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Methods for Studying Enzymatic Reactions 59
(a) Competitive inhibition
E + S ES E + P
K i
+ I
EI
1/v
inc. [I]
K M(app) = K M
1 + [I]
1/v max
unchanged
K i
1/[S]
(b) Non-competitive inhibition
E + S
ES
E + P
K i + I
EI + S
K i
+ I
EIS
inc. [I]
1/v
v max(app) = v max
1 + [I]
K i
−1/K M unchanged
1/[S]
Figure 4.6 Reversible inhibition.
All of the above kinetic data can be obtained by steady state kinetics, which
can be observed conveniently over a 1–10-minute assay period. However, if one
is able to examine an enzymatic reaction before the attainment of steady state,
then individual enzymatic rate constants can be measured directly using presteady-state
kinetics. If the turnover number for an enzyme is 10 s 1 then under
saturating conditions a molecule of substrate will be converted to product in
0.1 s. Therefore, in order to examine a single catalytic cycle one must examine
the enzymatic reaction in the range 0–100 ms. This can be done using a stopped
Xow apparatus, shown in simpliWed form in Figure 4.8.