acetyl CoA: if oxaloacetate is used up for other reactions such as amino acid synthesis, then the citrate synthesis from oxaloacetate + acetyl CoA will not occur, and acetyl CoA will accumulate. the buildup of acetyl CoA will then activate pyruvate carboxylase to replenish oxaloacetate so that the TCA cycle can get started. (thus higher Acetyl CoA stimulates the replenishing of oxaloacetate)
22. glutamate replenishes alpha-ketoglutarate by transamination or glutamate dehydrogenase activity. carbon skeletons of caline and isoleucine replenish succinyl CoA. other amino acids are degraded to fumarate and oxaloacetate, mainly in the liver.

biochem: mark's medical biochem chapter 22- glycolysis

this chapter introduces glycolysis and its role in cellular energy production. glycolysis is basically the first step in breaking down glucose for use in energy production. in aerobic glycolysis, one molecule of glucose is broken down into two molecules of pyruvate, in the process forming 2 molecules of NADH and 2 molecules of ATP. in anaerobic glycolysis, one molecules of glucose produces lactate and 2 molecules of ATP. the reactions for aerobic glycolysis are as follows:

1. glucose is phophorylated to glucose-6-phosphate via hexokinase.
2. glucose 6-phophate is isomerized to fructose 6-phosphate via phosphoglucose isomerase.
3. fructose 6-phosphate is phosphorylated to fructose 1,6 bisphosphate via phosphofructokinase-1.
4. fructose 1,6 bisphosphate is cleaved into two triose phosphates via an aldolase.
5. the triose phophates isomerize into glyceraldehyde 3-phosphate (G3P).
6. G3P is oxidized by G3P dehydrogenase, which reduces NAD+ to NADH2 and transfers a high energy acyl phosphate bond onto G3P, forming 1,3 bisphophoglycerate.
7. 1,3 bisphosphoglycerate phosphorylates ADP via phosphoglycerate kinase and becomes 3 phosphoglycerate.
8. 3-phosphoglycerate is isomerized to 2-phosphoglycerate via phosphoglyceromutase.
9. 2-phosphoglycerate loses an H2O and becomes phosphoenolpyruvate (PEP) via enolase.
10. PEP phosphorylates another ADP via pyruvate kinase and becomes pyruvate.

reactions 1 through 4 are known as the preparative phase, in which glucose is basically phosphorylated twice and cleaved into two 3 carbon molecules. reactions 5-10 are known as the ATP generating phase, because the phosphate groups from the 3 carbon molecules are then transferred onto ADP. after the first phosphate group is transferred in reaction 7, reactions 8 and 9 basically transform the remaining phosphate group from a low energy to a high energy one so that it can also be transferred to ADP.

in reaction 6, NAD+ is used as an electron acceptor during the oxidation of G3P. thus, the NADH2 must be constantly reoxidized to NAD+ in order to maintain a source for transferring electrons. in aerobic glycolysis, this problem is solved by shuttling NADH2's electrons across the mitochondrial membrane (shuttling is needed because the membrane is impermeable to NAD), where they can be used to power oxidative phosphorylation. the most common shuttle is the G3P shuttle: NADH2 reduces DHAP to G3P via G3P dehydrogenase. G3P then diffuses across the mitochondrial membrane and transfers the electrons to FAD, forming FADH2, which can then donate its electrons to coenzyme Q in the electron transport chain. the other shuttle mechanism is the malate aspartate shuttle, in which NADH2 reduces oxaloacetate to malate, which is then shuttled across the mitochondrial membrane via a translocase, and then donates its electrons to mitochondrial NAD+, reforming oxaloacetate. oxaloacetate is then transaminated to aspartate, which can then be shuttled back out the mitochondria via another translocase, and once it is back, transaminated back into oxaloacetate and ready to accept electrons from NADH2 again.

the net ATP production from the G3P shuttle is 3 moles of ATP per mole of glucose, whereas the malate aspartate shuttle produces 5 moles of ATP. combining this with the ATP produced in the ATP-generating phase of glycolysis (2 ATP per glucose), and taking into account the pyruvate, which can be further oxidized in the mitochondria to produce an additional 12.5 ATP (25 ATP per glucose): this means in aerobic glycolysis, total ATP production for each molecule of glucose is either 3+2+25=30 when the G3P is in use or 5+2+25=32 when the malate aspartate shuttle is in use.

when oxygen is not readily available or mitochondria are not present (as in red blood cells, where oxidative metabolism might interfere with hemoglobin's interaction with oxygen, or in the eye, where mitochondria would deflect incoming light), or if cellular demands for energy exceeds that which is produced via aerobic processes, then anaerobic glycolysis is used. in order to regenerate the NAD+ to serve as the electron acceptor in reaction 6, NADH2 reduces pyruvate into lactic acid, which dissociates into lactate and proton (which has the potential to decease body pH). pyruvate therefore cannot be further oxidized and therefore the energy production in anaerobic glycolysis is quite lower, requiring tissues to work 15 times as fast to produce the same energy output. whereas in aerobic glycolysis the total energy production can be up to 32 moles of ATP per mole of glucose, in anaerobic glycolysis, the total energy production is only 2 moles of ATP (from the ATP-generating phase of glycolysis)

questions
1. describe the process of glycolysis and what it produces.
2. what are the two phases of glycolysis and what happens in each?

3. what is the first step in glycolysis and what does it produce?
4. what are the next steps in the preparative phase of glycolysis?
5. describe the oxidation of G3P.
6. what is substrate level phosphorylation?
7. where is the first phosphorylation of ADP?
8. how is the low energy phosphate bond in 3 phosphoglycerate transformed into a high energy phosphate bond that can be transferred to ADP?
9. what is the final reaction of glycolysis?
10. what is the total free energy change from glycolysis?
11. why does NADH need to be reoxidized and what are the two pathways in which this is accomplished?
12. what are the two fates for pyruvate?

13. why are reducing equivalent shuttles required?
14. describe the G3P shuttle.
15. describe the malate-aspartate shuttle.
16. describe anaerobic glycolysis.
17. what is the ATP production of the G3P vs. the malate-aspartate shuttle?
18. describe the difference of ATP production possible in aerobic vs. anaerobic glycolysis.
19. how is anaerobic glycolysis related to body pH?
20. when do cells or tissues use anaerobic glycolysis vs. aerobic?


answers
1. glycolysis is the splitting of glucose into pyruvate and production of 4 ATP molecules per molecule of glucose.
2. the preparative phase, in which glucose is phosphorylated twice by ATP and cleaved into two triose phosphates, and the ATP-generating phase, in which the triose phosphates produce 2 ATP each.

3. phosphorylation of glucose yields glucose-6-phosphate via hexokinases.
4. isomerization of glucose 6-phosphate via phosphoglucose isomerase to fructose 6-phosphate, phosphorylation to fructose 1,6 bisphosphate via phosphofructokinase-1 (PFK-1), and cleavage into two triose molecules (glyceraldehyde 3-phosphate) via aldolase.
5. G3P is oxidized by glyceraldehyde-3-phosphate dehydrogenase, which has a cysteine residue near the active site which accepts a high energy acyl phosphate and transfers it to G3P. in this process, NADH and a high energy phosphate bond are formed.
6. the formation of a high energy phosphate bond without oxygen via direct transfer of a phosphate bond from a high energy intermediate.
7. 1,3 bisphosphoglycerate transfers its phosphate group to ADP via phosphoglycerate kinase, forming 3 phosphoglycerate.
8. the low energy phosphate bond in 3-phosphoglycerate is first shifted to the middle carbon via phosphoglyceromutase, and then transformed into a higher energy phosphate bond via enolase, which removes a water molecule and turns the compound into phosphoenolasepyruvate.
9. transfer of the high energy phosphate bond phospho enol pyruvate to ADP, turning the compound into pyruvate.
10. -22kcal
11. NADH needs to be reoxidized in order to continually accept electrons during the oxidation of G3P via G3P dehydrogenase. when oxygen is available, reducing equivalents are shuttled across the mitochondrial membrane to the electron chain. when oxygen is not available, NADH transfers its electrons to pyruvate, which is then reduced to lactate.
12. pyruvate can be reduced to lactate in anaerobic glycolysis, or can be converted into acetyl coenzyme A and further oxidized in aerobic glycolysis.

13. because the mitochondrial membrane is impermeable to NADH.
14. the G3P shuttle is the main shuttle mechanism in which cytosolic NAD+ is regenerated by G3P dehydrogenase, which transfers the electrons from NADH2 to DHAP, forming G3P. G3P then diffuses across the mitochondrial membrane and donates its electrons to FAD, forming FADH2 and reforming DHAP, which can then diffuse back and be reused in the reaction.
15. the malate-aspartate shuttle is the other way that NAD+ is regenerated: NADH transfers its electrons to oxaloacetate, reducing it to malate, which is then transported across the mitochondrial membrane via a translocase. in the membrane, the malate reduces NAD to NADH2, which can then be used in the electron transport chain. the oxaloacetate that is formed is transaminated to aspartate, which is transported back across the mitochondrial membrane via another translocase, and transaminated back into oxaloacetate in the cytosol.
16. anaerobic glycolysis occurs when the the oxidative capacity of a cell is limited. in this reaction, the electrons from NADH2 are donated to pyruvate, creating lactic acid, catalyzed by the enzyme lactate dehydrogenase.
17. the G3P shuttle produces 1.5 moles of ATP per NADH2, whereas the malate-asparate shuttle produces 2.5 moles of ATP per NADH2.
18. in anaerobic glycolysis, pyruvate is reduced to lactate and thus cannot produce any more ATP besides the two moles (per glucose) that is produced during glycolysis. in aerobic glycolysis, in addition to these two ATP's, the pyruvate is oxidized and used in the mitochondria to produce 25 ATP (per glucose), and the NADH produced can add another 3 (if using the G3P pathway) or 5 (if using the malate aspartate pathway) ATP molecules.
19. lactic acid is produced from the reduction of pyruvate in anaerobic glycolysis via lactate dehydrogenase. at the normal intracellular pH of 7.35, lactic acid dissociates into lactate and H+, which is then transferred out of the cell and eventually into the blood, where it can influence body pH.
20. tissues that have a low ATP demand and low O2 availability. also, blood cells have undergo anaerobic glycolysis because aerobic metabolism might interfere with the loading and unloading of O2 from hemoglobin. also, in other tissues with mitochondria and O2 availability, anaerobic glycolysis is used when the cell's energetic demands are not met by aerobic processes.

11.16.08 biochem: lippincott's chapter 6- bioenergetics and oxidative phosphorylation

this chapter was a short introduction to free energy and oxidative phosphorylation. it probably oversimplified the process and left out some details but was good to read just to get an overview of the main ideas. free energy is introduced as a measure of the driving force or "energetic feasibility" of reactions, concerned with the bond energies and entropies of only the reactants and products (and contrasted this with kinetics, which is concerned with the pathway and rate of reaction). if the change in free energy from reactants to products is negative, then the reaction is energetically favorable, "exergonic", and occurs spontaneously - and the opposite case for a positive change in free energy.

standard free energy is just the free energy of a reaction at standard conditions: 1M concentration for products and reactants, pH 7. free energy change can then be related to actual concentration of reactants / products via the equation G=Gº+RTln(B/A) where B=products and A=reactants. the equation implies that if the concentration of the products is greater than reactants, then B/A will be greater than 1, making the second term in the equation negative, allowing the free energy change to be negative (spontaneous) even if the standard free energy change is positive (non spontaneous). at equilibrium, the change in free energy = 0, making the equation Gº=-RTln(B/A), and B/A represent the static equilibrium concentrations... equivalent to Keq. thus Gº=-RTln(Keq) and this yields another set of predictions: if Keq is greater than 1, then Gº is negative. if Keq is less than 1, then Gº is positive. if Keq is 1, then Gº = 0.

the idea of energetic "coupling" is introduced: a endergonic reaction can proceed by being coupled with an exergonic one via a common intermediate. we then turn to the production of ATP via the electron transport chain, which can be summarized as the synthesis of ATP in the matrix of the mitochondria, powered by a H+ gradient across the inner membrane, which is created by a chain of membrane complexes that pump H+ into the mitochondrial matrix in response to electron transfers from high energy metabolic intermediates. electrons are transferred between "complexes" which are groupings of membrane proteins that work in tandem to either accept and donate electrons.

the first step in the chain is the reduction of NAD+ to NADH plus H+, via donation of a hydride ion from random dehydrogenases. NADH plus H+ can then donate its electrons to complex 1, also named NADH dehydrogenase, which then transfers the electrons to FMN to make FMNH2. FMNH2 can then donate its electrons to coenzyme Q, which is a "quinone derivative" with a long hydrophobic tail, which then transfers them to complex III. complexes III through V are cytochromes, which have a heme group made up of a porphyrin ring with a Fe atom in the middle (which is reduced from ferric Fe3+ to ferrous Fe2+ during electron transfers). electrons are passed from coenzyme Q to cytochrome "b+c1" to "c" to "a+a3".

the mitchell hypothesis is introduced as an explanation of the actual mechanism for the powering of the ATP synthesis. it explains that during transfer of electrons between each complex, H+ ions are pumped into the intermembrane space of the mitochondria and the resulting pH, electrical, concentration gradient is what powers the ATP synthase protein embedded in the inner mitochondrial membrane. this happens when H+ ions flow in through the "F0" domain of the ATP synthase protein, which causes conformational changes in the "F1" domain which activates its catalytic activity and allows it to phosphorylate ADP, resulting in synthesis of ATP in the matrix. oligomycin is a drug that blocks the H+ from entering the F0 domain, which prevents dissipation of the H+ complex, making it energetically unfavorable for the electron transport reactions to occur (since they have to work against a higher concentration gradient) and thereby blocking ATP synthesis.

"uncoupling" is then introduced in reference to uncoupling the H+ gradient from ATP synthesis activity. uncoupling proteins are sometimes present in the inner mitochondrial membrane, especially in the brown adipose cells of hibernating animals or babies, which allow H+ to leak back into the mitochondrial matrix without powering the ATP synthase, instead producing heat. synthetic uncouplers have the same effect by generally increasing the permeability of the membrane to protons by way of molecular "proton carriers" than can traverse the inner mitochondrial membrane and dissipate the H+ gradient.

questions

how do the studies of bioenergetics and kinetics differ?
what is the free energy formula? what is enthalpy? what is entropy?
what is Gº?
what is indicated by a negative, positive, and zero value for change in free energy?
how does G relate to the concentration of the reactants?
how does Gº relate to Keq?

what is reaction coupling and how does it relate to ATP?
what is the bond energy for the first two phosphate bonds of ATP?

describe the electron transport chain.
where does electron transport take place?
where do the electrons in the electron transport chain ultimately flow to?
describe the inner mitochondrial membrane.
what is inside the matrix of the mitochondrion?
what are the "complexes" in the electron transport chain?

describe the formation of NADH
describe the next step involving complex 1
describe complex 2's role in the electron transport chain
what is coenzyme Q's role in the electron transport chain
what are the basic structure of cytochromes?
how do cytochromes accept and donate electrons?
describe the pathway of the electrons from coenzyme Q through the cytochromes.

what are site specific inhibitors and what do they result in?
what are the two specific molecular methods of electron transport in the electron transport chain?

what are standard reduction potentials and how are they related to the electron transport chain?
how is G related to E?

what is the mitchell hypothesis?
according to the mitchell hypothesis, what drives ATP synthase activity?
describe the action of H+ on ATP synthase
describe the actions of oligomycin

what are uncoupling proteins and what are their ultimate effect in mitochondria?
what are the actions of synthetic uncouplers and what is one example?

how is ADP replenished in the mitochondria?

answers

bioenergetics is the study of the energetic feasibility of reactions that is concerned with the free energy of only the reactants and products, while kinetics is more concerned about the rates of reaction as well as the pathways between reactants and products.

change in free energy = change in enthalpy - (temperature) * (change in entropy). change in enthalpy is a measure of the difference of total bond energies between the reactants and products. change in entropy represents the change of order of randomness between reactants and products.

Gº is the standard free energy which is just the free energy at standard conditions: pH 7, [1M].

negative delta G indicates an exergonic, spontaneous reaction where the products are at a lower energy than the reactants. positive delta G indicates an endergonic, non-spontaneous reaction in which the products are at a higher energy than the reactants. zero delta G represents an equilibrium state, where the rate of formation of product is equal to the rate of backreaction for formation of reactant.

the concentrations of the reactants vs the products is related to the free energy change via this equation: G=Gº+2.303RTlog(B/A) where B=products and A=reactants

@ equilibrium, G=0, and thus the G=Gº+RTln(B/A) equation becomes: Gª=-RTln(Keq). this equation allows for some simple predictions: if Keq=1, then Gº=0. if Keq is greater than 1, then Gº > 0.

highly endergonic reactions harness the energy of highly exergonic reactions through coupling via common intermediates. ATP is the most common example of this, providing copious amounts of energy by the breaking of its high energy phosphate bonds.

7.3 kcal/mol of energy is released when the phosphate groups in ATP are released.



the electron transport chain is the the series of reactions in which the high energy reduced intermediate coenzymes NADH2 and FADH2 (reduced by the oxidation of glucose) donate their electrons to a "specialized set of electron carriers" which then produce ATP from ADP. it takes place inside the inner mitochondrial membrane and the ultimate receptor is O2.

the inner mitochondrial membrane is rich with proteins, most of which participate in the electron transport chain. it is also impermeable to small ions and molecules which are important in metabolism; they need specialized carriers to be transported into the inner space. the inner membrane is also folded with cristae which increase the inner surface area. proteins transport cristae.

inside the mitochondrial matrix is: a gel like solution made up of 50% protein, including enzymes related to oxidation of pyruvate, krebs cycle, amino acids, fatty acids, NAD, FAD, ADP, pi.

the "complexes" 1-5 are functional subdivisions of the proteins involved in the electron transport chain based on the coordinated action of donating or accepting electrons to and from "mobile electron carriers" such as coenzyme Q or cytochrome c.



the first step in the chain is the reduction of NAD to NADH plus free proton via a dehydrogenase that donates a hydride ion.

the free proton plus the hydride ion of NADH are donated to NAD hydrogenase, complex 1, which has a tightly bound coenzyme FMN which accepts the 2 hydrogen atoms and becomes FMN2.

complex II is succinate dehydrogenase, transferring two electrons, from the formation of fumarate from succinate, to FAD to form FADH2.

coenzyme Q is the next electron acceptor, taking FMNH2's two hydrogen atoms, as well as FADH2's two hydrogen atoms, from complex 1 and 2, respectively.

cytochromes are made up of a heme group made of a porphyrin ring containing an atom of iron.

the Fe3+ (ferric) ion in the porphyrin ring is reduced to Fe2+ (ferrous) form when it accepts electrons.

electrons passed from Coenzyme Q to cytochromes b+c1 (complex III), c, and a+a3 (complex iv)

site specific inhibitors are molecules that bind to certain components of the electron transport chain and block electron flow by preventing the oxidation/reduction reaction to occur. this causes all components upstream to be fully reduced and the downstream components to be fully oxidized.

the two ways that electrons are transferred in the chain: as hydride ions, in the case of FADH2 and FMNH2, and as free electrons, in the case of cytochromes.

standard reduction potentials are a measure of the potential for a reduction reaction to take place, ie a measure of the tendency of the reductant to lose electrons. it is related to the electron transport chain because each reduction (transfer, or donation of electrons) is always accompanied by an oxidation reaction (loss of electrons)

G = -nFE, where n = number of electrons transferred, F = faraday constant, and E = standard reduction potential.

the mitchell hypothesis (also known as the chemiosmotic hypothesis) is a theory of how the free energy released during electron transport powers the formation of ATP from ADP and pi.

the mitchell hypothesis says that the proton gradient produced by electron transport (where more H+ ions in the intermembrane space than in the matrix) powers the ATP synthase.

H+ proton reenters the F0 domain of the ATP synthase and causes rotation, which causes conformational change in the F1 domain which catalyzes synthesis of ATP.

oligomycin binds to the F0 domain of ATP synthase and prevents any H+ ion to enter, preventing dissipation of the H+ gradient, which stops electron transport, which stops ATP synthase activity.

uncoupling proteins are proteins on the inner mitochondrial wall that allow H+ to enter back into the matrix without production of ATP. UCP1 is one uncoupling protein that is responsible for fatty acid oxidation and heat production in the brown adipocyte cells of hibernating mammals.

synthetic uncouplers produce the same effect, allowing electron transport to transpire without establishing the proton gradient necessary for ATP synthesis, by way of increasing the membrane permeability to protons. aspirin can have this effect in high doses.

ADP + Pi are transported actively back into the mitochondria via an "adenine nucleotide carrier", which also exports ATP into the cytosol of the cell.