Introduction to Enzyme and Coenzyme Chemistry - E-Library Home

58 Chapter 4
Lineweaver−Burk Plot:
Eadie−Hofstee Plot:
1 1 K M 1
= v +
v = v
v max v max − K M v
max [S]
[S]
1/v
v
v max
slope −K M
slope K M /v max
1/v max
1/[S]
Figure 4.5 Graphical methods for determination of K M and k cat . v, reaction velocity; [S], substrate
concentration.
v/[S]
observed for competitive inhibition is shown in Figure 4.6. If the rate of the
enzyme reaction is measured at varying substrate concentrations but Wxed
inhibitor concentrations, apparent K M values ((K M ) app ) can be measured at
varying inhibitor concentrations. If plotted on a Lineweaver–Burk plot a series
of straight lines are obtained, intersecting on the y-axis. Thus the v max is
unaVected by competitive inhibition, since at high substrate concentrations
the substrate can competitively displace the inhibitor.
Non-competitive reversible inhibition is observed when an inhibitor,
I, binds to another part of the enzyme active site leading to a non-productive
EIS complex. This type of inhibition, illustrated in Figure 4.6, typically occurs
in multi-substrate reactions when the inhibitor, I, binds to the binding
site of the co-substrate. For a full discussion of these and other types of inhibition
in multi-substrate reactions reference to texts on enzyme kinetics is
recommended.
Irreversible inhibition occurs when an inhibitor Wrst binds at the active
site, then reacts with an active site group to form a covalent bond (E2I). The
active site is then irreversibly blocked by the inhibitor and is permanently
inactivated. Irreversible inhibitors usually contain electrophilic functional
groups such as halogen substituents or epoxides. The kinetic characteristic
associated with irreversible inhibition is that it is time-dependent. This is
because as time goes by more and more enzyme will be blocked irreversibly
by conversion of the reversible EI complex to E2I. Since [S] >> [E] then in
practice this is a unimolecular reaction, so the observed kinetic behaviour
follows unimolecular reaction kinetics. Thus, if enzyme activity (i.e. v max )is
plotted versus time, an exponential decrease of activity is observed versus time
(Figure 4.7).