Implication by site-directed mutagenesis of Arg 314 and Tyr 316 in the coenzyme site of pig mitochondrial NADP-dependent isocitrate dehydrogenase

Abstract

Sequence alignment of pig mitochondrial NADP-dependent isocitrate dehydrogenase with eukaryotic (human, rat, and yeast) and Escherichia coli isocitrate dehydrogenases reveals that Tyr 316 is completely conserved and is equivalent to the E. coli Tyr 345, which interacts with the 2 ′-phosphate of NADP in the crystal structure [Hurley et al., Biochemistry 30 (1991) 8671–8678]. Lys 321 is also completely conserved in the five isocitrate dehydrogenases. Either an arginine or lysine residue is found among the enzymes from other species at the position corresponding to the pig enzyme Arg 314. While Arg 323 is not conserved among all species, its proximity to the coenzyme site makes it a good candidate for investigation. The importance of these four amino acids to the function of pig mitochondrial NADP-isocitrate dehydrogenase was studied by site-directed mutagenesis. Mutants (R314Q, Y316F, Y316L, K321Q, and R323Q) were generated by a megaprimer polymerase chain reaction method. Wild-type and mutant enzymes were expressed in E. coli and purified to homogeneity. All mutant and wild-type enzymes exhibited comparable molecular weights indicative of the dimeric enzyme. Mutations do not cause an appreciable change in enzyme secondary structure as revealed by circular dichroism measurements. The kinetic parameters ( V max and K M values) of K321Q and R323Q are similar to those of wild-type, indicating that Lys 321 and Arg 323 are not involved in enzyme function. R314Q exhibits a 10-fold increase in K M for NADP as compared to that of wild-type, while they have comparable V max values. These results suggest that Arg 314 contributes to the affinity between the enzyme and NADP. The hydroxyl group of Tyr 316 is not required for enzyme function since Y316F exhibits similar kinetic parameters to those of wild-type. Y316L shows a 4-fold increase in K M for NADP and a decrease in V max as compared to wild-type, suggesting that the aromatic ring of the Tyr of isocitrate dehydrogenase contributes to the affinity for coenzyme, as well as to catalysis. The K i for NAD of R314Q, Y316F, and Y316L is comparable to that of wild-type, indicating that the Arg 314 and Tyr 316 may be located near the 2 ′-phosphate of enzyme-bound NADP.