Lactate Dehydrogenase Prof. Dr. Laila Fadda

Formation of Acetyl CoA and lactate from Pyruvate Acetyl Co A Pyruvate dehydrogenase complex O2O2 O2O2

1. It is an enzyme which catalyzes the reaction Lactate  Pyruvate 2. This reaction helps the re-oxidation of NADH, H+ into NAD+ 3. It has 5 isoenzymes: LD1, LD2, LD3, LD4 and LD5. 4. Medical importance: 5. Estimation of the activity of lactate dehydrogenase enzyme helps in the diagnosis of heart and liver diseases: a) LD1: Elevated in some heart diseases e.g. myocardial infraction. b) LD5: Elevated in some liver diseases as acute viral hepatitis.

Regulation of glycolvsis: u The rate of glycolysis is regulated by controlling of the 3 irreversible enzymes (key enzymes). These enzymes catalyze what is called committed reactions of the pathway. These enzymes are glucokinase (hexokinase), phospho-fructokinase-1 and pyruvate kinase.

1. Hormonal regulation: a) Insulin: Stimulates synthesis of all key enzymes of glycolysis. It is secreted after meal (in response to high blood glucose level). b) Glucagon: Inhibits the activity of all key enzymes of glycolysis. It is secreted in response to low blood glucose level. 2. Energy regulation: a) High level of ATP inhibits PFK-1 and pyruvate kinase. b) High level of ADP and AMP stimulate PFK-1.

3. Substrate regulation: a) Glucose-6-phosphate inhibits hexokinase (and not glucokinase). b) Citrate inhibits phosphofructokinase-1. d) Fructose 1,6 bisphosphate stimulates pyruvate kinase.

Lactate: Sources and fate of lactate: a) Sources: u From glycolysis especially in RBCs due to absence of mitochondria and muscle during exercises due to oxygen lack.

2) Conversion into pyruvate: If oxygen gets available, lactate is converted into pyruvate, which proceeds into Krebs' cycle. 3) Lactate may be accumulated in muscles causing muscle fatigue. 4) Lactate may be excreted in urine and sweat.

Clinical aspects of glycolvsis: 1. There are many diseases associated with impaired glycolysis. They include: 1. Pyruvate kinase deficiency. 2. Hexokinase deficiency. 3. Lactic acidosis. 2) Pyruvate kinase (PK) deficiency: a) This leads to excessive hemolysis of RBCs leading to hemolytic anemia. b) Genetic deficiency of PK enzyme causes decrease the rate of glycolysis and decrease production of ATP. c) ATP is required for Na' -K' ATPase, which is important for stability of RBCs.

3. Hexokinase deficiency: Leads to hemolytic anemia due to decrease ATP production. The mechanism is similar to that of PK deficiency. 4. Lactic acidosis: a) Definition and mechanism of lactic acidosis: 1) It is the lowered blood pH and bicarbonate levels due to increased blood lactate above normal level. OH OH CH3-C -- COOH + NaHCO3 CH3-C-COONa + H2CO3 CO2 + H2O

Lactate+Sodium bicarbonate Sodium lactate+Carbonic acid 2) This depletes bicarbonate ↓ pH of blood Lactic acidosis which if severe may lead to coma. b) Causes of lactic acidosis: It results from increased formation or decreased utilization of lactate. 1) Increased formation of lactate as in severe muscular exercises.

2) Decreased utilization of lactate in tissues: it occurs in cases of anoxia or lack of oxygen. This is because oxygen is essential for conversion of lactate into pyruvate, which proceeds into acetyl CoA, and Krebs' cycle. u Tissue anoxia may occur in conditions that impair blood flow e.g. myocardial infraction, angina pectoris, respiratory disorders, and anemia. 3) Phenformin (cidophage): is oral hypoglycemic, causing excessive anaerobic oxidation of glucose and excess lactate production.

II) Mitochondrial pathway for glucose oxidation: A. Introduction: Complete oxidation of glucose occurs in both cytoplasm (glycolysis) and mitochondria (Krebs' cycle) In the presence of O2 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 CO2, H2O through Krebs' cycle.

B. Oxidative decarboxylation of pvruvate to acetvl coenzvme A (= acetyl CoA): 1. Enzyme: Pyruvate dehydrogenase (PHD) complex. a) This enzyme complex contains 4 subunits, which catalyze the reaction in 4 steps.

b) This enzyme needs 5 coenzymes (all are vitamin B complex derivatives): 1) Vitamin B1 = Thiamin diphosphate = TPP. 2) Lipoic acid = L -SH.-SH (reduced), L-s-s (oxidized). 3) Coenzyme A = CoASH. 4) Flavin adenine dinucleotide = FAD. 5) Nicotinamide adenine dinucleotide = NAD+

c) Location: PDH is located within the mitochondrial matrix. 1) Energy production: (3ATP): u Oxidative decarboxylation of pyruvate to acetyl CoA produces one molecule of NADH,H+. This produces 3 ATP molecules through respiratory chain phosphorylation

2) Regulation of oxidative decarboxylation (PHD): + Insulin _ Calcium _Acetyl CoA Pyruvate DH complex Pyruvate +NAD + _ NADH + H H2OH2O +CoASH _ATP

a) Factors stimulating [+] PHD: 1) Pyruvate. 2) CoASH. 3) NAD+ 4) Insulin hormone. b) Factors inhibiting [-] PHD: 1) NADH,H+ 2) ATP. 3) Acetyl CoA. 4) Calcium ions.

u Kreb's cycle: [also known as citric acid cycle (CAC) or tricarboxylic acid cycle (TCA) or catabolism of acetyl CoA]: 1. Definition: TCA is a series of reactions in which acetyl CoA is oxidized into CO2 + H2O and energy. 2. Location: Mitochondria.

The Citric Acid (Krebs) Cycle consists of eight steps Fig. 9.11

Tricarboxylic Acid Cycle For oxidation of Acetyl CoA. Common pathway for final oxidation of proteins, carbohydrates and lipids. Occurs in mitochondrial matrix of all cells. ReactionsAcetyl CoA + 3 NAD + + FAD + GDP + Pi + 2H 2 O   2CO NADH + FADH 2 + GTP + 2H + + CoA

3. Steps: a) The enzymes of TCA cycle are present in the mitochondrial matrix either free or attached to the inner surface of the mitochondrial membrane. b) The cycle is started by acetyl CoA (2 carbons) and oxaloacetate (4 carbons) to form citrate (6 carbons). It ends by oxaloacetate (4 carbons). The difference between the starting compound (6 carbons) and the ending compound (4 carbons) is 2 carbons that are removed in the form of 2 CO2. These 2 carbons are derived from acetyl CoA. For this reasons acetyl CoA is completely catabolized in TCA and never gives glucose.

4. Energy production of TCA: (Energy catabolism of acetyl CoA): a) Oxidation of one molecule of acetyl CoA in TCA produces 12 ATP molecules, 11 by respiratory chain phosphorylation and 1 by Substrate level phosphorylation as follows:

No. of ATPMethod of ATP productionEnzyme 3 ATP Oxidation of NADH+H by respiratory chain phosph-orylation Isocitrate degydrogenase 3 ATP Oxidation of NADH+H by respiratory chain phosphorylation α-Ketoglutarate degydrogenase 1 ATPSubstrate level phosphorylationSuccinyl CoA thiokinase 2 ATP Oxidation of FADH by respiratory chain phosphorylation Succinate degydrogenase 3 ATP Oxidation of NADH+H by respiratory chain phosphorylation Malate degydrogenase 12 ATPTotal =

b) Energy production of complete oxidation of one molecule of glucose: Glucose oxidation 36 or 38 ATP Pyruvate oxidation 15ATP. Acetyl CoA 12 ATP

5. Oxidative decarboxylation of α-ketoglutarate to succinyl CoA It is similar to the conversion of pyruvate to acetyl CoA. a) Enzymes:α- ketoglutarate dehydrogenase complex. b) Coenzymes: 5: TPP, Lipoic acid, CoASH, FAD and NAD+. 6. Functions (significance) of TCA: The cycle is amphibolic i.e. it has catabolic (breakdown) and anabolic (formation) functions.

Energy: 12 ATP Catabolic functions: Oxidation of carbohydrate, lipids and proteins Anabolic function: Formation of: u Amino acids u Glucose u Heme u Fatty acid and cholesterol u CO 2 and over-controlling in his relation with the patient.

a) Production of energy (12 ATP). b) Catabolic functions: TCA is the final common pathway for oxidation of carbohydrates, fats and proteins (amino acids). c) Anabolic functions: Formation of: 1) Amino acids: -Ketoglutarate Transamination Glutamate. Oxaloacetate Transamination Aspratate.

2) Glucose: e.g. Ketoglutarate Gluconeogenesis Glucose. 3) Heme synthesis: Succinyl CoA Heme. 4) Fatty acid and cholesterol: Citrate (diffuse to cytoplasm) → Oxaloacetate + Acetyl CoA → Fatty acid and cholesterol.

5) CO 2 produced is used in the following (CO 2 fixation) reactions: u Pyruvate + CO 2 GIVE Oxaloacetate by Gluconeogenesis give Glucose. u Ammonia + ATP +CO 2 give Carbamoyl phosphate give Urea and pyrimidine u Formation of C6 of purine u Synthesis of H 2 CO 3 / HCO, buffer.

The electron transport chain

8. Regulation of citric acid cycle: u The key enzymes are citrate synthase dehydrogenase and - ketoglutarate dehydrogenase: a) Citrate synthase: 1) Stimulated by acetyl CoA, oxaloacetate, ADP and NAD­+. 2 ) Inhibited by long chain acyl CoA, citrate, succinyl CoA, ATP and NADH, H+

b) Isocitrate dehydrogenase and -ketoglutarate dehydrogenase: 1) Stimulated by NAD+, ADP. 2) Inhibited by NADH, H' and ATP

10. Energy production at substrate level in glucose oxidation: a) The removal of hydrogen atoms from a compound is accompanied by a release of energy. If this energy is captured in phosphate or sulfate bonds, it will produce high-energy compounds. b) The high energy compounds formed by glucose oxidation are: 1) Glyceraldhyde-3-p 1,3 BPG (phosphate bond). 2) Phosphoglycerate phosphoenol pyruvate (phosphate bond). 3) Pyruvate Acetyl CoA (sulfate bond(. 4) -Ketoglutarate Succinyl CoA (sulfate bond).