|Lecture Notes: 6 October||
Last time we stopped with:
KM = [S]([Et] - [ES]) / [ES] , where KM is the Michaelis-Menten constant.
Substituting for [ES]: vi/k3 = [Et] [S] / (KM + [S]),
and vi= k3[Et] [S] / (KM + [S]),
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].
Double Reciprocal or Lineweaver-Burke Plot: Need in form: y = ax + b , so take reciprocals of both sides and have
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.
In Competitive inhibition S & I are mutually exclusive, E can bind to one OR the other.
Plots: (text Box 6-2 figure 1)
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)
Classically assume binding to same site, but other possibilities also.
- steric hindrance between S & I in different sites.
- overlapping sites for S & I.
- Partial sharing of sites.
- Conformational change of enzyme with binding of either such that other can not bind.