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 kcat compared to wild-type ADH [Chen et al. (1991) Eur. 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, and 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 Km(app)NADP+ and a 3-fold higher kcat with NADP+ compared to wild-type ADH; binding of NAD+ to the mutant was unchanged and kcat with NAD+ was lowered by about 30%. For the A46R mutant, the ratio of kcat/Km 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 A 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 Km(app)NADP+ and 8-fold higher kcat with NADP+ as a cofactor compared to wild-type ADH; binding of NAD+ to this double mutant was lowered by 5-fold and kcat was increased by 1.5-fold. As a result, kcat/Km 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; kcat decreases slightly in A46R with NAD+ and remains unchanged or increases in the other mutants.
Chen, Z; Tsigelny, I; Lee, WR; Baker, ME; Chang, SH
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