Equilibrium substrate binding studies of the malic enzyme of pigeon liver. Equivalence of nucleotide sites and anticooperativity associated with the binding of L-malate to the enzyme-manganese(II)-reduced nicotinamide adenine dinucleotide phosphate ternary complex.

Abstract

Malic enzyme (ME) from pigeon liver is a tetrameric protein containing apparently identical subunits. In the present study, equilibrium dialysis and fluorescence titration techniques are employed to determine the binding parameters of nucleotide cofactors, malate, and the inhibitor oxalate. ME binds NADP+ or NADPH at four independent and equivalent sites with dissociation constants of 1.33 microM (pH 7.5, 4 degrees C) and 0.29 microM (pH 7.0, 5 degrees C), respectively, showing "all-of-the-sites" reactivity. The affinity of both nucleotides decreases with increasing temperature, yielding delta Hdissociation values of 11.4 kcal/mol for E-NADP+ and 8.9 kcal/mol for E-NADPH, thus implicating the involvement of polar forces in the binding process. The affinity of NADP+ is independent of pH between 6.1 and 8.4 whereas that of NADPH is highly pH dependent and decreases approximately 63-fold from pH 6.0 to pH 8.0. The pH profile suggests the participation of a protonated enzyme group(s) (pK = 7.2-7.5) in NADPH binding, probably a histidine residue. The affinity of NADP+ is enhanced ca. twofold by pyruvate, in the presence of Mn2+ (50-100 microM) saturating only two "tight" metal sites [Hsu, R. Y., Mildvan, A. S., Chang, G. G., & Fung, C. H. (1976) J. Biol. Chem. 251, 6574]. Binding of Mn2+ at weak metal sites (KD congruent to 0.9 mM) prevents this change. Malate binds free ME or binary E-Mn2+ and E-NADP+ (H) complexes weakly with dissociation constants of greater than or equal to 2 mM. The affinity is significantly increased by Mn2+ and NADPH in the ternary E-Mn2+-NADPH complex, yielding two "tight" (KD = 22-30 microM) and two "weak" (KD = 250-400 microM) malate sites per enzyme tetramer as the result of either preexisting nonidentity or negative cooperativity between intitially identical sites. The transition-state inhibitor oxalate binds ME tightly (KD = 65 microM) at the two tight malate sites, showing "half-of-the-sites" stoichiometry. The binding parameters are unaffected by Mn2+, whereas the affinity of this inhibitor is enhanced 3.5-fold by saturation with NADPH. Further evidence for the half-of-the-sites reactivity of the affinity label bromopyruvate [Pry, T. A., & Hsu, R. Y. (1978) Biochemistry 17, 4024] is obtained by sequential modification of the four putatively identical SH groups of ME with bromopyruvate, 5,5'-dithiobis(2-nitro-benzoic acid), and K14CN. The modified enzyme has a structure of E4(S-pyr)2(S-14CN)2 and is "inactive" in the reaction with malate. In contrast, the E(S-14CN)4 derivative prepared in the absence of bromopyruvate is completely active. The oxidative decarboxylase reaction is inhibited by high concentrations (greater than or equal to 0.3 mM) of malate in the presence of tightly bound Mn2+. Direct binding studies show a parallel increase in the affinity of NADPH, confirming our previous notion [Reynolds, C. H., Hsu, R. Y., Matthews, B., Pry, T. A., & Daibits the rate-limiting NADPH release step.