Enzymes 2 - kinetics Spencer Flashcards
chemical kinetics is the study of…
rates of chemical reactions
enzyme kinetics is the study of…
rates of enzyme catalyzed reactions
A -> P
what is the rate v
what is the order
what are the units of the rate constant
v = ∆P / dt = -∆A / dt = k[A]
1st order rxn
k = s^-1
what are the units of reaction rate, or velocity v
M/s
moles per second
A + B -> P
what is the rate v
what is the order
what are the units of the rate constant
v = ∆P/dt = -∆A/dt = -∆B/dt = k[A][B] 2nd order (1st in A, 1st in B) k = M^-1 s^-1
A = P what is the rate v what is the order what are the units of the rate constant equilibrium constant
v = ∆P / dt = -∆A / dt = k1[A] - k2[P]
1st order rxn
k = s^-1
Keq = k1/k2 = [P]/[A]
what is the equilibrium constant for a reversible reaction
Keq = [products]/[reactants]
= kforward/kreverse
for 1-substrate enzyme kinetics, what do the following graphs look like
[P] vs t
v0 vs [S]
- [P] vs t is hyperbolic, with slope (v0) initially linear, and greater [P] plateaus with greater [S]initial
- v0 vs [S] is hyperbolic
what is the general reaction equation for michelis-menten kinetics
k1 k3
E + S ES -> E + P
k2
what is the michelis constant
Km = (k2+k3)/k1
when [S] = Km, v0 = .5Vm
michelis menten equation
v0 = Vm [S] / Km + [S]
what are 5 assumptions of michelis-menten kinetics
- t = 0 (initial velocities only)
- k4 = 0 at t ~ 0 (no back reaction from P)
- negligible S depletion [S]»_space; [E]
- ES complex formed
- [ES] is at steady state
[S]»_space; Km, v0 =
v0 = Vm = k3[E]t
all active sites saturated
zero order… v0 independent of S
[S] = Km, v0 =
v0 = 1/2 Vm
50% active sites filled
[S] «_space;Km, v0 =
v0 = VmS / Km = k3EtS / Km
v0 is proportional to [S]
Vm =
k3[E]t
all active sites saturated
zero order… v0 independent of S
what are the units of a first order rate constant
s^-1
what are the units of a second order rate constant
M^-1 s^-1
michelis-menten Kd =
k2/k1 = [reactants]/[products] = [E][S]/[ES]
Km = Kd when…
k2»_space; k3
how can you increase Vm?
increase [E]t
Vm = k3Et
mechelis-menten kcat =
k3
catalytic ability of the enzyme
michelis-menten turnover number =
kcat = k3
catalytic ability of the enzyme
the catalytic ability of a michelis-menten enzyme =
kcat = k3 = turnover number
catalytic efficiency =
kcat / Km
~ how well enzyme reacts with substrate
Km is a measure of…
the strength of the ES interaction
what is the catalytic efficiency of a “perfect enzyme” ?
10^8 to 10^9
limited only by rate of diffusion
what does the v0 vs [S] plot look like for a michelis-menten enzyme?
hyperbolic
how can you linearize the michelis-menten equaiton?
take the inverse
1/v0 = Km/Vm x 1/[S] + 1/Vm
lineweaver-burke equation
1/v0 = Km/Vm x 1/[S] + 1/Vm
y-axis on lineweaver-burke plot =
1/v0
x-axis on lineweaver-burke plot =
1/[S]
slope on lineweaver-burke plot =
Km/Vm
y-intercept on lineweaver-burke plot =
1/Vm
x-intercept on lineweaver-burke plot =
-1/Km
reactions with same lineweaver-burke slope…
have same Km/Vm (same catalytic efficiency)
how can you tell catalytic efficiency from a lineweaver-burke plot?
smaller slope = greater catalytic efficiency
slope = Km/Vm ~ the inverse of kcat / Vm
reactions with same lineweaver-burke x-intercept
same Km
reactions with same lineweaver-burke y-intercept
same Vm
advantage of lineweaver-burke plot
can easily compare Vm, Km, and Km/Vm
lineweaver-burke plot does not work well for…
low or high [S]
distorts errors
if 2 enzymes have same catalytic efficiency kcat/Km, do you pick the one with higher kcat or lower Km?
the one with higher kcat (don’t know why, spencer just says)
2 mechanisms of bi-substrate reactions
sequential
ping-pong
2 types of sequential bi-substrate reactions
ordered sequential
random sequential
how does a sequential bi-substrate reaction work?
all substrates are bound before any products are released
how does an ordered sequential bi-substrate reaction work?
E always binds substrate A, then B, then transformation occurs (EAB -> EPQ), then release of P, then Q, left with E
how many rate constants are involved in a sequential bi-substrate reaciton?
10 (5 forward, 5 back)
k1 through 5 and k-1 through -5
howehow does a random sequential bi-substrate reaction work?
E can bind A or B first and release P or Q first, order does not matter
how does a ping-pong bi-substrate reaction work?
E + A = EA = FP = F + P
F + B = FB = EQ = E + Q
which bi-substrate reaction mechanism is characterized by a substituded enzyme intermediate, sequential or ping-pong mechanism?
ping-pong E +A = EA = FP = F + P F + B = FB = EQ = E + Q vs E+A = EA+B = EAB = EPQ = EQ+P = E+Q
what is the catalytic efficiency of a “perfect enzyme” ?
k1
because
Km perfect = kcat/k1 (no k2)
so k1 = kcat/Km = catalytic efficiency
what are the units of Km?
M
in kinetics, what is the difference between K and k?
K units are M = [products] / [reactants]
k units are s^-1 or M^-1 s^-1 etc = rate
what are the units of kcat?
s^-1
what are the units of Ki?
M
what are the units of k1?
M^-1 s^-1
