ADDING A POSITIVE CHARGE AT RESIDUE-46 OF DROSOPHILA ALCOHOL-DEHYDROGENASE INCREASES COFACTOR SPECIFICITY FOR NADP(+)

We previously reported that the D39N mutant of Drosophila alcohol dehydrogenase (ADH), in which Asp-39 is replaced with asparagine, has a 60-fold increase in affinity for NADP(+) and a 1.5-fold increase in k(cat) compared to wild-type ADH [Chen et al. (1991) fur. J. Biochem. 202, 263-267] and proposed that this part of ADH is close to the 2'-phosphate on the ribose moiety of NADP(+). Here we report the effect of replacing Ala-46 with an argine residue, an A46R mutant, on binding of NADP(+) to ADH and its catalytic efficiency with the NADP(+) cofactor, and a modeling of the three-dimensional structure of the NAD(+)-binding region of ADH. The A46R mutant has a 2.5-fold lower K(m(app)NADP(+)) and a 3-fold higher k(cat) with NADP(+) compared to wild-type ADH; binding of NAD(+) to the mutant was unchanged and k(cat) with NAD(+) was lowered by about 30%. For the A46R mutant, the ratio of k(cat)/K-m of NAD(+) to NADP(+) is 85, over ten-fold lower than that for wild-type ADH. Our model of the 3D structure of the NAD(+)-binding region of ADH shows that Ala-46 is over 10 Angstrom from the ribose moiety of NAD(+), which would suggest that there is little interaction between this residue and NAD(+) and explain why its mutation to arginine has little effect on NAD(+) binding. However, the positive charge at residue 46 can neutralize some of the coulombic repulsion between Asp-39 and the 2'-phosphate on the ribose moiety of NADP(+), which would increase its affinity for the A46R mutant. We also constructed a double mutant, D39N/A46R mutant, which we find has a 30-fold lower K(m(app)NADP(+)) and 8-fold higher k(cat) with NADP(+) as a cofactor compared to wild-type ADH; binding of NAD(+) to this double mutant was lowered by 5-fold and k(cat) was increased by 1.5-fold. As a result, k(cat)/K-m for the double mutant was the same for NAD(+) and NADP(+). The principle effect of the two mutations in ADH is to alter its affinity for the nucleotide cofactor; k(cat) decreases slightly in A46R with NAD(+) and remains unchanged or increases in the other mutants.