Carbohydrate Metabolism I

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1 Carbohydrate Metabolism I

2 Outline Glycolysis Stages of glycolysis Regulation of Glycolysis

3 Carbohydrate Metabolism Overview

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5 Enzyme Classification Dehydrogenase - oxidizes substrate using cofactors as electron acceptor or donor (pyruvate dehydrogenase) Reductase - adds electrons from some reduced cofactor (enoyl ACP reductase) Kinase - phosphorylates substrate (hexokinase) Hydrolases - uses water to cleave a molecule Phosphatase - hydrolyzes phosphate esters (glucose-6- phosphatase) Esterase (lipase) - hydrolyzes esters (those that act on lipid esters are lipases) (lipoprotein lipase) Thioesterases - hydrolyzes thioesters Thiolase - uses thiol to assist in forming thioester (βketothiolase) Isomerases - interconversions of isomers (example aldose to ketose) (triose phosphate isomerase)

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7 Glycolysis What is glycolysis? Ten step metabolic pathway to convert glucose into two molecules of pyruvate and two molecules each of NADH and ATP. All carbohydrates to be catabolized must enter the glycolytic pathway. Glycolysis is central in generating both energy and metabolic intermediaries.

8 Glycolysis Glycolysis is the sequence of reactions that metabolizes one molecule of glucose to two molecules of pyruvate with the concomitant net production of two molecules of ATP - energy The process is anaerobic, O 2 is not required - an ancient pathway. Pyruvate is further processed: Anaerobically through fermentation, Aerobically by complete oxidation to CO 2 generating more ATP

9 Stages of Glycolysis An energy investment phase. Reactions, 1-5. Glucose to two glyceraldehyde 3-phosphate molecules. Two ATPs are invested. An energy payoff phase. Reactions 6-10, two glyceraldehyde 3-phosphate molecules to two pyruvate plus four ATP molecules. A net of two ATP molecules overall plus two NADH.

10 Stages of Glycolysis 1. 3 stages 2. 3.

11 Glycolysis - stage 1 Energy used, none extracted

12 Glycolysis - stage 3

13 Phase I Energy Investment Glucose is phosphorylated. Glucose enters a cell through a specific glucose transport process. It is quickly phosphorylated at the expense of an ATP. The investment of an ATP here is called priming. Enzymes hexokinase or glucokinase

14 Glucose phosphorylation: step 1

15 Hexokinase found in all cells of every organism low specificity for monosaccharides (simple sugars) i.e., other monosaccharides can be phosphorylated by hexokinase. relatively high affinity for glucose, K m = 0.1 mm inhibited by its product, glucose 6-phosphate

16 Induced fit in hexokinase Conformation changes on binding glucose, the two lobes of the enzyme come together and surround the substrate

17 Glucokinase found in liver high K m (~10 mm) for glucose not inhibited by glucose-6-phosphate most effective when glucose level in blood is high, i.e., right after meal

18 Formation of fructose-6-phosphate: step 2 by phosphoglucose isomerase Conversion of an aldose to a ketose

19 Formation of fructose 1,6-bisphosphate: step 3 by phosphofructokinase (PFK): an allosteric enzyme that regulates the pace of glycolysis.

20 Cleavage of six-carbon sugar: step 4 by aldolase

21 End of First Phase: Production of two glyceraldehyde 3- phosphate molecules from one glucose molecule with the expenditure of two ATPs. Therefore: the energy yields of the following steps are multipled by two. Second Phase:

22 Glycolysis - stage 2 No energy used or extracted Two 3-carbon fragments are produced from one 6-carbon sugar

23 Glycolysis: stage 2 (or 1) Salvage of three-carbon fragment: step 5 By Triosephosphate isomerase

24 Glycolysis: stage 2 allows interconversion of two triose phosphate products of aldolase cleavage only glyceraldehyde phosphate can be used further in glycolysis. aldose-ketose isomerization similar to phosphoglucoisomerase rxn allows dihydroxyacetone phosphate to be metabolized as glyceraldehyde 3-phosphate reversible, G = +7.5 kj/mole, this is important in gluconeogenesis

25 The loop closes off the active site on substrate binding Triosephosphate isomerase Central core of 8 parallel strands, surrounded by 8 helices. This structural motif, called an barrel,is also found in three other glycolytic enzymes.

26 Glycolysis - stage 3 Energy extracted 2x2 ATP The oxidation of three-carbon fragments yields ATP

27 Formation of 1,3-Bisphosphoglycerate: step 6 by Glyceraldehyde-3-phosphate dehydrogenase Done in two steps

28 Two-process reaction Aldehyde Acid

29 Formation of 1,3-Bisphosphoglycerate: step 6 by Glyceraldehyde-3-phosphate dehydrogenase First high energy compound generated = beginning of payoff. product is an acylphosphate, a fused carboxylicphosphoric acid anhydrate, which has a very high free energy of hydrolysis. reversible rxn, G = +6.3 kj/mole because this fused group retains some of the energy produced by the oxidation of the aldehyde to the carboxylic acid.

30 Formation of 1,3-Bisphosphoglycerate: step 6 by Glyceraldehyde-3-phosphate dehydrogenase reaction produces important reducing compound NADH = nicotinamide adenine dinucleotide, reduced form core NAD + is recycled and not used up in metabolism H

31 Glyceraldehyde 3-phosphate dehydrogenase Active site configuration

32 Formation of ATP from 1,3-Bisphosphoglycerate: step 7 by phosphoglyceratekinase High phosphoryl-transfer potential first substrate level phosphorylation, yielding ATP

33 Rearrangement: step 8 shifts phosphate from position 3 to 2

34 An enol phosphate is formed: step 9 Dehydration elevates the transfer potential of the phosphoryl group, which traps the molecule in an unstable enol form Enol: molecule with hydroxyl group next to double bond

35 the energy is locked into the high energy unfavorable enol configuration by phosphoric acid ester upon later hydrolysis of phosphate: H high energy low energy O O -C=C- -C-C- This energy is recovered the next step.

36 Formation of Pyruvate and ATP: step 10 second substrate level phosphorylation yielding ATP highly exergonic reaction, irreversible

37 Formation of Pyruvate and ATP: step 10 reaction is so exergonic because the enol in PEP is transformed to a keto in pyruvate drives several previous reactions. pyruvate is the primary product of glycolysis pyruvate kinase is a highly regulated enzyme

38 Summary of Energy Relationships for Glycolysis Input = 2 ATP glucose + ATP glucose-6-p fructose-6-p + ATP fructose 1,6 bisphosphate Output = 4 ATP + 2 NADH 2 glyceraldehyde 3-P + 2 Pi + 2 NAD + 2(1,3-bisphosphoglycerate) + 2 NADH 2 (1,3 bisphosphoglycerate) + 2 ADP 2(3-P-glycerate) + 2 ATP 2 PEP + 2 ADP 2 pyruvate + 2 ATP Net = 2 ATP and 2 NADH

39 Maintaining Redox Balance NAD + must be regenerated for glycolysis to proceed Glycolysis is similar in all cells, the fate of pyruvate is variable

40 Diverse fates of pyruvate To citric acid cycle

41 Fate of Product of Glycolysis - Pyruvate Pyruvate is at a central branch point in metabolism. Recall: Aerobic pathway through citric acid cycle and respiration; this pathway yields far more energy NADH + O 2 NAD + + energy Pyruvate + O 2 3 CO 2 + energy

42 Fate of Product of Glycolysis- Pyruvate Two anaerobic pathways: to lactate via lactate dehydrogenase to ethanol via ethanol dehydrogenase Note: both use up NADH produced so only 2 ATP per glucose consumed

43 Fate of pyruvate Anaerobic condition - Lactate Fermentation Note: uses up all the NADH (reducing equivalents) produced in glycolysis.

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45 Alcoholic fermentation pathway is active in yeast second step helps drive glycolysis second step is reversible reverse is ethanol oxidation, eventially yields acetate, which ultimately goes into fat synthesis ethanol acetaldehyde acetate humans have alcohol dehydrogenase in liver which mainly disposes of ethanol acetaldehyde is reactive and toxic

46 Fate of pyruvate Anaerobic condition alcoholic fermentation pyruvate decarboxylase - irreversible alcohol dehydrogenase - reversible Note: NADH used up

47 Regulation of Glycolysis Three irreversible kinase reactions primarily drive glycolysis forward: hexokinase or glucokinase phosphofructokinase pyruvate kinase These enzymes regulate glycolysis as well

48 Regulation of glycolysis

49 Hexokinase and Glucokinase Hexokinase Phosphorylation of glucose Inhibited by its product, glucose 6- phosphate, as a response to slowing of glycolysis Glucokinase liver enzyme with high K m for glucose so most effective when glucose levels are very high not inhibited by glucose 6- phosphate sensitive to high glucose in circulation from recent meal it decreases high level of glucose in blood by taking glucose into liver

50 Phosphofructokinase rate limiting for glycolysis an allosteric multimeric regulatory enzyme measures adequacy of energy levels Inhibitors: ATP and citrate, high energy Activators: ADP, AMP, low energy and fructose 2,6 bisphosphate

51 Phosphofructokinase ATP inhibits phosphofructose activity by decreasing fructose 6-phosphate binding AMP and ADP reverse ATP inhibition fructose 2,6 bisphosphate is a very important regulator, controlling the relative flux of carbon through glycolysis versus gluconeogenesis it also couples these pathways to hormonal regulation.

52 Allosteric regulation of phosphofructokinase step 3 Decreases affinity for F 6-phosphate AMP diminishes, and citrate enhances the inhibitory effect of ATP

53 Pyruvate kinase an allosteric tetramer: PEP + ADP pyruvate + ATP inhibitors: ATP, acetyl CoA and fatty acids (alternative fuels for TCA cycle) activator: fructose 1,6-bisphosphate ( feedforward ) phosphorylation (inactive form) and dephosphorylation (active form) under hormone control also highly regulated at the level of gene expression ( carbohydrate loading )

54 Glycolytic pathway tightly controlled Pathway is regulated to meet two major needs: 1. Production of ATP 2. Provision of building blocks for biosynthesis Hexokinase, phosphofructokinase, and pyruvate kinase serve as control sites (their reactions are virtually irreversible) 1. Their activities are regulated by reversible binding of allosteric effectors (milliseconds), or by covalent modification (seconds) 2. The amounts of the enzymes are varied by the regulation of transcription (hours)