Critical role of Lys212 and Tyr140 in porcine NADP-dependent isocitrate dehydrogenase

Lys(212) and Tyr(140) are close to the enzyme-bound isocitrate in the recently determined crystal structure of porcine NADP-specific isocitrate dehydrogenase (Ceccarelli, C., Grodsky, N. B., Ariyaratne, N., Colman, R. F., and Bahnson, B. J. ( 2002) J. Biol. Chem. 277, 43454 43462). We have constructed mutant enzymes in which Lys(212) is replaced by Gln, Tyr, and Arg, and Tyr(140) is replaced by Phe, Thr, Glu, and Lys. Wild type and mutant enzymes were each expressed in Escherichia coli and purified to homogeneity. At pH 7.4, the specific activity is decreased in K212Q, K212Y, and K212R, respectively, to 0.01 - 9% of wild type. The most striking change is in the pH-V-max curves. Wild type depends on the deprotonated form of a group of pK(aes) 5.7, whereas this pK(aes) is increased to 7.4 in neutral K212Q and to 8.3 in K212Y. In contrast, the positive K212R has a pK(aes) of 5.9. These results indicate that ( by electrostatic repulsion) a positively charged residue at position 212 lowers the pK of the nearby ionizable group in the enzyme-substrate complex. Lys(212) may also stabilize the carbanion formed initially on substrate decarboxylation. The Tyr(140) mutants have specific activities at pH 7.4 that are reduced to 0.2 - 0.5% of those of wild type, whereas their K-m values for isocitrate and NADP are not increased. Most notable are the altered pH-V-max profiles. V-max is constant from pH 5.3 to 8 for Y140F and Y140T and increases as pH is decreased for Y140E and Y140K. These results suggest that in wild type enzyme, Tyr(140) is the general acid that protonates the substrate after decarboxylation and that the carboxyl and ammonium forms of Y140E and Y140K provide partial substitutes. Relative to wild type, the Y140T enzyme is specifically activated 106-fold by exogenous addition of acetic acid and 88-fold by added phenol; and the K212Q enzyme is activated 4-fold by added ethylamine. These chemical rescue experiments support the conclusion that Tyr(140) and Lys(212) are required for the catalytic activity of porcine NADP-dependent isocitrate dehydrogenase.