Lecture 6-8: Enzyme kinetics

Question 1

what effects rate of enzymatic reactions

Answer 1

enzyme conc
substrate conc
effectors/inhibitors
temperature = collision rate

Question 2

why study enzyme kinetics

Answer 2

1. quantitive description of biocatalysis
2. determination of order of binding of substrates
3. elucidate acid-base catalysis
4. understand catalytic mechanism
5. find effective inhibitors
6. understand regulation of activity

Question 3

how to do kinetic measurements

Answer 3

1. purify enzyme + mix with substrate
2. record disappearance of substrate or product formation as a function of time - the velocity of the reaction
3. plot initial velocity (Vo) vs substrate concentration
4. change substrate concentration and repeat

Question 4

Effect of substrate concentration on Vo

Answer 4

Leonor Michaelis and Maud Menten

Michaelis-Menten plot
x-axis = conc of substrate
y axis = initial velocity Vo

10 or 12 time courses
skip dead time between mixing and start of experiment
where the graph decrease in a linear way = time to calculate initial velocity

1/2 Vmax
Vmax = theoretical maximal enzyme rate
1/2 Vmax
Km = substrate conc @ 1/2Vmax = Kd

Question 5

Vmax

Answer 5

theoretical maximal enzyme rate

never reaches graph asymptotes to it

Question 6

Km

Answer 6

measure of the affinity of the enzyme for the substrate

Km = substrate conc @ 1/2Vmax

Question 7

steady state MM Michealis-menten equation

Answer 7

rectangular hyperbola

Vo = (Vmax 8[S])/([Km]+[S])

Question 8

Saturation kinetics

Answer 8

at high [S]: Vo does not depend on [S] but on Vmax

Question 9

how did M&M get to the equation

Derivation of enzyme kinetics equations

Answer 9

1. start with a model equation
2. identify constraints and assumptions =

A. total enzyme concentration is constant
mass balance eq for enzyme
Etot = [E] + [ES}
enzyme total = free enzymes and enzymes bound to a substrate
Stot = [S] + [ES]
conc. of enzyme is 10 fold lower than substrate
B. steady state assumption
the rate of ES formation = rate of ES breakdown
C. enzyme and substrates are purifies
D. what is the actual measured rate
overall rate of product formation
Vnet = dP/dt = k[ES]
change in product of change over time

3. carry out algebra or graph theory for complex reactions

rate of product formation (V) = V = [ES]k3
rate of ES complex formed V=[E][S]K1
and breakdown = V = [ES}K2 + [ES]K3
therefore:

[E][S]K1= [ES}K2 + [ES]K3

4. simplest model mechanism
   one reactant one product no inhibitors