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 inthe 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 and on and on…

One solution – “Cardiac enzymes”

Enzymes

Definition: Biological catalysisQualities• Efficient• Specific

• Stereo-specific - they can tell the difference between isomers

• Regulated • Saturable• Inhibitable

Substrate versus product)(sproductsubstrate enzyme

2222 223

OOHOHFe

catalase

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…

MechanismEnzymes 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 rateEA 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

pH – alters enzyme structure by altering chargeTemperature – 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 directlyMetal ions – like magnesium in some ATPases.

Michaelis-Menten KineticsShows saturation at high substrate concentrationsVmax – rate at saturation for a given enzyme concentration in moles per unit timeKm – Michaelis constant – substrate concentration that gives ½ maximal velocity

SKSVV

m max

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 pH 8.9.”

Competitive inhibitorsMany drugs (like Cipro and anti-HIV drugs) are enzyme inhibitorsTwo 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?

Noncompetitive inhibitors

Noncompetitive inhibitors bind somewhere besides the active site.They alter the behavior of the enzyme in a manner analogous to allosteric regulationAlter Vmax.

What will happen to V ifyou push the substrateconcentration very high?

RegulationAllosteric 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 enzymeThese enzymes usually have catalytic and regulatorydomainsCan have multiple domainsor subunits for different regulators

RegulationAllostericCooperativity

• 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 cascadeAssociation-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 lowCreatine phosphate is regenerated when ATP is abundant

CrCrCr-PCr-P

ATPATPADPADP

CK

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?

Creatine kinase isozymesThe enzyme is dimeric Two different polypeptide chains (M and B) are differentially expressed in tissuesCombine 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 muscleCK-MB >5% of total CPK strongly suggests myocardial infarction

Total CK activity is determined by a simple enzyme assay (phosphocreatine + ADP ATP)CK-MB mass is determined by a two-antibody “sandwich” assay.

Determining CK-MB (mass) / CK (activity)

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anti-CK-B coated tube

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Tagged anti-CK-M

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POSITIVE

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Substrate