Carbohydrate Metabolism Glycolysis 10262020 1 A Map of

Carbohydrate. Metabolism (Glycolysis) 10/26/2020 1

A Map of The Major Metabolic Pathways in A Typical Cell The Diagram was taken from “Biochemistry” by Voet & Voet (2 nd edition, 1995, John Wiley & 10/26/2020 6 Sons, pg. 413)

By the end of this session you should be able to: Learning Objectives • Outline three stages of glycolysis. • Describe the steps of glycolysis between glucose and pyruvate and recognize all the intermediates and enzymes and the cofactors that participate in the reactions. • Mention ATP-generating reactions. • Illustrate the regulation of glycolysis. 10/26/2020 7

Major Pathways of CHO Metabolism CHO metabolism in mammalian cells can be classified into: 1. Glycolysis: Oxidation of glucose to pyruvate (aerobic state) or lactate (anaerobic state) 2. Krebs cycle: After oxidation of pyruvate to acetyl Co. A, acetyl Co. A enters the Krebs cycle for the aim of production of ATP. 3. Hexose monophosphate shunt: Enables cells to produce ribose-5 -phosphate and NADPH. 4. Glycogenesis: Synthesis of glycogen from glucose, when glucose levels are high 5. Glycogenolysis: Degradation of glycogen to glucose when glucose in short supply. 6. Gluconeogenesis: Formation of glucose from noncarbohydrate sources. 10/26/2020 Glucose is the major fuel of most organisms. The major pathways of CHO metabolism either begin or end with glucose. Dr. Mohamed Z Gad 8

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Cellular energetic C H O + 6 O → 6 CO +6 H O + 686 kcal ( in Lab. ) 6 12 6 2 2 2 ( in the cell)C 6 H 12 O 6 + 6 O 2 → 6 CO 2 +6 H 2 O + 38 ATP 1 ATP + H 2 O → ADP+ Pi + 7. 3 kcal 38 x 7. 3 = 278 kcal 278 /686 = 40% efficiency 10/26/2020 10

flavin adenine dinucleotide (FAD) 12

Glycolysis (Embden-Meyerhof Pathway) Glycolysis occurs in all human cells. Glycolysis is believed to be [glycolysis: from the Greek glyk-, sweet, and lysis, splitting] among the oldest of all the biochemical pathways. Aerobic: Glucose Pyruvate Anaerobic: Glucose Lactate (or ethanol & acetic acid) Glycolysis (10 reactions in 3 stages, all in cytoplasm) 1) Priming stage: D-Glucose + 2 ATP D-fructose 1, 6 -biphosphate + 2 ADP + 2 H+ 2) Splitting stage : D-Fructose 1, 6 -biphosphate 2 D-Glyceraldehyde 3 -phosphate 3) Oxidoreduction – Phosphorylation stage: 2 D-Glyceraldehyde 3 -phosphate + 4 ADP + 2 Pi + 2 H+ 2 Lactate + 4 ATP --------------------------------------Sum: Glucose + 2 ADP + 2 Pi ----- 2 Lactate + 2 ATP + 2 H 2 O (Anaerobic) Glucose + 2 ADP + 2 Pi + 2 NAD+ ---- 2 pyruvate + 2 ATP + 2 NADH + 2 H+ + 2 H 2 O (Aerobic)13 10/26/2020 Dr. Mohamed Z Gad

Common Abbreviations & Alternative Names 10/26/2020 14 14

1) Priming Stage • Glucose (and other hexoses) are phosphorylated immediately upon entry into the cell. Phosphorylation prevents transport of glucose out of the cell and increases the reactivity of oxygen in the resulting phosphate ester. • Several isoenzymes of hexokinase with different Km values for glucose are located in different tissues. Brain hexokinase has a particularly low Km for glucose. • The major enzyme for phosphorylating glucose in liver is glucokinase. • Steps catalyzed by hexokinase & 10/26/2020 PFK-1 are irreversible. Dr. Mohamed Z Gad 15

Differences between Glucokinase & Hexokinase Glucokinase ² Present in all tissues ² Liver only ² Low Km for glucose (<0. 1 ² Higher Km for glucose m. M) ² Strongly inhibited by G 6 P ² Not inhibited by G 6 P ² Non-inducible enzyme, not ² Inducible, synthesis induced by affected by diabetes or insulin & repressed in diabetes insulin ² Level of enzyme is not ² Depends on glucose concentration affected by fasting or high CHO diet ² Act on glucose, fructose and ² Glucose only galactose 10/26/2020 Dr. Mohamed Z Gad 16

2) Splitting Stage • The reaction catalysed by aldolase is the reverse of aldol condensation. • Although the cleavage of F 1, 6 BP is energetically unfavourable, rapid removal of the product drives the reaction forward. • Of the two products of the aldolase reaction, only GAP (or G 3 P) serves as a substrate for the next reaction in glycolysis. • To prevent the loss of the other threecarbon unit, triose phosphate isomerase catalyses the interconversion of DHAP & G 3 P. Because of this reaction, the original molecule of glucose has now been converted to two molecules of 10/26/2020 Dr. Mohamed Z Gad G 3 P. 17

3) Oxidoreduction – Phosphorylation Stage • G-3 -P dehydrogenase is a tetramer, each subunit contains 1 binding site for G 3 P & another for NAD+ (NAD+ is permanently bound to the enzyme). • G 3 P 1, 3 -BPG 3 PG and PEP Pyruvate are examples of “substrate– level” phosphorylation. • 3 -PG 2 -PG is mediated by an intermediate [2, 3 -BPG]. Most cells have low amounts of 2, 3 -BPG except in RBCs, which act as allosteric modifier of Hb-O 2 binding. • PEP Pyruvate is an “irreversible reaction” due to free energy loss associated with tautomerization of the 10/26/2020 enol to the more stable keto form. Dr. Mohamed Z Gad 18

What is meant by “substrate–level phosphorylation” ? ADP is converted to ATP by the direct transfer of a phosphoryl group from a high energy compound. 10/26/2020 19

Why glycolysis under anaerobic conditions proceed to lactate and not just stop at pyruvate formation ? The reaction of lactate dehydrogenase is essential in anaerobic glycolysis , as it is the mean for reoxidizing NADH formed in the G-3 -P dehydrogenase step to reenter into the glycolysis cycle. In aerobic glycolysis reoxidation takes place in mitochondria by the respiratory chain. 10/26/2020 20

Overall pathway of Glycolysis & ATP Formation Number of ATP generated from glycolysis Enzyme Aerobic Anaerobic Hexokinase -1 ATP PFK-1 -1 ATP G-3 -P dehydrog. +6 ATP ---- Phosphoglycerate kinase +2 ATP Pyruvate kinase +2 ATP Sum +8 ATP +2 ATP 10/26/2020 21

Study Question ? ? ? Louis Pasteur, the great 19 th century French chemist and microbiologist, was the first scientist to observe the following phenomenon. “Cells that can oxidize glucose completely to CO 2 and H 2 O utilize glucose more rapidly in the absence of O 2 than in its presence”. It would appear that O 2 inhibits glucose consumption. Thus, another definition for Pasteur effect is: inhibition of glucose utilization and lactate accumulation by the initiation of respiration (O 2 consumption)… Can you explain why ? Louis Pasteur (1822 - 1895) The reason behind this phenomenon is that complete oxidation of glucose under aerobic conditions yield much more ATP (~38 ATP) than anaerobic glycolysis (~2 ATP). Thus it is anticipated that the rate of glucose consumption will be 1920 times faster under anaerobic condition to meet the metabolic demand in a way equivalent to aerobic conditions. 10/26/2020 Dr. Mohamed Z Gad 22

Regulation of Glycolysis Rate of glycolysis is controlled primarily by allosteric regulation of the 3 key enzymes (irreversible steps), hexokinase, PFK-1, and pyruvate kinase. • PFK-1 is the major regulatory Enzyme Activator Inhibitor enzyme of glycolysis. In the liver Hexokinase AMP, ADP, Pi G-6 -P only, PFK-1 is activated by fructose 2, 6 -diphosphate (F-2, 6 -DP). PFK-1 F-6 -P, AMP, NADH, • PFK-2, the enzyme that synthesize the activator F-2, 6 -DP, is itself a regulatory enzyme. It is inhibited by Pyruvate citrate & ATP and by kinase phosphorylation. The reverse reaction is catalyzed by fructose-2, 6 diphosphatase(F-2, 6 -DPase). F-2, 6 -DP (liver only) AMP, DP H+ , citrate, ATP F-1, 6 - ATP, acetyl Co. A, phosphorylation • Hormones also regulate glycolysis e. g. , glucagon inhibits glycolysis by repressing the synthesis of F-2, 6 -DP. Insulin promotes glycolysis by stimulating the synthesis of F-2, 6 -DP. 10/26/2020 Dr. Mohamed Z Gad 23

Study Question ? ? ? What effects do fluoride and magnesium have on glycolysis ? 10/26/2020 24

Comments on Glycolysis ü Glycolysis is the only pathway that produce ATP in absence of O 2. ü The best known inhibitors of the glycolytic pathway include: Arsenate and iodoacetate: inhibit glyceraldehydes-3 -phosphate dehydrogenase which has cysteine residue in the active site. Fluoride: a potent inhibitor of enolase. Thus, fluoride is usually added to blood samples to inhibit glycolysis before estimation of blood glucose. ü Magnesium: required for kinase reactions by forming Mg-ATP complex. ü Accumulation of lactate is responsible for muscle fatigue and cramps observed under heavy exercise (anaerobic glycolysis). ü In RBCs, glycolysis is the major source of ATP since RBCs lack • mitochondrial oxidation. 10/26/2020 25