9780521516358book.pdf

value is obtained from a secondary plot (Fig. 15.6). The reaction is carried out for a

range of substrate concentrations in the presence of a series of fixed inhibitor concen-

trations and a Lineweaver–Burk plot for each inhibitor concentration constructed.

Secondary plots of the slope of the primary plot against the inhibitor concentration or

of the apparentKm,(K^0 m) (which is equal toKm(1þ[I]/Ki) and which can be calculated

from the reciprocal of the negative intercept on the I/[S] axis) against inhibitor

concentration, will both have intercepts on the inhibitor concentration axis ofKi.

Sometimes it is possible for two molecules of inhibitor to bind at the active site. In

these cases, although all the primary double reciprocal plots are linear, the secondary

plot is parabolic. This is referred to asparabolic competitive inhibitionto distinguish

it from normallinear competitive inhibition.

Non-competitive

––^1

v 0

Uninhibited

––^1

[S]

Competitive

Uncompetitive

(a)

v 0

––^1

––^1

[S]

(b)

Substrate inhibition v 0

––^1

––^1

[S]

(c)

Positive

cooperativity

No

cooperativity

Negative

cooperativity

Fig. 15.5Lineweaver–Burk plots showing (a) the effects of three types of reversible inhibitor, (b) substrate

inhibition and (c) homotropic cooperativity.

(a)

0.2

0 0.02 0.04 0.06 0.08

0.4

0.6

0.8

1.0

1.2

––^1

v 0

––^1

[S](μM

–1)

(b)

0.1

0.2

0.3

0.4

0.5

–100 1020304050

Intercept

[I] (μM)

• Ki

Ki= 6 μM

[I] = 45 μM[

I] = 35

μM

[I] = 25 μM

[I] = 15 μM

Uninhibited

[I] = 5 μM

Fig. 15.6(a) Primary Lineweaver–Burk plots showing the effect of a simple linear non-competitive inhibitor at a

series of concentrations and (b) the corresponding secondary plot that enables the inhibitor constantKito be

calculated.

593 15.2 Enzyme steady-state kinetics