Humboldt State University ® Department of Chemistry

Richard A. Paselk

Chem 431

Biochemistry

Fall 2008

Lecture Notes: 6 October

© R. Paselk 2008
 
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Enzyme Kinetics, cont.

Last time we stopped with:

KM = [S]([Et] - [ES]) / [ES] , where KM is the Michaelis-Menten constant.

Now a couple of tricks: Solve for [ES]: [ES] = [Et] [S] / (KM + [S])

and recall that k3[Et] = Vmax and therefore vi = k3[ES], and dividing both sides by k3 we get vi/k3 = [ES].

Substituting for [ES]: vi/k3 = [Et] [S] / (KM + [S]),

and vi= k3[Et] [S] / (KM + [S]),

But maximum possible velocity must = k3[Et] = Vmax

So, two forms of the M-M equation Which is known as the Michaelis-Menten Equation.

For simple, one-substrate enzymes then, have Michaelis-Menten Equation as a model for enzyme activity.

two forms of the M-M equation

Note predicted consequences of model:

 

M-M kinetics plot

Turnover Number

The rate constant (First order) for the breakdown of the [ES] complex, kcat (k3), is also known as the turnover number, that is the maximum number of substrate molecules processed/active site (moles substrate/mole active site): kcat=Vmax / [E]total. Note that this is best determined under saturating conditions. (text Table 6-08) At very low concentrations of [S] can find the second-order rate constant for the conversion of E + S E + P: vo = (kcat / KM)([E][S].

Linear plots for enzyme kinetic studies

Double Reciprocal or Lineweaver-Burke Plot: Need in form: y = ax + b , so take reciprocals of both sides and have

. (text Box 6-01 figure 6-1)

L-B double reciprocal plot for enzyme kinetics

Other linear plots are also available, and are better in terms of statistics (L-B one of worst, best quality points [high concentration] have least influence on slope, while low precision points [low concentration] are more spread out, and have a large moment, with a strong influence on the slope and the KM intercept ­ this is not as much of a concern now with computer statistical packages, but you still have to understand the statistics). We will see others in the laboratory discussion.

FYI - The Eadie-Hofstee Plot

One common plot is shown below. Note that the data points are distributed much more evenly over the plot giving better statistics for the slope. In addition the value of KM is obtained from the slope, giving better precision.

Eadie-Hofstee plot

Enzyme Kinetics and Inhibition

What's exciting about enzyme inhibition?

Three major types of inhibition:

Competitive Inhibition:

In Competitive inhibition S & I are mutually exclusive, E can bind to one OR the other.

Plots: (text Box 6-2 figure 1)

M-M plot for competitive inhibition

We can model this inhibition with chemical equations, keeping in mind that S & I are mutually exclusive, E can bind to one OR the other: (text Figure 6-15a)

 

Model: competitive inhibition equilibria model; and: MM equation for competitive inhibition; where equilibrium expression for inhibitor binding, Ki.

Classically assume binding to same site, but other possibilities also.

  1. steric hindrance between S & I in different sites.
  2. overlapping sites for S & I.
  3. Partial sharing of sites.
  4. Conformational change of enzyme with binding of either such that other can not bind. 

 


Pathway Diagrams

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Last modified 7 October 2008