Glycolysis 1. Lecture 25. Key Concepts

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1 Glycolysis 1 Lecture 25 Key Concepts Overview of the Glycolytic Pathway Glycolysis generates a small amount of ATP Preview of the ten enzyme-catalyzed reactions of glycolysis Stage 1: ATP Investment Reaction 1: Hexokinase Reaction 2: Phosphoglucose isomerase Reaction 3: Phosphofructokinase Reaction 4: Aldolase Stage 2: Formation of GAP Reaction 5: Triose phosphate isomerase Stage 3: ATP Earnings Reaction 6: Glyceraldehyde-3-P dehydrogenase Reaction 7: Phosphoglycerate kinase (substrate level phosphorylation) Reaction 8: Phosphoglycerate mutase Reaction 9: Enolase Reaction 10: Pyruvate kinase (substrate level phosphorylation)

2 Overview of the Glycolytic Pathway Glycolysis is considered one of the core metabolic pathways in nature for three primary reasons 1. glycolytic enzymes are highly conserved among all living organisms, suggesting it is an ancient pathway. 2. glycolysis is the primary pathway for ATP generation under anaerobic conditions and in cells lacking mitochondria such as erythrocytes. 3. metabolites of glycolysis are precursors for a large number of interdependent pathways, including mitochondrial ATP synthesis. Pathway Questions 1. What does glycolysis accomplish for the cell? Generates a small amount of ATP which is critical under anaerobic conditions. Generates pyruvate, a precursor to acetyl CoA, lactate, and ethanol (in yeast). 2. What is the overall net reaction of glycolysis? Glucose + 2NAD + + 2ADP + 2 Pi 2 pyruvate + 2NADH + 2H + + 2ATP + 2H 2 O Gº = kj/mol

3 Pathway Questions 3. What are the key regulated enzymes in glycolysis? Hexokinase, Phosphofructokinase, Pyruvate kinase 4. What are examples of glycolysis in real life? A deficiency in the hexokinase-related enzyme, glucokinase, leads to a rare form of diabetes, which is caused by the inability of liver and pancreatic cells to phosphorylate glucose inside cells when blood glucose levels are elevated. The complete oxidation of glucose to CO 2 and H 2 O is summarized by the reaction: Glucose (C 6 H 12 O 6 ) + 6O 2 6CO 2 + 6H 2 O Gº = -2,840 kj/mol G = -2,937 kj/mol

4 Glucose CO 2 + H 2 O Fermentation

5 Lactobacillus Convert glucose into lactic acid and CH 4 and H 2 Glycolysis has 3 Stages ATP Investment GAP formation and TIM ATP Earnings

6 2 ATP used Fructose-1,6-bisphosphate Net result: 2 molecules of GAP

7 Each molecule of GAP 4 ATP gained Net: 2 ATP Glycolysis produces a small amount of ATP More importantly: glycolysis produces Pyruvate What is the most important product of glycolysis?

8 What is the most important intermediate metabolite in glycolysis? The six carbons and six oxygens present in glucose are stoichiometrically conserved by glycolysis in the two molecules of pyruvate that are produced

9 Important features of glycolysis Ten enzymatic reactions primarily bond rearrangements phosphoryl transfer reactions isomerizations an aldol cleavage an oxidation a dehydration There is no net loss of carbon or oxygen atoms. For every mole of glucose entering glycolysis, two moles of glyceraldehyde-3-p (GAP) are metabolized to pyruvate, generating in the process a net 2 ATP and 2 NADH.

10 Free energy changes for the ten glycolytic reactions STAGE 1: ATP INVESTMENT Stage 1 of glycolysis includes three enzymatic reactions that accomplish two tasks: 1) Use ATP as the phosphate donor to create phosphorylated compounds that are negatively charged and cannot diffuse out of the cell 2) Prepare the backbone of glucose to be cleaved

11 Reaction 1: Phosphorylation of glucose by hexokinase or glucokinase hexokinase is found in all cells glucokinase is present primarily in liver and pancreatic cells Hexokinase binds glucose through an induced fit mechanism that excludes H2O from the enzyme active site and brings the phosphoryl group of ATP into close proximity with the C6 carbon of glucose

12 Hexokinase is feedback inhibited by glucose-6-p which binds to a regulatory site in the amino terminus of the enzyme Reaction 2: Isomerization of glucose-6-p to fructose-6-p by phosphoglucose isomerase Phosphoglucose isomerase (phosphohexose isomerase) interconverts an aldose (glucose-6-p) and a ketose (fructose-6-p) through a multi-step pathway that involves opening and closing of the ring structure

13 Reaction 3: Phosphorylation of fructose-6-p to fructose-1,6-bp by phosphofructokinase Reaction 3 is the second ATP investment reaction in glycolysis and involves the coupling of ATP hydrolysis to a phosphoryl transfer reaction that is catalyzed by the enzyme phosphofructokinase STAGE 2: BACKBONE CLEAVAGE AND GAP FORMATION Stage 2 of glycolysis includes two enzymatic reactions that produce two molecules of glyceraldehyde-3-phosphate from every molecule of glucose that enters glycolysis

14 Reaction 4: Cleavage of fructose-1,6-bp into glyceraldehyde-3-p and dihydroxyacetone-p by aldolase The splitting of fructose-1,6-bp into the triose phosphates glyceraldehyde-3-p and dihydroxyacetone-p is the reaction that puts the lysis in glycolysis (lysis means splitting) Reaction 5: Isomerization of dihydroxyacetone-p to glyceraldehyde-3-p by triose phosphate isomerase Glyceraldehyde-3-P, not dihydroxyacetone-p, is the substrate for reaction 6 in the glycolytic pathway. The enzyme triose phosphate isomerase converts the ketose dihydroxyacetone-p to the aldose glyceraldehyde-3-p in an isomerization reaction.

15 The TIM barrel structure was named for this enzyme (triose phosphate isomerase) Why is this protein considered to be a kinetically perfect enzyme? STAGE 3: ATP EARNINGS Three key features of stage 3 reactions need to be pointed out : 1. two substrate level phosphorylation reactions catalyzed by the enzymes phosphoglycerate kinase and pyruvate kinase generate a total of 4ATPs (net yield of 2ATP) in stage 2 of glycolysis. 2. an oxidation reaction catalyzed by glyceraldehyde-3-p dehydrogenase generates 2 NADH molecules that can be shuttled into the mitochondria to produce more ATP by oxidative phosphorylation. 3. reaction 10 is an irreversible reaction that must be bypassed in gluconeogenesis by two separate enzymatic reactions catalyzed by pyruvate carboxylase and phosphoenolpyruvate carboxykinase

16 Reaction 6: Oxidation and phosphorylation of glyceraldehyde-3-p to form 1,3-bisphosphoglycerate by glyceraldehyde- 3-P dehydrogenase The glyceraldehyde-3-p dehydrogenase reaction is a critical step in glycolysis because it uses the energy released from oxidation of glyceradehyde-3-p to drive a phosphoryl group transfer reaction using inorganic phosphate (Pi) to produce 1,3-bisphosphoglycerate. glyceraldehyde-3-p dehydrogenase Requires NAD + NAD + must be continually replenished within the cytosol aerobically in the mitochondrial matrix by the electron transport chain anaerobically in the cytosol by the enzyme lactate dehydrogenase which converts pyruvate to lactate anaerobically in yeast using the enzyme alcohol dehydrogenase which converts pyruvate to CO 2 and ethanol

17 1,3-bisphosphoglycerate has a standard free energy of hydrolysis that is higher than ATP hydrolysis This difference in free energies is harnessed by the enzyme phosphoglycerate kinase in reaction 7 to drive the synthesis of ATP by a mechanism called substrate level phosphorylation. Reaction 7: Substrate level phosphorylation to generate ATP in the conversion of 1,3-bisphosphoglycerate to 3- phosphoglycerate by phosphoglycerate kinase Phosphoglycerate kinase catalyzes the payback reaction in glycolysis because it replaces the 2 ATP that were used in stage 1 to prime the glycolytic pathway. Remember, this occurs twice for every glucose that entered glycolysis.

18 The molecular structure of phosphoglycerate kinase is similar to hexokinase in that it has two lobes (jaws) that each bind one of the substrates (ADP-Mg 2+ or 1,3- bisphosphoglycerate) leading to a large conformational change in the enzyme that brings the substrates close together and excludes H 2 O from the active site. Reactions 6 and 7 are coupled reactions! (Rxn 6) Glyceraldehyde-3-P + Pi + NAD + 1,3-bisphosphoglycerate + NADH + H + Gº = +6.3 kj/mol G = -1.3 kj/mol (Rxn 7) 1,3-bisphosphoglycerate + ADP 3-phosphoglycerate + ATP Gº = kj/mol G = +0.1 kj/mol (Coupled) Glyceraldehyde-3-P + Pi + ADP + NAD + 3-phosphoglycerate + ATP + NADH + H + Gº = kj/mol G = -1.2 kj/mol Actual change in free energy for each of these two reactions is very close to zero, and therefore both reactions are in fact reversible inside the cell. This is important for gluconeogenesis.

19 Reaction 8: Phosphoryl shift in 3- phosphyglycerate to form 2- phosphoglycerate by phosphoglycerate mutase The purpose of reaction 8 is to generate a compound, 2-phosphoglycerate, that can be converted to phosphoenolpyruvate in the next reaction, in preparation for a second substrate level phosphorylation that generates ATP earnings in step 10. mechanism of this highly reversible reaction requires a phosphoryl transfer from a phosphorylated histidine residue (His-P) located in the enzyme active site BPG can diffuse out of active site. Remember that it (BPG) is important in regulation of Hemoglobin binding of oxygen

20 Reaction 9: Dehydration of 2- phosphoglycerate to form phosphoenolpyruvate by enolase Standard free energy for this reaction is relatively small ( Gº = +1.7 kj/mol), meaning that the overall metabolic energy available from 2-phosphoglycerate and phosphoenolpyruvate is similar. But, Traps the phosphate group in an unstable enol form, resulting in a dramatic increase in the phosphoryl transfer potential of the triose sugar. Standard free energy change for phosphate hydrolysis in 2-phosphoglycerate is Gº = -16 kj/mol, whereas for phosphoenolpyruvate it is an incredible Gº = -62 kj/mol! Reaction 10: Substrate level phosphorylation to generate ATP in the conversion of phosphoenolpyruvate to pyruvate by pyruvate kinase The second of two substrate level phosphorylation reactions in glycolysis that couples energy released from phosphate hydrolysis ( Gº = -62 kj/mol) to that of ATP synthesis ( Gº = kj/mol). Unlike phosphoenolpyruvate, pyruvate is a stable compound in cells that is utilized by many other metabolic pathways as will be described later.