Storage mechanisms and control in carbohydrate metabolism Storage

Storage mechanisms and control in carbohydrate metabolism

Storage mechanisms and control in carbohydrate metabolism • How is glycogen produced and degraded? • How does gluconeogenesis produce glucose from pyruvate? • How is carbohydrate metabolism controlled? • why is glucose sometimes diverted through the pentose phosphate pathway?

How is glycogen produced and degraded? Glycogen metabolism • Glycogen is the major storage form of glucose mainly in the liver and muscle. • Glycogen composed of glycosyl units linked by α-1, 4 glycosidic bond with α-1, 6 branches occurring about every 8 -10 glycosyl units. • The synthesis, glycogenesis and degradation, glycogenolysis occur via different pathway – Glycogenesis and glycogenolysis are both cytosolic processes

Glycogenesis • Definition and location – Glycogenesis is the pathway for the formation of glycogen from glucose in muscle and in liver in the fed state, when insulin/glucagon ratio is high. • Glycogen is synthesized from α- D- glucose • The process requires energy ATP and UTP • The process occur in the cytosol

How is Glycogen formed from Glucose? • Not exact reversal of glycogen breakdown to glucose • Glycogen synthesis requires energy • Energy supplied by hydrolysis of UTP • Glucose-1 -phosphate reacts with UTP to make UDPG • Pyrophosphate is also formed • UDPG is then added to a growing chain of glycogen, catalyzed by glycogen synthase

How is Glycogen formed from Glucose? (Cont’d) • Coupling of UDPG formation with hydrolysis of pyrophosphate drives formation of UDPG to completion

Reaction Catalyzed by Glycogen Synthase

Hexokinase Glucose + ATP Glucose-6 -P Glucokinase in liver phosphoglucomutase UTP + Glucose-1 -P UDPG pyrophosphorylase UDPG + PPi Glycogen(n+1) + UDP pyrophosphatase H 2 O Glycogen synthase Glycogen primer(n) 2 Pi

Glycogenolysis • Definition and location – Glycogenolysis is the degradation of glycogen to glucose-6 -phosphate and glucose in muscle and liver respectively – Glycogenolysis is not the reverse of the glycogenesis but is a separate pathway

How does Glycogen Breakdown take place?

How does Glycogen Breakdown take place? (Cont’d) • Three reactions play roles in the conversion of glycogen to glucose-6 - phosphate • In the first reaction – Glycogen is cleaved by phosphate to give -D-glucose-1 phosphate – Cleavage reaction is phosphorlysis and not hydrolysis – No ATP is involved in reaction – Reaction is catalyzed by glycogen phosphorylase

How does Glycogen Breakdown take place? (Cont’d) • In the second reaction, glucose-1 -phosphate is isomerized to glucose-6 -phosphate • This reaction is catalyzed by phosphoglucomutase

How does Glycogen Breakdown take place? (Cont’d) • Complete breakdown requires debranching enzymes to degrade the -1, 6 linkages • α -1, 4 to α- 1, 4 glucan transferase Amylo-1, 6 glucosidase debranching enzymes

Glycogen chain (glucose units)n Pi Glycogen Phosphorylase G-1 -P + glycogen chain (glucose units)n-1 phosphoglucomutase Glucose-6 -Phosphate H 2 O Glucose + Pi Glucose-6 phosphatase

Control of Glycogen Metabolism • Glycogen phosphorylase is a major control point in the synthesis and breakdown of glycogen • Glycogen phosphorylase activity can be allosterically controlled, as well as, controlled through covalent modification

Control of Glycogen Metabolism (Cont’d) • The activity of glycogen synthase is subject to the same type of covalent modification as glycogen phosphorylase, however, the response is opposite • In addition: – Hormonal signals (glucagon or epinephrine) stimulate its phosphorylation (by enzyme called c. AMP-dependent protein kinase) – After phosphorylation, glycogen synthase becomes inactive at the same time the hormonal signal is activating phosphorylase – Glycogen synthase can be phosphorylated by several other enzymes including phosphorylase kinase – Dephosphorylation is by phosphoprotein phosphatase

Insulin activates Glycogen Glucagon inhibits Glycogen synthase UDPG + PPi H 2 O 2 Pi Insulin inhibits Glycogen phosphorylase UDPG pyrophosphorylase glycogenesis G-1 -P phosphoglucomutase UTP H 2 O G-6 -P ADP Glucose-6 phosphatase Glucokinase ATP Glucose + Pi Uptake Release Blood glucose glycogenolysis UDP Glucagon activates Pi

Summary • Glycogen is the storage form of glucose in animals, including humans. • Glycogen releases glucose when energy demands are high • Glucose polymerizes to form glycogen when the organism has no immediate need for the energy derived from glucose breakdown • Glycogen metabolism is subject to several different control mechanisms, including covalent modification and allosteric effects

Practice Exercise 2: Which of the following organs is primarily responsible for controlling blood glucose levels? (a) Brain (b) Muscle (c) Spleen (d) Liver Exercise 2: Which of the following enzymes is called the glucose sensor for the body? • (a) Glycogen phosphorylase a (b) Glucokinase (c) Adenylate cyclase (d) Protein phosphatase I

Practice (count’d) • Exercise 2: Both glycogen synthase and glycogen phosphorylase are allosterically regulated by glucose. (a) No Response (b) True (c) False • Exercise 3: The addition of glucose to a cell causes an increase in glycogen synthesis and a decrease in glycogen breakdown. (a) No Response (b) True (c) False

Gluconeogenesis • Definition – The formation of glucose from non-carbohydrate precursors is called gluconeogenesis – The major non-carbohydrate substrate for gluconeogenesis are the: • • Lactate Glycerol Glucogenic amino acids Intermediate of citric acid cycle • Location – Liver – Kidney • Certain enzymes required in gluconeogenesis are present only in these organs

Substrate for gluconeogenesis Lactate

Synthesis of glucose from glycerol

Synthesis of glucose from proteins Mechanism by which breakdown of muscle proteins supplies the liver with a source of pyruvate for gluconeogenesis during starvation. In addition, many other amino acids are converted to pyruvate or citric acid cycle intermediates which are metabolized to oxaloacetate to an intermediate of gluconeogenesis

Gluconeogenesis • Gluconeogenesis: The synthesis of glucose from pyruvate – Gluconeogenesis is not the exact reversal of glycolysis; that is, pyruvate to glucose does not occur by reversing the steps of glucose to pyruvate – Three irreversible steps in glycolysis - Phosphoenolpyruvate to pyruvate + ATP (PK) - Fructose-6 -phosphate to fructose-1, 6 -bisphosphate (PFK) - Glucose to glucose-6 -phosphate (H/GK) – Net result of gluconeogenesis is reversal of these three steps, but by different reactions and using different enzymes

Gluconeogenesis Pathway of gluconeogenesis compared with glycolysis Special reactions and enzymes of gluconeogenesis pathway are shown in yellow boxes Remaining reactions which are common to glycolysis and gluconeogenesis are shown in red. The entry points of substrate are shown in blue

Gluconeogenesis Summary of Gluconeogenesis Pathway: Gluconeogenesis enzyme names in yellow.

Oxaloacetate is an Intermediate • In first step, Pyruvate is carboxylated to oxaloacetate – Requires biotin (CO 2 carrier) – Pyruvate carboxylase is subject to allosteric control; it is activated by acetyl-Co. A

Gluconeogenesis (Cont’d) – Next, decarboxylation of oxaloacetate is coupled with phosphorylation by GTP to give PEP – The net reaction of carboxylation/decarboxylation is Pyruvate + ATP + GTP PEP + ADP + Pi

Pyruvate Carboxlyase Reaction

Role of Sugar Phosphates • Other different reactions in gluconeogenesis relative to glycolysis involve phosphate-ester bonds bound to sugar-hydroxyl groups being hydrolyzed – Fructose-1, 6 -bisphosphatase is an allosteric enzyme, inhibited by AMP and activated by ATP – This reaction is a control point in the pathway

Role of Sugar Phosphates (Cont’d) • Another reaction is the hydrolysis of glucose-6 phosphate to glucose and Pi • Reaction catalyzed by glucose-6 -phosphatase

Control of Carbohydrate Metabolism • Allosteric control: fructose-2, 6 -bisphosphate (F 2, 6 P) – An allosteric activator of phosphofructokinase (PFK) – An allosteric inhibitor of fructose bisphosphatase (FBPase) – High concentration of F 2, 6 P stimulates glycolysis; a low concentration stimulates gluconeogenesis – Concentration of F 2, 6 P in a cell depends on the balance between its synthesis (catalyzed by phosphofructokinase-2) and its breakdown (catalyzed by fructose bisphosphatase-2) – Each enzyme is controlled by phosphorylation/dephosphorylation

Synthesis and Breakdown of F 2, 6 P

Mechanisms of Metabolic Control

Substrate Cycling • Substrate cycling – Oopposing reactions can be catalyzed by different enzymes and each opposing enzyme or set of enzymes can be regulated independently Fructose-6 -Phosphate + ATP ---> Fructose-1, 6, -bisphosphate + ADP Fructose-1, 6, -bisphosphate + H 2 o ---> Fructose-6 -Phosphate + Pi Both Reactions are exergonic, and the net reaction is ATP +H 2 O <---> ADP + Pi

The Cori Cycle: How Different Organs Share Carbohydrate Metabolism • The Cori cycle – Under vigorous anaerobic exercise, glycolysis in muscle tissue converts glucose to pyruvate; NAD+ is regenerated by reduction of pyruvate to lactate – Lactate from muscle is transported to the liver where it is reoxidized to pyruvate and converted to glucose – The liver shares the stress of vigorous exercise

The Cori Cycle

Major Control Points in Carbohydrate Metabolism • First and last steps in glycolysis are major control points in glucose metabolism • Hexokinase – Inhibited by high levels of glucose 6 -phosphate – When glycolysis is inhibited through phosphofructokinase, glucose 6 -phosphate builds up, shutting down hexokinase • Pyruvate kinase (PK) is an allosteric enzyme – Inhibited by ATP and alanine – Activated by fructose-1, 6 -bisphosphate • PK isoenzymes have 3 different subunits – M predominates in muscle, L in liver, and A in other tissues – Native PK is a tetramer – Liver isoenzymes are subject to covalent modification

Control of liver pyruvate kinase by phosphorylation Phosphorylated pyruvate kinase (less active) H 2 O - + Low blood glucose level Dephosphorylated pyruvate kinase (more active) P Phosphoenolpyruvate + ADP H + F 1, 6 BF ATP Alanine ATP Pyruvate + ATP

Summary • A number of control mechanisms operate in carbohydrate metabolism. These include allosteric effects, covalent modification, substrate cycles, and genetic control • In the mechanism of substrate cycling, the synthesis and the breakdown of a given compound are catalyzed by two different enzymes

Pentose phosphate pathway PPP • Definition – The PPP is an alternative route for metabolism of glucose – PPP formation of pentose phosphate (R-5 -P) for nucleotide biosynthesis in the quantities that the cell requires – PPP provides NADPH for reductive biosynthesis by oxidation of G 6 P • Location – The enzymes of PPP are present in cytosol – The pathway is found in all cells

Glucose is Sometimes Diverted through the PPP • The PPP is an alternative to glycolysis, and differs in several ways • In glycolysis, ATP production is important, in PPP, this is not the case • As the name implies, five-carbon sugars, including ribose, are produced from glucose • Oxidizing agent is NADP+; it is reduced to NADPH, which is a reducing agent in biosyntheses • Begins with two oxidation steps (NADP+) to give ribulose-5 phosphate • Following this, a series of carbon-shuffling steps occur during which three-, four-, five-, six-, and seven-carbon monosaccharide phosphates are produced

Difference between glycolysis and pentose phosphate pathway glycolysis • Oxidation occur utilizing NAD+ as a H-acceptor • Aerobic as well as anaerobic process • CO 2 is not produced at all • ATP is generated, where it is a major function • Ribose phosphate are not generated • 0 -90% of glucose oxidized by glycolysis Pentose phosphate pathway • Oxidation occur utilizing NADP as a H-acceptor • Anaerobic process • CO 2 is characteristic product • ATP is not generated • Ribose phosphate are generated • 10 -20% of glucose oxidized by PPP

The pentose phosphate pathway • The reactions of the PPP are divided into two phases: – Phase I: Oxidative irreversible phase • G-6 -p is oxidized with the generation of 2 molecules of NADPH and 1 molecule of PP with liberation of one molecule of CO 2 – Phase II: Non-oxidative reversible phase • The pentose phosphate is converted to intermediates of glycolysis

Phase I: Oxidative irreversible phase • In the first phase, G-6 -P undergoes dehydrogenation and decarboxylation to give pentose, ribulose-5 - phosphate (Ru 5 P) with generation of two molecules of NADPH § Dehydrogenation of G-6 -P to 6 -phosphogluconate catalyzed by glucose-6 -phosphate dehydrogenase § Oxidative decarboxylation of 6 - phosphogluconate to Ru 5 P and co 2 is catalyzed by 6 -phosphgluconate dehydrogenase

Oxidative reactions of the PPP

Phase II: Non-oxidative reversible phase • the second phase, consist of two stages Stage one: Isomerization and epimerization of Ru 5 P serves as substrate for two different enzymes: § Ribulose-5 -phosphate-3 -epimerase →(Xu 5 P) § Ribose-5 -phosphate ketoisomerase →(R 5 P) Stage two: Involves carbon-carbon bond cleavage and formation Ru 5 P is converted back to G-6 -P by a series of reactions

Nonoxidative reactions of the PPP

Group Transfer Reactions The carbon-shuffling reaction are catalyzed by: Transketolase for the transfer of two-carbon units Transaldolase for the transfer of three-carbon units

Group Transfer Reactions (count’d) • Transketolase reaction – It transfers two carbon unit with keto group from Xu 5 P to R 5 P to form a 7 carbon sugar, sedoheptulose 7 phosphate (S 7 P) and glyceraldehyde 3 phosphate (GA 3 P) – Transketolase enzyme will transfer the group from a donor ketose to an aldose acceptor • Transaldolase reaction – It transfer three carbon unit from S 7 P to GA 3 P to form F 6 P – The donor is a ketose and acceptor is an aldose • Second transketolase reaction – In the second transketolase reaction a 2 C unit is transferred from Xu 5 p to erythrose 4 P (E 4 P) to form F 6 P and GA 3 P

Control of the PPP • Control of the PPP is maintained by: – G 6 P can be channeled into either glycolysis or the pentose phosphate pathway – G 6 P channeling into glycolysis, if ATP needed – G 6 P channeling into the pentose phosphate pathway, if NADPH or R 5 P are needed • If NADPH needs exceeds that of R 5 P → GAP and F 6 P consumed through glycolysis, oxidative phosphorylation or recycled by gluconeogenesis • If R 5 P needs exceeds that of NADPH → GAP and F 6 P use in the synthesis of R 5 P by reversal of the transaldolase and transketolase reactions

Relationship between PPP and glycolysis

Overview of glucose metabolism Glycogen breakdown Glycogen synthesis Glucose 6 P Ribose 5 phosphate Pentose phosphate pathway Glucose Glycolysis Gluconeogenesis Pyruvate Amino acid Lactate Acetyl-Co. A Citric acid cycle

Summary of Gluconeogenesis Pathway: Gluconeogenesis enzyme names in red. Glycolysis enzyme names in blue.