Introduction to Enzyme and Coenzyme Chemistry - E-Library Home

74 Chapter 4
O
D
H
ketosteroid
isomerase
SLOW
HO
EnzB-D
FAST
O
D
H
k H
k D
= 6.2
O
H
D
ketosteroid
isomerase
SLOW
HO
D
EnzB-H
FAST
O
D
k H
= 1.1
k D
Figure 4.26 Substrate kinetic isotope eVects in the ketosteroid isomerase reaction.
abstracted is replaced by deuterium, and a primary kinetic isotope eVect of 6.2
is observed on k cat . However, with 4S-[4- 2 H]-ketosteroid a secondary kinetic
isotope eVect of 1.1 is observed, due to re-hybridisation of C-2 in the ratedetermining
step.
If an enzymatic reaction is carried out in 2 H 2 O rather than 1 H 2 O a solvent
kinetic isotope eVect is observed if there is a proton transfer from water or a
water-exchangeable group in the rate-determining step. For example, in the
ketosteroid isomerase reaction there is a D 2 O solvent isotope eVect of 1.6 on
k cat , due to replacement of the phenolic proton of Tyr-14 by deuterium, as
shown in Figure 4.27.
D 2 O solvent isotope eVects also arise where there is base-catalysed attack
of water in a rate-determining step. However, D 2 O solvent isotope eVects can
in some cases arise for a number of reasons far removed from active site
catalysis, so they need to be interpreted with caution. Finally, kinetic isotope
eVects are not restricted only to cleavage of C2H bonds: rate-determining
cleavage of C2O, C2N and C2C bonds can be studied using 18 O-, 15 N- and
13 C-labelled substrates. In these cases the diVerence in zero point energy between
heavy isotopes is very much smaller, so the observed eVects are typically
Tyr 14
O
D
O
k H2 O
k D2 O
= 1.6
H
− O
O
Asp 38
Figure 4.27 D 2 O solvent isotope eVect in the ketosteroid isomerase reaction.