Role of metal cofactors in enzyme regulation. Differences in the regulatory properties of the Escherichia coli nicotinamide adenine dinucleotide phosphate specific malic enzyme, depending on whether magnesium ion or manganese ion serves as divalent cation

Abstract

A number of differences in the kinetic and physical properties of the Escherichia coli nicotinamide adenine dinucleotide phosphate (NADP+) dependent malic enzyme have been found, depending upon whether Mg2+ or Mn2+ served to fulfill the divalent cation requirement. The velocity-NADP+ and velocity-cation saturation curves exhibit a simple hyperbolic response in the presence of either metal cofactor, but the affinity for NADP+ (and malate) as well as the Vmax is increased in the presence of Mn2+. The high affinity of the enzyme for Mn2+ coupled with the increased affinity for substrates indicates that Mn2+ is the preferred cofactor in vitro. With either Mg2+ or Mn2+ as cation, the velocity-malate saturation curves in the absence of effectors are complex at pH 7.45, indicating varying combinations of apparent positive and negative cooperative behavior. Greater initial positive cooperative behavior between malate binding sites is observed with Mg2+ as cation. The enzyme appears to be equally sensitive to inhibition by the allosteric inhibitors reduced nicotinamide adenine dinucleotide (NADH) and oxaloacetic acid (OAA) in the presence of either cation, but the interaction between malate binding sites, in the presence of effectors, varies significantly with the choice of metal cofactor. The inhibitor NADH increases the interaction between malate binding sites in the presence of Mn2+ but has little effect on subunit interaction in the presence of Mg2+. The inhibitor OAA increases the interaction between malate binding sites in the presence of both cations, with increased positive cooperativity observed with Mn2+ but increased negative cooperativity with Mg2+. The kinetic data can be explained by a model involving sequential ligand-induced conformational changes of the enzyme, resulting in a mixture of apparent positive and negative cooperative behavior. Alternative explanations involving different classes of noninteracting binding sites or different enzyme forms are also considered. The metal cofactors, Mg2+ and Mn2+, appear to stabilize two distinct conformational states of the enzyme which differ in response to varying substrate and effector concentrations. Altered conformational states of the enzyme in the presence of the two cations are further substantiated by proteolytic digestion studies with the homogeneous enzyme. The results are strikingly similar to previous results reported on the nicotinamide adenine dinucleotide (NAD+) dependent malic enzyme and the NAD+-dependent isocitrate dehydrogenase, supporting the suggestion that metal cofactors function as regulatory entities.