Control of coenzyme binding to horse liver alcohol dehydrogenase

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.