Enzymes and heart attacks
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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. Symptoms Angina pectoralis

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Enzymes and heart attacks

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Enzymes and heart attacks

Enzymes and heart attacks


Myocardial infarction

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 infarction1

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


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

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

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. 


Things for optimal activity

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.


Michaelis menten kinetics

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

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

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 inhibitors

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?


Noncompetitive inhibitors

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?


Regulation

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


Regulation1

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 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

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 isozymes

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

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


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