Homoisocitrate dehydrogenase (HIcDH) catalyzes the Mg2+- and K+-dependent oxidative decarboxylation of homoisocitrate to α-ketoadipate using NAD as the oxidant. A recent consideration of the structures of enzymes in the same family as HIcDH, including isopropylmalate and isocitrate dehydrogenases, suggests all of the family members utilize a Lys-Tyr pair to catalyze the acid-base chemistry of the reaction (Aktas, D. F., and Cook, P. F. (2009) Biochemistry submitted.). Multiple sequence alignment indicates the active site Lys-Tyr pair consists of lysine-206 and tyrosine-150. Therefore, the K206M and Y150F mutants of HIcDH were prepared and characterized in order to test the potential roles of these residues as acid-base catalysts. The V/Et values of the K206M and Y150F mutant enzymes at pH 7.5 are decreased by about 2400- and 680-fold, respectively, compared to wild type HIcDH; the Km for HIc does not change significantly. V/Et and V/KMgHIc
Et for the K206M mutant enzyme are pH independent below pH 6 and decrease to a constant value above pH 7, while V/KNAD
Et is independent over the pH range 6.2 to 9.5. In the case of the Y150F mutant enzyme, V/Et and V/KNAD
Et are pH independent above pH 9.5 and decrease to a constant value below pH 8. This behavior can be compared to wild type enzyme, where V/Et decreases at high and low pH giving pKa values of about 6.5 and 9.5. Data were interpreted in terms of a group with a pKa of 6.5 that acts as a general base in the hydride transfer step, and a group with a pKa of 9.5 that acts as a general acid to protonate C3 in the tautomerization reaction (Lin, Y., Volkman, J., Nicholas, K. M., Yamamoto, T., Eguchi, T., Nimmo, S. L., West, A. H., Cook, P. F. (2008) Biochemistry 47, 4169–4180.). Solvent deuterium isotope effects on V and V/KMgHIc were near unity for the K206M mutant enzyme, but about 2.2 for the Y150F mutant enzyme. The dramatic decreases in activity, the measured solvent deuterium isotope effects and changes in the pH dependence of kinetic parameters compared to wild type, are consistent with K206 acting as a general base in the hydride transfer step of the wild type enzyme, but as a general acid in the Y150F mutant enzyme, replacing Y150 in the tautomerization reaction. In addition, Y150 acts as a general acid in the tautomerization reaction of the wild type enzyme, and replaces K206 as the general base in the hydride transfer step of the K206M mutant enzyme.