Glycolysis Part 2 BCH 340 lecture 4
Regulation of Glycolysis There are three steps in glycolysis that have enzymes which regulate the flux of glycolysis These enzymes catalyzes irreversible reactions of glycolysis I. The hexokinase (HK) II. The phoshofructokinase (PFK) III. The pyruvate kinase They are regulatory enzymes which are regulated by the level of ATP in the cell
I- Phosphofructokinase-1 (PFK-1): The most important regulatory enzyme which catalyzes the first irreversible reaction unique to the glycolytic pathway (the committed step) Allosteric enzyme inhibited by elevated level of ATP, which:is the end product of glycolysis as well as it is substrate for PFK-1
I- Phosphofructokinase-1 (PFK-1): o Sigmoidal dependence of reaction rate on [fructose-6-p] is seen. o At high [ATP], PFK has lower affinity for the other substrate, fructose-6-p. ATP binds to inhibition site of PFK, and thereby decreases the activity of enzyme.
I- Phosphofructokinase-1 (PFK-1): AMP, present at significant levels only when there is extensive ATP hydrolysis, antagonizes effects of high ATP. AMP, ADP and Fructose 2, 6 biphosphate act as allosteric activators of PFK-1.
II- Hexokinase It is allosterically inhibited by its product Glucose 6 phosphate. In liver, glucokinase is inhibited by Fructose 6P and ATP (acts as a competitive inhibitor of this enzyme)
III- Pyruvate Kinase It is allosterically inhibited by ATP. ATP binding to the inhibitor site of PK decreases its ability to bind to PEP the substrate. It is also inhibited by Acetyl Coenzyme A and long chain fatty acid because they are source rich ATP which inhibits PK.
Hormonal regulation of glycolysis Insulin and Glucagon (secreted by the pancreas) are the main endocrine that modulate blood glucose levels and they act antagonistically Insulin is secreted in hyperglycemia and after carbohydrates feeding, it causes: 1. Induction for synthesis of glycolytic key enzyme 2. Activation of protein phosphatase 1 producing dephosphorylation and activation of glycolytic key enzymes
Hormonal regulation of glycolysis Glucagon is secreted in hypoglycemia or in CHO deficiency and it affects liver cells mainly as follows: 1. It acts as repressor of glycolytic key enzymes (PFK1, Pyruvate kinase, glucokinase) 2. It produces phosphorylation of specific enzymes leading to inactivation of glycolytic key enzymes
Hormonal regulation of glycolysis
Inhibitors of glycolysis 2-deoxyglucose: inhibits hexokinase Mercury and iodoacetate: inhibit glyceraldehyde-3-p dehydrogenase Fluoride: inhibits enolase by removal of Mg 2+ as Mg fluoride Arsenate: is uncoupler of oxidation and phosphorylation, it forms 1-arseno-3-phosphoglycerate which interferes with ATP formation at substrate level
Pasteur Effect It is the inhibition of glycolysis by the presence of oxygen Explanation: Aerobic oxidation of glucose produces increased amount of ATP and citrate. Those inhibit PFK1.
Mitochondrial pathway for glucose oxidation (TCA cycle) BCH 340 lecture 5
Under aerobic conditions, pyruvate (the product of glycolysis) passes by special pyruvate transporter into mitochondria which proceeds as follows: 1. Oxidative decarboxylation of pyruvate into acetyl CoA. 2. Acetyl CoA is then oxidized completely to CO 2, H 2 O through Krebs' cycle G first stage cytosol glycolytic pathway Pyr Mitochodria second stage Pyr CH 3 CO~SCoA third stage CO2 + H 2 O+ATP TAC
Oxidative decarboxylation of Pyruvate to Acetyl CoA COO - C CH 3 pyruvate NAD + NADH + H + O + HSCoA H 3 C C~SCoA + CO 2 Pyruvate dehydrogenase complex Acetyl CoA Irreversible reaction catalyzed by a multi enzyme complex associated within the inner mitochondrial membrane known as Pyruvate dehydrogenase complex O
Pyruvate dehydrogenase complex This enzyme complex contains 3 subunits, which catalyze the reaction in 3 steps: Es E 1 pyruvate dehydrogenase E 2 dihydrolipoyl transacetylase E 3 dihydrolipoyl dehydrogenase HSCoA NAD +
Pyruvate dehydrogenase complex This enzyme needs 5 coenzymes (all are vitamin B complex derivatives) Thiamine pyrophosphate, TPP (VB 1 ) HSCoA (pantothenic acid) cofactors lipoic Acid NAD + FAD (VB 2 ) HSCoA NAD +
Regulation of Pyruvate dehydrogenase complex E and product accumulation: 1 2 allosteric inhibitors: ATP, acetyl CoA, NADH, FA Low levels E: allosteric activators: AMP, CoA, NAD +,Ca 2+ Pyruvate dehydrogenase (Active(active dephosphorylated form) form) Pi pyruvate dehydrogenase phosphatase H 2 O ATP pyruvate dehydrogenase kinase ADP 3 Ca 2+,insulin pyruvate dehydrogenase P (Inactive (inactive phosphorylated form) form) acetyl CoA, NADH ADP, NAD + Regulation of E1 by covalent modification through phosphorylation
Regulation of Pyruvate Dehydrogenase Irreversible reaction must be tightly controlled-- three ways Allosteric Inhibition Inhibited by products: acetyl-coa and NADH Inhibited by high ATP Allosteric activation by AMP Ratio ATP/AMP important
Covalent modification (hormonal regulation): Through Phosphorylation/dephosphorylation of E1 PDH exists in two forms: Phosphorylated (inactive): Protein kinase enzyme converts active into inactive enzyme Dephosphorylated (active): Phosphatase enzyme converts inactive into active NB: In vitro inhibition of PDH: Arsenic Mercury
Figure: Metabolic sources and fates of acetyl CoA Acetyl CoA is an important molecule in metabolism used in many biochemical reactions Acetyl CoA functions as: 1. input to Krebs Cycle, where the acetate moiety is further degraded to CO 2 2. donor of acetate for synthesis of FA, ketone bodies, & cholesterol citric acid cycle GLUCOSE PYRUVATE Acetyl CoA glycolysis pyruvate dehydrogenase lipogenesis -oxidation Fatty acids (Cytoplasm) CO 2 ketogenesis (liver only) In mammals, acetyl CoA is essential to the balance between CHO and fat metabolism Ketone bodies ketone oxidation Cholesterol cholesterol synthesis (endocrine glands) steroid hormones
Figure: Metabolic sources and fates of pyruvate and acetyl CoA gluconeogenesis GLUCOSE glycolysis alanine aminotransferase Alanine PYRUVATE pyruvate carboxylase lactate dehydrogenase pyruvate dehydrogenase Lactate Oxaloacetate Acetyl CoA lipogenesis Fatty acids citric acid cycle -oxidation (Cytoplasm) CO 2 ketogenesis (liver only) ketone oxidation cholesterol synthesis Ketone bodies Cholesterol (endocrine glands) steroid hormones
Kreb's cycle Also known as Citric Acid Cycle (CAC) Or Tricarboxylic Acid Cycle (TCA) Or Catabolism of Acetyl CoA (CAC)
Definition: TCA is a series of enzyme-catalyzed chemical reactions in which acetyl CoA is oxidized into CO 2, H 2 O and energy. Location: Occurs in the matrix of the mitochondrion = aerobically
Steps: o The enzymes of TCA are present in the mitochondrial matrix either free or attached to the inner surface of the mitochondrial membrane. o The cycle is started by acetyl CoA (2C) and oxaloacetate (4 C) to form citrate (6C). It ends by oxaloacetate (4C). o The difference between the starting compound (6C) and the ending compound (4C) is 2 carbons that are removed in the form of 2 CO 2. These 2 carbons are derived from acetyl CoA. For this reason acetyl CoA is completely catabolized in TCA and never gives glucose.
The cycle begins with the condensation of acetyl-coa and oxaloacetate to form citrate Non-equilibrium reaction catalyzed by citrate synthase Inhibited by: ATP NADH Citrate - competitive inhibitor of oxaloacetate
Aconitase then catalyzes the interconversion of citrate and isocitrate via dehydration and hydration Equilibrium reactions Results in interchange of H and OH
Isocitrate is then converted to α-ketoglutarate via oxidative decarboxylation, producing CO 2 Isocitrate dehydrogenated and decarboxylated to give - ketoglutarate Non-equilibrium reaction catalyzed by isocitrate dehydrogenase + NAD
Isocitrate is then converted to α-ketoglutarate via oxidative decarboxylation, producing CO 2 Results in formation of: o NADH + H + o CO 2 Stimulated by isocitrate, NAD +, Mg 2+, ADP, Ca 2+ Inhibited by NADH and ATP + NAD
The α-ketoglutarate is then converted to succinyl-coa via another oxidative decarboxylation, producing the second CO 2 Series of reactions result in decarboxylation, dehydrogenation and incorporation of CoASH Non-equilibrium reactions catalyzed by -ketoglutarate dehydrogenase complex Stimulated by Ca 2+ Inhibited by NADH, ATP, Succinyl CoA TPP lipoate FAD
Succinyl CoA is then converted to succinate, accompanied by the formation of a GTP (or ATP) Equilibrium reaction catalyzed by succinate thiokinase Results in formation of GTP and CoA-SH Nucleoside diphosphate kinase interconverts GTP and ATP by a readily reversible phosphoryl transfer reaction: GTP + ADP GDP + ATP
Succinate is then converted to fumarate by dehydrogenation Succinate dehydrogenated to form fumarate Equilibrium reaction catalyzed by succinate dehydrogenase Only Krebs enzyme contained within inner mitochondrial membrane Results in formation of FADH 2
Fumarate is then converted to malate via hydration Equilibrium reaction catalyzed by fumarase
The cycle ends by the regeneration of oxaloacetate from L-malate Malate dehydrogenated to form oxaloacetate Equilibrium reaction catalyzed by malate dehydrogenase Results in formation of NADH + H +
Glucose glycolysis PDH ------ Pyruvate fatty acids, ketone bodies Acetyl CoA CoA Figure: Reactions of the citric acid cycle NADH ------ NAD + Malate Oxaloacetate Citrate -------- cis Aconitate Isocitrate -Ketoglutarate NAD ------ + NADH, ------ CO 2 CoA, NAD + ------ Fumarate Succinyl ------CoA NADH, ------ CO 2 FADH ------ 2 FAD Succinate GTP GDP ATP ------ ADP
Products of Krebs Cycle 2 CO 2 3 NADH 1 ATP 1 FADH 2 ATP Yield Each NADH energizes 3 ATP Each FADH 2 energizes 2 ATP Double this list for each glucose
The amphibolic nature of Citric acid cycle By transamination, oxaloacetate is converted to aspartate By transamination α-ketoglutarate is converted to glutamate This pathway is utilized for the both catabolic reactions to generate energy as well as for anabolic reactions to generate metabolic intermediates for biosynthesis
What are the key regulated enzymes in citrate cycle?
What are the key regulated enzymes in citrate cycle? Pyruvate dehydrogenase not a citrate cycle enzyme but it is critical to flux of acetyl-coa through the cycle; this multisubunit enzyme complex is inhibited by acetyl-coa, ATP and NADH. Citrate synthase catalyzes the first reaction in the pathway and can be inhibited by citrate, succinyl-coa, NADH and ATP; inhibition by ATP is reversed by ADP.
What are the key regulated enzymes in citrate cycle? (Cond ) Isocitrate dehydrogenase - catalyzes the oxidative decarboxylation of isocitrate by transferring two electrons to NAD + to form NADH, and in the process, releasing CO 2, it is activated by ADP and Ca 2+ and inhibited by NADH and ATP α-ketoglutarate dehydrogenase - functionally similar to pyruvate dehydrogenase in that it is a multisubunit complex, requires the same five coenzymes and catalyzes an oxidative decarboxylation reaction that produces CO 2, NADH and succinyl-coa; it is activated by Ca 2+ and AMP and it is inhibited by NADH, succinyl- CoA and ATP
Inhibitors of TCA Fluoroacetyl CoA: it combines with oxaloacetate giving rise to fluorocitrate which inhibits aconitase enzyme Malonic acid: inhibits succinate dehydrogenase (competitive inhibition) Arsenate and Mercury : inhibit Pyruvate dehydrogenase and α-ketoglutarate dehydrogenase complex by reacting with sulphydral group of lipoic acid leading to accumulation of pyruvic lactic acid and α- ketoglutarate with acidosis