Post on 06-Feb-2016
description
Enzymes and heart attacks
2009-2010
Enzymes:
“Helper” Protein molecules
Flow of energy through life
Life is built on chemical reactions
Chemical reactions of life
Processes of life• building molecules
• synthesis
• breaking down molecules
• digestion
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Nothing works without enzymes!
How important are enzymes?• all chemical reactions in living organisms require
enzymes to work
• building molecules
• synthesis enzymes
• breaking down molecules
• digestive enzymes
• enzymes speed up reactions
• “catalysts”
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enzyme
enzymeWe can’t live without enzymes!
Examples
synthesis
digestion
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enzyme
enzyme
Enzymes are proteins
Each enzyme is the specific helper to a specific reaction• each enzyme needs to be the right shape for the job
• enzymes are named for the reaction they help
• sucrase breaks down sucrose
• proteases breakdown proteins
• lipases breakdown lipids
• DNA polymerase builds DNA
Oh, I get it!They end in -ase
Enzymes aren’t used up
Enzymes are not changed by the reaction• used only temporarily• re-used again for the same reaction with other molecules• very little enzyme needed to help in many reactions
enzyme
substrate product
active site
It’s shape that matters!
Lock & Key model• shape of protein allows
enzyme & substrate to fit
• specific enzyme for each specific reaction
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Enzyme vocabulary
Enzyme• helper protein molecule
Substrate• molecule that enzymes work on
Products• what the enzyme helps produce from the reaction
Active site• part of enzyme
that substrate molecule fits into
What affects enzyme action
Correct protein structure• correct order of amino acids
• why? enzyme has to be right shape
Temperature• why? enzyme has to be right shape
pH (acids & bases)• why? enzyme has to be right shape
Order of amino acids
Wrong order = wrong shape = can’t do its job!
DNA
DNA
chain ofamino acids
chain ofamino acids
foldedprotein
foldedprotein
right shape!
wrong shape!
Temperature
Effect on rates of enzyme activity• Optimum temperature
• greatest number of collisions between enzyme & substrate
• human enzymes • 35°- 40°C (body temp = 37°C)
• Raise temperature (boiling)
• denature protein = unfold = lose shape• Lower temperature T°
• molecules move slower • fewer collisions between enzyme & substrate
37°
Temperature
temperature
reac
tion
rat
eWhat’s happening here?!
humanenzymes
How do cold-blooded creatures do it?
pH Effect on rates of enzyme activity• changes in pH changes protein shape
• most human enzymes = pH 6-8
• depends on where in body
• pepsin (stomach) = pH 3
• trypsin (small intestines) = pH 8
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pH
pH
reac
tion
rat
e
20 1 3 4 5 6 8 9 10
stomachpepsin
intestinestrypsin
What’s happening here?!
11 12 13 14
2009-2010
For enzymes…What matters?
SHAPE!
2009-2010
Let’s build some
Enzyme Models!
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 catalysis
Qualities• 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…
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
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
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
SK
SVV
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 inhibitors
Many 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 regulation
Alter 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
Regulation
AllostericCooperativity
• 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
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 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
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