The rate of association of NAD(+) with wild-type horse liver alcohol dehydr
ogenase (ADH) is maximal at pH values between pK values of about 7 and 9, a
nd the rate of NADH association is maximal at a pH below a pK of 9. The cat
alytic zinc-bound water, His-51 (which interacts with the 2'- and 3'-hydrox
yl groups of the nicotinamide ribose of the coenzyme in the proton relay sy
stem), and Lys-228 (which interacts with the adenosine 3'-hydroxyl group an
d the pyrophosphate of the coenzyme) may be responsible for the observed pK
values, In this study, the Lys228Arg, His51Gln, and Lys228Arg/His51Gln (to
isolate the effect of the catalytic zinc-bound water) mutations were used
to test the roles of the residues in coenzyme binding. The steady state kin
etic constants at pH 8 for the His51Gln enzyme are similar to those for wil
d-type ADH. The Lys228Arg and Lys228Arg/His51Gln substitutions decrease the
affinity for the coenzymes up to 16-fold, probably due to altered interact
ions with the arginine at position 228. As determined by transient kinetics
, the rate constant for association of NAD(+) with the mutated enzymes no l
onger decreases at high pH. The pH profile for the Lys228Arg enzyme retains
the pK value near 7. The His51Gln and Lys228Arg/His51Gln substitutions sig
nificantly decrease the rate constants for NAD(+) association, and the pH d
ependencies show that these enzymes bind NAD+ most rapidly at a pH above pK
values of 8.0 and 9.0, respectively. It appears that the pK of 7 in the wi
ld-type enzyme is shifted up by the H51Q substitutions, and the resulting p
H dependence is due to the deprotonation of the catalytic zinc-bound water.
Kinetic simulations suggest that isomerization of the enzyme-NAD(+) comple
x is substantially altered by the mutations. In contrast, the pH dependenci
es for NADH association with His51Gln, Lys228Arg, and Lys228Arg/His51Gln en
zymes were the same as for wild-type ADH, suggesting that the binding of NA
D(+) and the binding of NADH are controlled differently.