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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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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

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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

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Y

Y

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Y

Tagged anti-CK-M

anti-CK-B coated tube

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