Enzymes and heart attacks

1. Enzymes and heart attacks

2. Myocardial infarction • Acute myocardial infarction is the rapid development of myocardial necrosis caused by a critical imbalance between the oxygen supply and demand of the myocardium. • 500,000-700,000 deaths inthe US annually.

3. Symptoms Angina pectoralis Dyspnea Nausea and/or abdominal pain Anxiety Lightheadedness and syncope Cough Nausea and vomiting Diaphoresis One problem - Differential diagnosis Pericarditis Aortic Dissection Cholecystitis and Cholelithiasis Laryngeal spasm Anxiety attack and on and on and on… One solution – “Cardiac enzymes” Myocardial infarction

4. Enzymes • Definition: Biological catalysis • Qualities • Efficient • Specific • Stereo-specific - they can tell the difference between isomers • Regulated • Saturable • Inhibitable • Substrate versus product

5. Types of enzymes • All enzymes end in the suffix “_______ase” • Different versions of the same enzyme (often made by alternative splicing) are called isoenzymes or isozymes • General classes of enzymes • Polymerases – nucleic acid synthesis • Transferases – transfer a functional group • Hydrolases – hydrolytic cleavage • Proteases – hydrolytic cleavage of protein chains • Kinases – add phosphate groups to compounds • … and many, many more…

6. Mechanism • Enzymes work by lowering activation energy • If you don’t understand free energy changes, see Box 5A in your book • ∆G is a measure of the ability of a reaction to go forward, but not necessarily the rate • EA is the activation energy. • The rate at which a reaction proceeds is directly proportional to the number of molecules reaching the transition state - that is, those that reach EA. 

7. Things for optimal activity • pH – alters enzyme structure by altering charge • Temperature – increases activity by moving molecules closer to the activation energy, and by making ∆G slightly more negative… until the enzyme "denatures" • Coenzymes – like biotin in amino group transfer – bind reversibly but participate directly • Metal ions – like magnesium in some ATPases.

8. Michaelis-Menten Kinetics • Shows saturation at high substrate concentrations • Vmax – rate at saturation for a given enzyme concentration in moles per unit time • Km – Michaelis constant – substrate concentration that gives ½ maximal velocity

9. How do you measure this crap? • Things you need: • The enzyme • The substrate • A way of measuring either the disappearance of substrate, or the appearance of product, usually photometrically.

10. Other commonly reported values • Turnover • rate at saturation for 1 enzyme molecule (reactions catalyzed per second per molecule) • “Units” • are defined by convention, but are something of an industry standard.  For example… • “One unit of creatine kinase is defined as the amount necessary to catalyze the conversion of one micromole of creatine to creatine phosphate per minute at 25°C and pH 8.9.”

11. Competitive inhibitors • Many drugs (like Cipro and anti-HIV drugs) are enzyme inhibitors • Two major kinds of inhibitors: competitive and noncompetitive. • Competitive inhibitors bind to the active site of the enzyme. • Alter Km but not Vmax. • What will happen to V ifyou push the substrateconcentration very high?

12. Noncompetitive inhibitors • Noncompetitive inhibitors bind somewhere besides the active site. • They alter the behavior of the enzyme in a manner analogous to allosteric regulation • Alter Vmax. • What will happen to V ifyou push the substrateconcentration very high?

13. Regulation Allosteric regulation • A regulatory molecule binds to a site separate from the active site (like small molecules to repressors in operons) • Induced conformational changes regulate the activity of the enzyme • These enzymes usually have catalytic and regulatorydomains • Can have multiple domainsor subunits for different regulators

14. Regulation Allosteric Cooperativity • One substrate aids or impedes the catalysis of another • Implies multiple catalytic subunits. Covalent modification • Adding/removing groups – like phosphate groups by kinases • Cleaving bonds – converting proenzymes to enzymes - like in the blood clotting cascade Association-dissociation of subunits • One protein binds to another, thereby activating the enzymatic activity of one of them.

15. Creatine kinase • Creatine phosphate acts as a backup for rapid ATP regeneration in active tissues • Creatine phosphate is in energetic equilibrium with ATP • Creatine kinase (CK) catalyzes the transfer of phosphate between creatine and ATP/ADP • Provides rapid regeneration of ATP when ATP is low • Creatine phosphate is regenerated when ATP is abundant ADP ATP CK Cr-P Cr

16. Application: Cardiac enzymes • enzymes released from injured myocardium. • Creatine kinase (CK) is the one usually assayed • If CK is found in the blood stream, this implies that the myocardium may have been damaged • Problems: • Tells you little about the time course or severity • Lets you spot really small infarcts. • What else?

17. Creatine kinase isozymes • The enzyme is dimeric • Two different polypeptide chains (M and B) are differentially expressed in tissues • Combine at random to give three isozymes: • CK-MM (primarily muscle) • CK-MB (hybrid) • CK-BB (primarily brain) • The CK-MB has its highest concentration in heart muscle • CK-MB >5% of total CPK strongly suggests myocardial infarction

18. Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y POSITIVE Substrate Determining CK-MB (mass) / CK (activity) • Total CK activity is determined by a simple enzyme assay (phosphocreatine + ADP  ATP) • CK-MB mass is determined by a two-antibody “sandwich” assay. Y Y Y Y Y Y Y Y Y Y Tagged anti-CK-M anti-CK-B coated tube