BC10M: Introductory Biochemistry, 2006 Semester 2.

1. BC10M: Introductory Biochemistry, 2006 Semester 2. • Thursday 17 Mar. • Lecture 25 • Gluconeogenesis • PPP • Calvin cycle • Andrew Pearson

3. Glycogen synthesis proceeds by the sequential addition of glucose units to the C-4 end of a chain. The glucose-carrier is UDP-glucose. UDP-glucose is formed from uridine triphosphate and glucose 1-phosphate, with the release of PP which is hydrolysed to drive the reaction. Glucose 1-P is formed from G 6-P by phosphoglucose isomerase.

4. Glycogen degradation is stimulated in: Glycogen synthesis is stimulated in:

5. Gluconeogenesis: the synthesis of glucose in liver (&kidney) Gluconeogenesis (Gneog) is the new formation of glucose from smaller carbon-containing biochemicals, and is partly responsible for converting atmospheric CO2 into starch in plants, which we eat. It is necessary in animals to supply glucose to the blood when the nutritional status is one of fasting: dietary glucose has all been consumed and the liver stores of glucose in the form of glycogen have been depleted. The pathway is activated in response the signal which indicates that blood [glucose] is low: glucagon. We have already come across the control point: hepatic glucagon receptors activate adenylate cyclase to form cAMP which in turn activates cAMP-dependent protein kinase which amongst other things, phosphorylates and inactivates pyruvate kinase in gluconeogenic tissues.

6. The reactions of the Gneog pathway occurring in liver and kidney can be regarded as being essentially a reversal of glycolysis, since the two pathways use the same “Michaelis-Menten enzymes”. The 3 glycolysis enzymes that are not M-M are “by-passed”.

7. Gneog showing the M-M enzymes; common carbon inputs

8. Gneog really starts in the mito matrix of liver and kidney.

9. Mitochondrial oxaloacetate is an intermediate in the Krebs cycle. Under normal circumstances, the amount of oxaloacetate in the mitochondrion is not changed: two carbons enter the TCA cycle as acetyl CoA and two leave as 2 CO2. Putting more acetyl CoA into the cycle cannot increase the amount of oxaloacetate present, it can only increase the rate of its formation and usage together: the cycle cannot “grow wider” but it can go faster, up to a Vmax. If oxaloacetate is removed from the mitochondrion to enter gluconeogenesis, then it must be replaced from another source, or the TCA cycle will have too few intermediates to operate efficiently.

10. If there were a pathway which could convert acetyl CoA directly to oxaloacetate, that would be ideal for the obese, struggling with an excess of fat. The Glyoxylate pathway exists in yeasts & those plants that need to convert the fats stored in their seeds during germination to glucose for metabolism before photosynthesis can take over. Animals have no Glyoxylate pathway and cannot convert fatty acids to glucose.

11. Apart from the glucogenic amino acids, other sources of carbon for gluconeogenesis can be considered: lactate/pyruvate and glycerol. Lactate released by muscle into the blood can be returned to pyruvate in the liver and kidney by their isoenzymes of lactate dehydrogenase. Pyruvate produced in this way can be converted by pyruvate carboxylase to oxaloacetate and on up through gluconeogenesis to glucose. Pyruvate carboxylase: pyruvate + ATP + HCO3-Þ oxaloacetate + ADP + Pi

12. Conversion of Mitochondrial Oxaloacetate to Cytosolic Phosphoenolpyruvate Oxaloacetate can only cross the inner membrane of the mitochondrion very slowly, there are two alternate routes to accomplish this objective.

13. Conversion ofPhosphoenolpyruvate to Fructose 1,6-bisphosphate The same enzymes are used for these reactions as are used in glycolysis: Enolase Phosphoglycerate Mutase Phosphoglycerate Kinase Glyceraldehyde 3-P dehydrogenase Triose phosphate isomerase Aldolase

14. Gneog showing the M-M enzymes; common carbon inputs

15. Fructose 1,6-bisphosphatase. It is strongly inhibited by AMP, a situation which would arise if the liver were not able to maintain its own ATP levels. Fructose 1,6-bisphosphatase is also strongly inhibited by the powerful PFK1 stimulant: fructose 2,6-bisphosphate, which as well as inhibiting in its own right also increases the inhibition by AMP.

16. Phosphoglucose isomerase This rapidly interconverts Glucose 6-P Û Fructose 6-P

17. Glucose 6-Phosphatase This is found only in liver and kidney, the only two tissues that can release glucose into the blood. Recall that glucose transporters will assist transport down the concentration gradient into the low [glucose] blood.

18. Pentose Phosphate Pathway (PPP), alias Hexose Monophosphate Shunt, alias Phosphogluconate Pathway. This is a set of enzymes in the cytosol of most cells that can carry out intermediary metabolism of some monosaccharides and simple derivatives. In some cells these enzymes perform a catabolic role like glycolysis: some bacteria without a complete glycolytic pathway rely on these enzymes for the fermentative generation of ATP. Muscle cells have very low levels of PPP activity, whereas normal erythrocytes have high levels.

19. Pentose Phosphate Pathway The two major outputs from this in animal cells are: ribose 5-phosphate for nucleic acid synthesis and NADPH for biosynthetic reduction or plasma membrane reduction and maintenance. Sadly, most textbooks describe all of the possible reactions of this matrix, and the first-time student is often hard put “to see the wood for the trees”.

20. Pentose Phosphate Pathway

21. Pentose Phosphate Pathway At the heart of this area of intermediary carbohydrate metabolism are two types of reaction: transketolases and transaldolases. Transketolase Reactions. These transfer 2-carbon ketol groups: from a variety of ketose sugars, such as ribulose & ribulose 5-phosphate, xylulose & xylulose 5-phosphate, fructose & fructose 6-phosphate, sedoheptulose & sedoheptulose 7-phosphate. The group is transiently held on a thiamin pyrophosphate cofactor, before being transferred onto the 1-carbon of an aldose sugar, which is always an aldehyde, such as glyceraldehyde 3-phosphate.

22. A typical transketolase reaction in the PPP is: Step 1. Xylulose 5-phosphate  glyceraldehyde 3-phosphate (5 carbon) (3 carbon) + TPP-Enz. + Ketol-TPP-Enz. (2 carbon) Step 2. Ketol-TPP-Enz. sedoheptulose 7-phosphate. (2 carbon) (7 carbon) + ribose 5-phosphate  + TPP-Enz (5 carbon)

23. Transaldolase Reactions. These transfer 3-carbon aldol groups:

24. A typical transaldolase reaction in the PPP is: Step 1. sedoheptulose 7-phosphate  erythrose 4-phosphate (7 carbon) (4 carbon) + lys-Enz. + Aldol-lys-Enz. (3 carbon) Step 2. Aldol-lys-Enz. fructose 6-phosphate. (3 carbon)  (6 carbon) + glyceraldehyde 3-phosphate + lys-Enz (3 carbon)

25. The oxidative branch of the PPP 1. Glucose 6-phosphate dehydrogenase. This enzyme is deficient in some individuals; this deficiency offers some protection against malaria caused by Plasmodium falciparum. Glucose 6-phosphate is present, to some extent, in the cytosol of all cells, being formed by one of the hexokinase isoenzymes. The enzyme transfers reducing equivalents and a proton from the 1-carbon of glucose to NADP+, forming 6-phosphoglucono-δ-lactone.

26. 1. Glucose 6-phosphate dehydrogenase. 2. Lactonase: hydrolysis of 6- phosphoglucono-δ-lactone. 3. 6-phosphogluconate dehydrogenase. 4. Phosphopentose isomerase Summary of route 1: Glucose 6-phosphate ribose 5-phosphate + CO2 +  + 2NADP+ 2NADPH + 2H+

27. bisphosphate rubisco Rubisco: ribulose 1,5 bisphosphate carboxylase.

30. The Calvin cycle uses transaldolase and transketolase reactions, as does the PPP. 3-phosphoglycerate is converted to hexoses in a pathway similar to gluconeogenesis.