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Lecture 12: Enzymes of Metabolism: An Introduction Reference: Lecture notes only, no reading assigned 1. As a review, list the 6 general classes of enzymes and name the two major types of regulation for metabolic enzymes. a. Classes of Metabolic Enzymes i. Oxidoreductases ii. Transferases iii. Hydrolases iv. Lyases v. Isomerases vi. Ligases b. Regulation for Metabolic Enzymes i. Allosteric modulators 1. Positive or activator 2. Negative or inhibitor ii. Covalent Modification 1. Mainly phosphorylation 2. Define in simple terms the function of pyruvate dehydrogenase and pyruvate carboxylase. a. Pyruvate dehydrogenase and pyruvate carboxylase are the two key enzymes that participate in the TCA cycle which occurs in the mitochondria b. PDH: it’s main job is to convert pyruvate to acetyl CoA so that it can enter the TCA cycle c. Pyruvate carboxylase: it converts pyruvate to oxaloacetate (OAA). When acetyl CoA levels are high, cell need OAA to keep the TCA cycle running 3. List the cofactors required for PDH activity. a. Thiamin pyrophosphate (Vitamin B1 derivative): involved in group transfers. Helps catalyze the decarboxylation reaction b. Lipoate: Energy Reactions. Lipoate hold the acetyl group and transfers it to acetyl Coa, which carry the acetyl group into the TCA cycle. c. FAD (riboflavin derivative): Energy reactions. Helps to regenerate lipoate for the next reaction. 4. Define in general terms the mechanism(s) of regulation of pyruvate dehydrogenase and pyruvate carboxylase (i.e., allosteric? phosphorylation?). a. Pyruvate dehydrogenase is regulated by covalent phosphorylation (it’s inactive when it is phosphorylated) i. Positive modulators/activators: NAD+, CoA ii. Negative modulators/inhibitors: NADH, acetyl CoA b. Pyruvate Carboxylase is activated by acetyl CoA i. High levels of acetyl CoA activate pyruvate carboxylase to convert pyruvate to OAA (cell signal) 5. Define in general terms the function of carnitine:palmitoyl transferase I and II and acetyl CoA carboxylase. Again, no real detail is needed since these pathways have not been covered. a. CPT I enzyme: exhanges carnitine for CoA bound to Fatty Acid (FA). Only carnitine FA can be transported in the mitochondrial matrix. It allows FA to be transported into the mitochondrial matrix to be oxidized. Once inside, FA is converted back into FA-CoA (the active form). b. Acetyl CoA carboxylase: is used to transform glucose into fats like palmitate and palmitoyl CoA 6. Explain why newly synthesized fatty acids are not immediately transported into mitochondria for oxidation. a. FA must first be bound to carnitine in order to enter the mitochondrial matrix to be oxidized b. FA are not immediately transported into the mito in order to prevent a “futile cycle” where energy is spent to make a molecule that is degraded right away. 7. Explain the term “committed step” for a metabolic pathway. a. A committed step is an irreversible enzymatic reaction b. In Fatty Acid synthesis, the enzyme acetyl CoA carboxylase is the committed step. It carboxylates acetyl CoA and changes ATP to ADP to form Malonyl CoA 8. Define in simple terms the goal of glycolysis and role of PFK-1 in this pathway. a. Glycolysis is the pathway for the anaerobic oxidation of glucose to pyruvate b. Glucose enters cell and gets phosphorylated trapping it in the cell. c. Glucose-6-P is isomerized to fructose-6-P (reversible) d. Conversion of fructose-6-P to fructose-2,6-P by allosteric regulation by PFK-1 enzyme (committed step) e. F-2,6-P must turn into pyruvate f. PRK-1 is required for glycolysis, deficiencies result in little or no mitochondria—muscle cramps after exercise 9. Explain the significance of HMG-CoA reductase and mevalonate in the cholesterol synthesis pathway. Include in your answer why HMG-CoA reductase catalyzes the committed step in the pathway. a. HMG-CoA reductase: reduces HMG-CoA to Mevalonate for cholesterol synthesis. It is a committed step b. HMG-CoA reductase catalyzes the committed step and controls it because while there are many uses for acetyl CoA and HMG-CoA, there is only one use for mevalonate (cholesterol) so its synthesis must be very controlled. 10. List and explain in very general terms the four regulation strategies for HMG-CoA reductase. i. Controlled by sterols- If sterols are high, HMG-CoA is degraded to stop production of mevalonate ii. Hormonal control causes reversible covalent phosphorylation of HMG-CoA iii. Activity depends on the availability of substrate, HMG-CoA iv. Statins are competitive inhibitors of HMG-CoA reductase 1. Bind with affinity to the HMG-CoA reducatase active site and block the active site so HMG-CoA cant bind for reaction 11. State in general terms the relationship between LDL particles, LDL receptors on cells and cholesterol production by cells. a. LDL particles carry cholesterol to the peripheral tissues b. Cells contain LDL receptors that bind the LDL particles and bring them into the cell, where they dump their cholesterol cargo. c. Since HMG-CoA reductase is sensitive to the level of sterols (cholesterol) in cells, this will down regulate HMG-CoA reductase. d. This activates storage of excess cholesterol. It is reesterified to form cholesterol ester droplets in the cytosol. 12. Describe in general terms the function of the enzymes ALT and AST, and explain why they are considered markers of liver disease/damage. a. ALT: alanine aminotransferase—transfers an amino group from alanine to alpha ketoglutarate to generate glutamate, which carries the nitrogen to be disposed b. AST: aspartate aminotransferase—transfers an amino group from oxaloacetate to glutamate to generate alphaketolutarate c. Liver cells contain a much greater concentration of ALT and AST than most other cells. The appearance of ALT or AST in blood is a sign of cellular damage, and in particular, liver damage. i. Hepatitis ii. Chronic alcoholism 13. Describe in general terms the structure and function of creatine kinase, and explain why it is considered a marker of cardiac muscle disease / damage. a. Creatine kinase structure: consists of two subunits, B and M i. 3 isozymes of CK: 1. BB (brain) 2. BM (heart only) 3. MM (skeletal and cardiac) b. Creatine kinase function: catalyzes the reaction of creatine into creatine phosphate and uses ATP into ADP c. It is considered a marker of cardiac muscle disease/damage because there is usually a little CK in the blood due to normal cell turnover. However, when cells are damaged (heart attack or heart disease), there is more than the normal amount of CK in the blood. i. The MM isozyme is found in the heart, and so an elevated level of CK-BM and CK-MM in the blood is indicative of heart cell damage.