Enzymes and heart attacks

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Enzymes and heart attacks
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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.

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Myocardial infarction

• One problem - Differential diagnosis
• Pericarditis
• Aortic Dissection
• Cholecystitis and Cholelithiasis
• Laryngeal spasm
• Anxiety attack
• and on and on and on
• One solution Cardiac enzymes

• Symptoms
• Angina pectoralis
• Dyspnea
• Nausea and/or abdominal pain
• Anxiety
• Lightheadedness and syncope
• Cough
• Nausea and vomiting
• Diaphoresis

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Enzymes

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

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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

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Mechanism

• Enzymes work by lowering activation energy
• If you dont 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. 

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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.

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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

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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.

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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 25C and pH 8.9.

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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?

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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?

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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

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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.

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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

ATP
ADP
CK
Cr
Cr-P
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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?

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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 gt5 of total CPK strongly suggests
  myocardial infarction

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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.

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Tagged anti-CK-M
anti-CK-B coated tube