Enzymes and heart attacks
Enzymes:
“Helper” Protein molecules
2009-2010
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
+
Nothing works without enzymes!
How important are enzymes?
• all chemical reactions in living organisms require
enzymes to work
• building molecules
enzyme
• synthesis enzymes
+
• breaking down molecules
• digestive enzymes
We can’t live
without enzymes!
• enzymes speed up reactions
• “catalysts”
enzyme
+
Examples
 synthesis
+
enzyme
 digestion
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
Oh, I get it!
• lipases breakdown lipids
They end
in -ase
• DNA polymerase builds DNA
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
substrate
active site
product
enzyme
It’s shape that matters!
Lock & Key model
• shape of protein allows
enzyme & substrate to
fit
• specific enzyme for
each specific reaction
2
1
3
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!
chain of
amino acids
DNA
folded
protein
right shape!
folded
protein
chain of
amino acids
DNA
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
Temperature
reaction rate
human
enzymes
37°
temperature
What’s
happening
here?!
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
pH
intestines
trypsin
What’s
happening
here?!
reaction rate
stomach
pepsin
0
1
2
3
4
5
6
pH
7
8
9
10
11
12
13
14
For enzymes…
What matters?
SHAPE!
2009-2010
Let’s build some
Enzyme Models!
2009-2010
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.
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
Fe3
2 H 2O2  2 H 2O  O2
• Efficient
catalase
• Specific
• Stereo-specific - they can tell the difference between
isomers
• Regulated
• Saturable
• Inhibitable
substrate enzyme
 product (s)
Substrate versus product
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
Vmax S 
V
K m  S 
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 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 if
you push the substrate
concentration 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 if
you push the substrate
concentration very high?
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 regulatory
domains
Can have multiple domains
or subunits for different
regulators
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 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
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
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)
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 Y
Y Y Y Y Y
Substrate
Y Y Y Y Y
Tagged anti-CK-M
Y Y Y Y Y
anti-CK-B coated tube
Y Y
Y Y Y Y Y
Y Y Y Y Y
Y Y Y Y Y
Y
POSITIVE