Enzymes
1. Catalysis, co-enzymes, kinetics
2. Catalytic mechanisms, enzyme regulation
Catalyst: speeds up a chemical reaction without being changed by the reaction Enzymes are biological catalysts
Each enzyme catalyzes a specific reaction
Most enzymes are made of protein (except for a few RNA enzymes)
Some enzymes require cofactors for activity
Some enzymes require cofactors for activity
Enzyme Commission classification: http://www.chem.qmul.ac.uk/iubmb/enzyme/
Enzymes catalyze reactions by providing a special environment for the reaction. The reaction takes place at a cavity or groove on the enzyme surface called the active site.
Reactants that bind to the active site are called substrates.
Active site is a cavity or groove on enzyme surface
In a reaction where S (substrate) goes to form P (product):
If enzyme E catalyzes this reaction:
E +S
ES
EP
E +P
the catalyzed reaction reaches equilibrium faster but the equilibrium between S and P is not changed.
Substrate and product are separated by a barrier: the transition state activation energy G
Catalysts speed up reactions by lowering the activation energy barrier
Go depends only on beginning and ending states, not pathway
Reaction of glucose with oxygen to form CO2 and H2O has the same Go whether in living cells (enzymes lower the activation energy) or in burning sugar cane fields (fire supplies the activation energy)
Equilibria and thermodynamics
S P
Keq
[P] _____ = [S]
equilibrium constant
Go = - R T ln Keq
substrate Kinetics: for a simple reaction, S
product
V = k [S]
molar concentration of A
Reaction rate ("velocity") V is measured as:
1) amount of S disappearing in a period of time or 2) amount of P appearing in a period of time
Proprotionality constant k is rate constant
Active site stabilizes transition state.
Binding energy GB lowers activation energy and contributes to enzyme specificity
Active sites
3-D cleft or crevice Side chains from different parts of sequence Relatively small part of whole enzyme surface Mostly non-polar, except for side chains that participate in catalysis Substrate binds using many relatively weak interactions Specificity results from these interactions
Contributions of active site to catalysis:
Entropy reduction: holds reacting groups still Desolvation: removes water from reacting groups Charge redistribution: stabilizes transient changes Induced fit: enzyme (or substrate) changes shape to fit transition state
Catalytic strategies
Covalent catalysis Covalent modification of enzyme by substrate General acid-base catalysis Proton donor or acceptor other than water Metal ion catalysis Charge effects on enzyme or substrate; stabilization of ES complex
Example of both covalent and general acid-base catalysis:
Evidence for active sites from measurement of reaction velocity, v
S
Substrate
P
Product
Product
initial velocity = v = P/t
equilibrium
Time
More substrate causes faster initial velocity
Product
S8 S 7 S6
increasing substrate
S5 S4 S3 S2 S1
Time
Reaction rate saturates at a maximum velocity
Vmax
S9
S1
Saturation at Vmax suggests formation of ES complex
ES complex proposed by Michaelis & Menten, 1913
Explains the V vs. S saturation curve
k1 E +S k-1 ES
k2
E +P
enzyme-substrate complex
Does the Michaelis-Menten kinetic model correctly predict the shape of the V vs. S saturation curve?
k1
E +S k-1 ES
k2
E +P
V = velocity of reaction = rate of appearance of P
V = k2 [ES]
Steady-state assumption: rate of formation of ES equals rate of breakdown of ES
k1
E +S
k-1
ES
k2
E +P
In steady-state:
rate of ES formation = rate of ES breakdown
k1 [E] [S] = k-1 [ES] + k2 [ES]
In steady-state: rate of ES formation = rate of ES breakdown
k1 [E] [S] = k-1 [ES] + k2 [ES] k1 [E] [S] = [ES] (k-1 + k2) k-1 + k2 [E] [S] ________ ________ = = KM [ES] k1
In steady-state: rate of ES formation = rate of ES breakdown
k1 [E] [S] = k-1 [ES] + k2 [ES] k1 [E] [S] = [ES] (k-1 + k2)
Michaelis constant
k-1 + k2 [E] [S] ________ ________ = = KM [ES] k1
Rearrange equation for KM and plug into rate equation to get equation for V in terms of [S]:
[E] [S] ________ [ES] = KM V = k2 [ES] [E] [S] V = k2 [ES] = k2 ________ KM
Rearrange equation for KM and plug into rate equation to get equation for V in terms of [S]:
[E] [S] ________ [ES] = KM V = k2 [ES] [E] [S] V = k2 [ES] = k2 ________ KM
Not very useful: we dont know [E]!
But we do know the total enzyme:
total enzyme = [E]T = [E] + [ES] [E] [S] [E]T = [E] + [ES] = [E] + ________ KM [E]T __________ = [E] 1 + [S] / KM
[E] [S] V = k2 [ES] = k2 ________ KM [E]T __________ = [E] 1 + [S] / KM [E]T [S] / KM V = k2 ____________ 1 + [S] / KM
(useless)
(extremely valuable!)
[E]T [S] / KM V = k2 ____________ 1 + [S] / KM [S] V = k2 [E]T _________ KM + [S]
What is the maximum velocity?
When every active site is filled with substrate, Vmax = k2 [ES]max = k2 [E]T
V = k2 [E]T
[S] ________ KM + [S]
V = Vmax
[S] _________ KM + [S]
What is KM?
KM is the concentration of substrate at which half the active sites are filled
If k-1 >> k2, KM is the dissociation constant for ES
k1 E +S ES k2 E +P
k-1
k-1 + k2 ________ = KM k1
What is Vmax?
Vmax is the rate of product formation at a high substrate concentration, where all the active sites are filled with substrate The turnover number of the enzyme, kcat, is the number of substrate molecules converted to product in a unit of time when the active site is saturated with substrate kcat = Vmax / [E]T
How good can an enzyme be?
[S] V = kcat [E]T _________ KM + [S]
At low substrate concentrations, KM >> [S]
kcat V = __ [E]T [S] KM
kcat/KM is approximately the rate constant at low [S]
