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Enzyme Kinetics I

Quick guide focusing on mathematical understanding (other parts not discussed in any length)

Review- describing change in math


To signify a change, the operator d (or one of the greek equivalents or ) is used. This can be read as change in. For example, dV means change in V. Because change happens over time, it is usually expressed with a dT denominator For example, dV/dT means change in v over change in time. If v changes by +6 units over 3 seconds, dV/dT= +2 units/second Any such change in a value over time is a rate

First/Second Order Reactions


First order reaction: the concentration of one reactant is multiplied by the rate constant to determine the rate of formation of product. Eg- the decay of the enzyme-substrate complex into enzyme and product:

+
= [].
d[] ,

Here,

d[]

(the rate in this example, velocity: v = [])

is also known as

Second Order Reactions


Second order reaction: the concentration of both reactants are multiplied together and then multiplied by the rate constant, in order to determine the rate of formation of product. Eg.- the formation of the ES complex from enzyme and substrate + So the formation of ES (ignoring for the moment the ways the ES complex degrades) is shown by
d[] 1

= 1 [].

Kd
The formation of the ES complex can also be described as the association of the enzyme and substrate. This has a reverse reaction which we havent yet discussed, the dissociation of the ES complex into individual enzyme and substrate molecules

(We wont discuss the decay of ES into E & P here). Change in [ES] includes two terms: one positive, for the formation of the complex (a 2nd order reaction), and one negative, for the dissociation of the complex (a 1st order reaction).

= 1 1 []

Kd cont
Kd is a way of expressing the balance of these reactions, defined as the rate constant of dissociation (k-1 above) divided by the rate constant of association (k1) How is this value experimentally determined? At equilibrium, there is no overall change in concentration of ES over time, because the forward and reverse reactions are in balance. = 1 1 = 0 (at equilibrium). So 1 = 1

Rearrange to get 1 /1 =

Same as the slide Simple kinetic definitions.

[] []

Transition State Theory


Involves modeling a catalyzed reaction using pseudo-first order kinetics. This leads us to the clusterfuck equation on the first page of 2 titled Transition state theory- the meaning of the rate constant. Firstly, v (velocity) = []/ (formation of product over time).

If = 1 [] (first order kinetics). Therefore v = 1 []

then

TST, cont.
expands upon and includes the transition state ( ) in the reaction equation. This gives us another way to express the velocity of the reaction: v = [ ]. We can also express the equilibrium constant (not a Kd) of the part of the reaction: = /[]
1

TST, cont. cont.


So we can substitute [] for in the equation v = and get v = [] From the text on the top right of the slide, we can get an equation for . With a commitment factor k of 1 (standard for simple reactions of the kind we will examine), then = (vibrational frequency, not velocity). So = kT/h (k=Boltzmann constant, T=temp K, h = Planck constant)

TST cont. cont. cont.


So, using v = [] and substituting kT/h for , we get: v = (kT/h) [] And because = , we can substitute / for to get v = (kT/h) [] Then since v = 1 [] (top equation in list) we can substitute for v in the above equation, and [S] terms drop out to give: 1 = (kT/h)
/

What use is that?


Assuming T is constant (say 298 K) We can get rid of all constants (k,T,h,R) in formula simply by replacing equals symbol with is proportional to (symbol: ) / 1 = (kT/h) becomes 1 As increases, rate of reaction decreases (as this graph -> of shape y=e-x shows).

The second page titled Transition State Theory- the meaning of the
Now, lets look at a similar.

1 = This is a formulation of the Arrhenius equation. A is not an amount in this equation. Its something called the pre-exponential factor or frequence factor. Has to do with number of collisions of molecules in solution of reaction. This value is usually defined experimentally.

Magnitude of Rate Acceleration


Arrhenius again. = is the number of catalytic collisions of molecules A is the overall number of collisions of molecules / is the probability that a collision will result in catalysis. There is a tenfold increase in rate for every decrease of 1.36 kcal/mol in . If is -1.36 velocity is 10x that of uncatalysed rxn If is -2.72 velocity is 100x that of uncatalysed, etc. Catalysed reactions generally 107 to 1014 times faster than uncatalysed. Be prepared to use the second equation on the corresponding page of notes to determine the magnitude of rate acceleration (kcat/kuncat)
/

Steady-state kinetic analysis the Michaelis-Menten equation


At steady state formation and decay of ES are equal, described by the formula 1 ][ = 1 + 1 ][ = (1 + )
[] []

1 + 1

= (by definition).

Steady-state kinetic analysis the Michaelis-Menten equation cont.


= =
[] []

= +
( ][ )[] []

[] = [] []( +[]) = [ ][] [] =


[ ][] +[]

Steady-state kinetic analysis the Michaelis-Menten equation cont. cont.


[] =
[ ][] +[]

v0 = [], therefore = v0/ v0 = v0 =


[ ][] +[] [] +[]

Vmax = [ ] This is the Michaelis-Menten equation.

Notes on Kcat and Km


Kcat is the catalytic constant, representing the maximum velocity of the enzyme when there is unlimited substrate. High Kcat = more efficient enzyme Km is a more nuanced guide to activity- the substrate concentration that gives Vmax. Low Km = more efficient enzyme These can be combined, Kcat/Km is the specificity constant- the gold standard of enzyme activity.