Histidine in the Nucleotide‐Binding Site of NADP‐Linked Isocitrate Dehydrogenase from Pig Heart

Abstract

Incubation of pig heart NADP-dependent isocitrate dehydrogenase [EC 1.1.1.42] with ethoxyformic anhydride (diethylpyrocarbonate) at pH 6.2 results in a 9-fold greater rate of loss of dehydrogenase than of oxalosuccinate decarboxylase activity. The rate constants for loss of dehydrogenase and decarboxylase activities depend on the basic form of ionizable groups with pK values of 5.67 and 7.05, respectively, suggesting that inactivation of the 2 catalytic functions results from reaction with different amino acid residues. The rate of loss of dehydrogenase activity is decreased only slightly in the presence of manganous isocitrate, but is reduced up to 10-fold by addition of the coenzymes or coenzyme analogs, such as 2''-phosphoadenosine 5''-diphosphoribose (Rib-P2-Ado-P). Enzyme modified at pH 5.8 fails to bind NADPH, but exhibits Mn-enhanced isocitrate binding typical of native enzyme, indicating that reaction takes place in the region of the nucleotide binding site. Kd for enzyme .cntdot. coenzyme-analog complexes were calculated from the decrease in the rate of inactivation as a function of analog concentration. In the presence of isocitrate, activating metals (Mn2+, Mg2+, Zn2+) decrease the Kd value for enzyme .cntdot. Rib-P2-Ado-P, while the inhibitor Ca2+ increases Kd. The strengthened binding of nucleitide produced by activating metal-isocitrate complexes may be essential for the catalytic reaction, reflecting an optimal orientation of NADP+ to facilitate hydride transfer. Measurements of ethoxyformyl-histidine formation at 240 nm and of incorporation of [14C]ethoxy groups in the presence and absence of Rib-P2-Ado-P indicate that loss of activity may be related to modification of approximately 1 histidine. The critical histidine appears to be located in the nucleotide binding site in a region distal from the substrate binding site.