Biomol4

Carbohydrate Metabolism Glycolysis, Gluconeogenesis Pentose phosphate Pathway

Citric Acid Cycle and

Oxidative Phosphorylation Department of Biochemistry

Carbohydrate metabolism -Glycolysis (for catabolism) Oxidation of glucose into pyruvate -Gluconeogenesis (for anabolism) Processes in glucose synthesis from nonhexose precursors -Pentose phosphate pathway (for catabolism and anabolism) Oxidative pathway of glucose for synthesis of specialized products needed by the cell, NADPH and ribose 5-phosphate for nucleic acids

Glycolysis Glycolysis : Greek, glykys = sweet, lysis = splitting -A series of enzyme-catalyzed oxidation of glucose (C6) yield two molecules of pyruvate (C3). -During the sequential reactions of glycolysis, the free energy is released from glucose oxidation, and conserved in the from of ATP and NADH. -Glycolysis is an universal pathway in glucose catabolism found in most cells to obtain energy.

Glycolysis

Glycolysis

The reasons why intermediates in glycolysis are phosphorylated. 4) pKa of phosphate group is low, giving each glyclytic intermediates a net negative charge, and can not diffuse out from the cell. 7) Phosphoryl groups are essential components in the enzymatic conservation of metabolic energy. 10) Binding of phosphate group to active sites of enzymes is a specific binding to enzymes in specific reaction.

Substrate-level phosphorylation The formation of ATP by phosphoryl group transfer from substrate such as 1,3-biphosphoglycerate and phosphoenolpyruvate. O O P O-

O C

O-

CHOH

+ ADP

CH2OPO32-

C

Mg2+

CHOH

C

O-

Phosphoglycerate Kinase

3-phosphoglycerate

C CH2

O O

O P O-

+ ADP

O-

Phosphoenolpyruvate

+ ATP

CH2OPO32-

1,3-biphosphoglycerate

O

O-

O

Mg2+ Pyruvate Kinase

C

O-

C

O

CH2

Pyruvate

+ ATP

The net gain of energy from glycolysis

Regulation of glycolysis •Louis Pasteur discovered that both rate and total amount of glucose consumption were many times greater under anaerobic condition than aerobic condition. •Later studies in muscles was clear that the ATP yields from glycolysis under anaerobic condition is 2ATP, much lower than the complete oxidation in aerobic condition (30 or 32ATP). •18 times as much glucose must therefore be consumed in anaerobic condition to yield same amount of ATP. •The regulation of glycolysis is achieved by two glycolytic enzymes, phosphofructokinase-1 and pyruvate kinase in ATP (allosteric regulation).

Trehalose

Lactose

Sucrose

Feeder pathways for Glycolysis

Degradation of glycogen and disaccharides

Glycogen of starch are degredrded by glycogen phosphorylase α-1-6 linkage

Glucose 1-phosphate

Glycogen Phosphorylase

Transferase Activity of Debranching Enzyme

Oligo (α1-6) to (α1-4) glucantransferase

α-1-6 Glucosidase Activity of Debrnaching Enzyme

Glycogen Phosphorylase

Fates of Pyruvate

Fermentation of pyruvate

Fermentation of pyruvate to lactate The fermentation of pyruvate is important to recycle the limited NAD+ to degrade glucose during burst phase of working in muscle.

2ATP

Gluconeogenesis •Biosynthesis of glucose is an absolute necessity in all mammals. •The brain and nervous system, erythrocyte, testes, renal medulla, and embryonic tissue require glucose as a fuel source. •The formation of glucose from nonhexose precursor is called gulconeogenesis. •Gluconeogenesis is not occurred in every tissue types, and occurred in glucogenic tissues, liver and kidney.

Gluconeogenesis

Three reactions of glycolysis are irreversible and can not be used in gluconeogenesis. •The conversion of glucose to glucose 6-phosphate by hexokinase •The phosphorylation of fructose 6-phosphate to fructose 1,6-biphosphate by phosphofructokinase-1 •The conversion of phosphoenolpyruvate to pyruvate by pyruvate kinase “Gluconeogenesis requires bypass pathway of these three reactions.’’

Precursors for gluconeogenesis

I Glycerol

Glycerol 3-phosophate

Dihydroxyacetone phosphate

II Carbon skeleton from amino acid degradation III Lactate IV Pyruvate

Oxaloacetate

Glucose

Three reactions of glycolysis are irreversible and can not be used in gluconeogenesis.

Gluconeogenesis

Gluconeogenesis

Alternative pathways from pyruvate to PEP

Transportation of oxaloacetate from mitochondria into cytosol via malate-aspartate shuttle

Cori cycle Blood Circulation

Glucose

Glucose

Liver Lactate

Muscle Glucose

Blood Circulation

NAD+

NADH + H+

2 Pyruvate Muscle

2 Lactate Muscle

Reciprocal regulation of glycolysis and gluconeogenesis Glucose 2ADP + 2Pi

4ADP + 4GDP +4Pi

2ATP

4ATP + 4GTP Pyruvate

•Both pathways required tight regulation. •No net conversion, both glycolysis and gluconeogenesis were allowed and the only net results is the utillization of two ATPs and two GTPs (futile cycle).

Reciprocal regulation of glycolysis and gluconeogenesis Regulatory enzymes I Phosphofructokinase (PFK-1) •Allosteric regulation by small molecules -activators: AMP, fructose 2,6-bisphosphate -inhibitors: ATP, citrate, H+ ion (prevention for producing lactate) ATP ADP+Pi •Fructose 2,6-bisphospshate PFK-2 is synthesized from fructose 6-phosphate Fructose 6-phosphate

Fructose 2,6-bisphosphate

Pi Fructose bisphosphatase 2 (FBPase2)

Reciprocal regulation of glycolysis and gluconeogenesis Regulatory enzymes PFK-2 is a bifunctional enzyme and both activities are catalyzed by the same polypeptide. Kinase

Phosphatase

•PFK-2 is control by phosphorylation. •Phosphorylated stage is active for phosphatase activity but inactive for kinase activity.

Reciprocal regulation of glycolysis and gluconeogenesis Lowered blood glucose Activation of protein kinase A

Releasing of glucagon Triggering cAMP cascade Acivation of FPBase 2

Phosphorylation of PFK-2

Lower glycolysis

Inhibition of PFK-2

Lower fructose 2,6-bisphosphate

Glycogen metabolism •Glycogen is mainly stored in the liver and skeleton muscle. •Muscle: source of energy during prolong muscle contraction •Liver: maintaining blood glucose level Glycogen degradation •Enzyme glycogen phosphorylase (break α1-4 glycosidic bond) •Enzyme glycogen debranching enzyme (α 1-6 glycosidic bond) •Product – glucose 1-phopshate •Glucose 1-phosphate is converted into glucose 6-phosphate by enzyme phosphoglucomutase, and enter to glycolysis.

Glycogen metabolism Glycogen synthesis -Starting compound: UDP-glucose -Enzyme: glycogen synthase (use UDP-glucose) -Glycogen synthase require primer compose of complex between glucose and protein (glycogenin) UTP + glucose 1-phosphate

UDP-glucose + PPi

UDP-glucose pyrophosphorylase

Branching enzyme -Enzyme: amylo (1-4→1-6) transglycosylase creates α1-6 bond

Glycogen metabolism Regulatory enzymes for glycogen metabolism I Glycogen phosphorylase II Glycogen synthase (require high energy compound, UTP) Both enzymes are required tight regulation in prevention of futile cycle with no net conversion. Glycogen Pi

UMP + PiPi

UTP glucose 1-phosphate

Glycogen metabolism Hormonal regulation Epinephrine Norepinephrine Glucagon

Activate

Adenylate cyclase

ATP

cAMP

cAMP-dependent protein kinase (inactive)

Phosphorylate target proteins (Phosphorylase kinase)

cAMP-dependent protein kinase (active)

Glycogen metabolism Epinephrine Norepinephrine Glucagon

ATP

Phosphorylase kinase

ADP

Phosphorylase kinase phosphorylase b (inactive)

Pi

phosphorylase a (active)

Protein phosphatase I

Glycogen metabolism Hormonal regulation Insulin (from β-cell of the pancreas) -lower blood glucose after feeding and stimulate glycogen synthesis Insulin-responsive protein kinase phosphorylate

phosphorylate

Protein phosphatase I

Protein phosphatase

inhibit phosphorylase

activate glycogen synthase

Glycogen metabolism Hormonal regulation Insulin activates protein phosphatase. Protein phosphatase Pi

glycogen synthase (inactive)

glycogen synthase (active)

Protein kinase ADP

ATP

Pentose phosphate pathway -Glucose 6-phosphate can be the intermediate of other catabolic fates, pentose phosphate pathway or phosphogluconate pathway or hexose monophosphate pathway. -This pathway leads to produce specialized products needed by the cell. -Specialized products produced by pentose phosphate pathway 10. NADPH for biosynthesis of fatty acids and steroids 11. Pentose (ribose 5-phosphate) for nucleic acid synthesis

Pentose phosphate pathway Glucose Glucose 6-phosphate Oxidation Oxidation Pyruvate

Citric Acid Cycle

Pentose Phosphate Pathway

Ribose 5-phosphate (Pentose)

NADPH

Biosynthesis of Biosynthesis of Nucleic Acid Fatty Acid and Steroid

Pentose phosphate pathway

Role of NADPH and glutathione in protecting cell membrane Glucose 6-phosphate dehydrogenase deficiency (G6PD)

Tricarboxylic acid cycle (TCA Cycle) or The citric acid cycle Respiration Broader sense: an physiological aspect referring to a multicellular organism’s uptake of O2 and release CO2 More narrow sense: a molecular process by which cells consume O2 and produce CO2 (Cellular respiration).

•The TCA cycle is a center of metabolism in with degradative pathways leading in and anabolic pathways leading out. •The pathway of further oxidation of pyruvate into CO2, and free energy is conserved in the reduced electron carriers, NADH, FADH2 and ATP.

Oxidation of proteins, lipids and carbohydrates into three stages of cellular respiration.

Oxidation of pyruvate into acetyl-CoA

O

C

O-

C O CH3

Pyruvate

CoA-SH

NAD+ NADH O S TPP, Lipoate C FAD Pyruvate dehydrogenase complex

C O

CoA

+ CO2

CH3

Acetyl-CoA

-The overall reaction is called oxidative decarboxylation (irreversible). -The carboxyl group is removed from pyruvate as a molecule of CO2. -The enzyme complex is composed of three enzymes.

The citric acid cycle -1948, Eugene Kennedy and Albert Lehninger showed that reactions of the citric acid cycle in eukaryotic cells take place in mitochondria. - They also found that electron transfer and ATP synthesis by oxidative phosphorylation occurs in inner membrane of mitochondria. - In most prokaryotic cells, the enzymes catalyzing reactions in the citric acid cycle are in cytosol and plasma membrane play a role analogous to that of the inner membrane of mitochondria in ATP synthesis.

TCA cycle

Products from single turn of the TCA Cycle The GTP formed by succinyl-CoA synthetase can donate phosphoryl group to ADP form ATP. Mg2+

GTP + ADP GDP + ATP Nucleoside diphosphate kinase

Glycolysis

TCA cycle

Intermediates in citric acid cycle are important biosynthetic precursors. The citric acid cycle serves in both catabolic and anabolic processes, amphibolic pathway.

The reactions that replenish TCA intermediates. As intermediates of the citric acid cycle are removed to serve as biosynthetic precursors, they are replenished by anaplerotic reaction.

Pyruvate carboxylase is an enzyme important to anaplerotic reaction. -The most important anaplerotic reaction in mammalian kidney and liver is the reversible carboxylation of pyruvate and CO2 to form oxaloacetate. -Pyruvate carboxylase is a regulatory enzyme, which is controlled by allosteric regulation, and is activated by excess amount of acetyl-CoA. -The pyruvate carboxylase reaction requires the vitamin biotin, which is prosthetic group of the enzyme. -Biotin acts as a specialized one-carbon group in the most oxidized form, CO2. -Sources of biotin; many foods and intestinal bacteria -The deficiency is rare, generally occurring when large quantities of raw eggs are consumed (avidin in egg whites bind tightly to biotin preventing for intestinal absorption).

Oxidative phosphorylation •1948, Eugene Kennedy and Albert Lehninger found that the site of oxidative phosphorylation is occurred in mitochondria. •The outer membrane of mitochondria: permeable to small molecules (Mr< 5000) and ions by passing through transmembrane proteins. •The inner membrane is impermeable to most small molecules and ions, including proton (H+) but it can be transported by specific transporters. •The inner membrane contains the component for ATP synthase.

Mitochondria

Universal electron acceptors -The electrons from catabolic pathways are kept in form of NADH, NADPH, FADH2 and FMNH2 (oxidized forms, NAD, NADP, FAD and FMN). -Both NADH and NADPH can not cross the inner mitochondrial membrane, but electrons can be shuttled across indirectly. -Flavoproteins: proteins contain FAD or FMN as a cofactor. The FAD or FMN binds tightly to protein, sometimes covalently attached to protein. -The electron carriers will release energy in electron transport chain.

Summary of the flow electrons and protons Higher [H+]

Lower [H+] Energy of electron transfer is conserved in a proton gradient.

Energy from electron transfer is conserved in a proton gradient (proton-motive force). Proton-motive force provides conserved energy in two forms. • Chemical potential energy From different concentration in two separated sites. 2) Electrical potential energy from separation of charge. The free-energy change from electrochemical gradient by ion pump is

∆G = RT ln(C2 / C1 ) + ZF∆E

= 2.3RT∆pH + ZF∆E Z = absolute value electrical charge (+1 for P+) F = Faraday constant

ATP synthesis I Peter Mitchell proposed chemiosmotic model for ATP synthesis mechanism. II The electrochemical potential (proton-motive force) drives the synthesis of ATP as protons flow passively back into the matrix.

FADH2 = 2 ATP

NADH = 3 ATP

The shuttle of cytosolic NADH reducing equivalent into mitochondrial matrix I Inner membrane of mitochondria is not permeable to NADH. II Special shuttle systems carry reducing equivalents from cytosolic NADH into mitochondria matrix by an indirect route. Shuttle systems 7) Malate-aspartate shuttle system: Liver, Kidney and heart muscle 9) Glycerol 3-phosphate shuttle system: Brain and muscle

Malate-aspartate shuttle system

NADH = 3 ATP

Glycerol 3-phosphate shuttle system

FADH2 = 2 ATP