Enzymes and Kinetics Objectives

Enzymes
What is catalytic power? How do you calculate it?
Substrate Speficity
Lock and key model
Induced fit model
Stereospecificity
Be able to determine the pro R and pro S hydrogen of prochiral molecules.
What are Cofactors? Coenzymes? Prosthetic Groups?
What is a holoenzyme? Apoenzyme?
Be able to interpret Reaction coordinate diagrams. Know what the activation energy is. Free energy
changes between substrates and products. Identify intermediates and transition states, the rate
determining step.
Know how enzymes increase the rates of the reaction.
Why is it bad to bind the substrate too tightly.
Why do enzymes bind the transition state tightly?
Know what transition state analogs are?
Enzymes Catalytic power
Transition state binding/stabilization.
Proximity effects

Kinetics
Know the stoichiometric rate equation.
What the elementary steps are.
The order of a reaction
The molecularity of the elementary steps.
Reaction Rates
The change in concentration of reactant or product as a function of time.
Zero-order.
Rate equation
1st order
rate equation
Enzyme Kinetics

Be able to write rate equations for enzyme intermediate concentrations

k1
E +S

ES

k3

E+P

k2
d[E ]
k2[ ES ] k3[ ES ] k1[ E ][ S ]
dt
d [ ES ]
k1[ E ][ S ] k2[ ES ] k3[ ES ]
dt
d [ P]
k3[ ES ]
dt
Know the steady state assumption.
The rapid equilibrium assumption.
The end result the Michealis Menten Equation.
Know this equation And the meaning of its parameters.

Understanding the Km
The "kinetic activator constant"
Km is a constant
Km is a constant derived from rate constants
Km is, under true rapid equilibrium conditions, equal to the dissociation constant of E from S , Ks
Small Km means tight binding; high Km means weak binding
Understanding Vmax
The theoretical maximal velocity
Vmax is a constant
Vmax is the theoretical maximal rate of the reaction - but it is NEVER achieved in reality
To reach Vmax would require that ALL enzyme molecules are tightly bound with substrate
Vmax is asymptotically approached as substrate is increased

The dual nature of the Michaelis-Menten equation

Combination of 0-order and 1st-order kinetics
When S is low, the equation for rate is 1st order in S
When S is high, the equation for rate is 0-order in S
The Michaelis-Menten equation describes a rectangular hyperbolic dependence of v on S!
The turnover number
A measure of catalytic activity
kcat, the turnover number, is the number of substrate molecules converted to product per enzyme
molecule per unit of time, when E is saturated with substrate.
If the M-M model fits, k2 = kcat = Vmax/Et
Values of kcat range from less than 1/sec to many millions per sec
The catalytic efficiency
Name for kcat/Km
An estimate of "how perfect" the enzyme is
kcat/Km is an apparent second-order rate constant
It measures how the enzyme performs when S is low
The upper limit for kcat/Km is the diffusion limit - the rate at which E and S diffuse together

Know the Lineweaver-Burk Plot. The equation describing the double riprocal plot. How to
extrapolate a Km and Vmax.
Know the four types of reversible enzyme inhibition.
Their patterns in the Lineweaver Burk plots.
Know how the different types of inhibitors effect the slope, y-intercept, x-intercept.

You should be able to take a data set, do the lineweaver burk plot, extrapolate the Km and Vmax,

Given an inhibitor, You should be able to take a data set, do the lineweaver burk plot, extrapolate
the Km and Vmax for the uninhibited enzyme, Identify which type of inhibitor by the inhibition
pattern and determine the Ki.

Bisubstrate reactions
Know the Cleland notation terminology.
Know mechanism of sequential ordered Bi Bi and random Bi Bi
Mechanism of Ping Pong
How to distinguish between ping pong and sequential mechanism by analyzing the lineweaver-burk plots
of 1/v versus 1/[A] at constant [B] and 1/v versus 1/[B] at constant [A].
Irreversible inhibition.

Enzyme Regulation
Know the mechanisms of enzyme regulation.
Availability
Proteolysis
Reversible covalent modification
Allosteric regulation
Cooperativity and substrate binding
Shape of allosteric enzymes kinetic curves.
The Hill equation

Catalytic mechanisms
Acid-base catalysis
Know difference between specific acid-base catalysis and general acid-base catalysis.
pH profiles for general acid-base catalysis.
Know the mechanism of RNAase. The roles of the two histidines. The intermediate in the enzyme
catalyzed reaction. Why RNAase doesnt hydrolyze DNA.
Covalent Catalysis
Nucleophilic catalysis
Mechanism of Shiff base formation.
Metal Ion Catalysis
Difference between metalloenzymes and metal activated enzymes.
Electrophilic catalysis.
Know the mechanism of carbonic anhydrase. How a coordinated Zinc lowers the pKa of a
water molecule.
Know mechanism of Serine proteases? Intermediates. Nucleophiles, general acid-bases.
Know the roles of the catalytic triad, oxyanion hole, specificity pocket. Know the specificity of
trypsin, chymotrypsin and elastase.
Know what an affinity label is?
Know what a zymogen is. How they are activated.