Enzymes and heart attacks Myocardial infarction Acute myocardial

Enzymes and heart attacks

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 in the US annually.

Myocardial infarction 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… One solution – “Cardiac enzymes”

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

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…

Mechanism Enzymes work by lowering activation energy • If you don’t understand free energy changes, see Box 5 A 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.

Things for optimal activity p. H – 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.

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

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.

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 p. H 8. 9. ”

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 if you push the substrate concentration very high?

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 if you push the substrate concentration very high?

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 regulatory domains Can have multiple domains or subunits for different regulators

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.

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

Application: Cardiac 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?

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

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 Y Y Y Y anti-CK-B coated tube Tagged anti-CK-M Substrate POSITIVE