Brown D A, Cook R A
Biochemistry. 1981 Apr 28;20(9):2503-12. doi: 10.1021/bi00512a022.
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.
已发现，取决于Mg2+还是Mn2+满足二价阳离子需求，大肠杆菌烟酰胺腺嘌呤二核苷酸磷酸（NADP+）依赖性苹果酸酶在动力学和物理性质上存在一些差异。在存在任何一种金属辅因子的情况下，速度-NADP+和速度-阳离子饱和曲线均呈现简单的双曲线响应，但在存在Mn2+的情况下，对NADP+（和苹果酸）的亲和力以及Vmax会增加。该酶对Mn2+的高亲和力以及对底物亲和力的增加表明，Mn2+是体外的首选辅因子。以Mg2+或Mn2+作为阳离子时，在pH 7.45且无效应物的情况下，速度-苹果酸饱和曲线很复杂，表明存在明显的正协同行为和负协同行为的不同组合。以Mg2+作为阳离子时，在苹果酸结合位点之间观察到更大的初始正协同行为。在存在任何一种阳离子的情况下，该酶似乎对变构抑制剂还原型烟酰胺腺嘌呤二核苷酸（NADH）和草酰乙酸（OAA）的抑制作用同样敏感，但在存在效应物的情况下，苹果酸结合位点之间的相互作用会因金属辅因子的选择而有显著差异。抑制剂NADH在存在Mn2+的情况下会增加苹果酸结合位点之间的相互作用，但在存在Mg2+的情况下对亚基相互作用几乎没有影响。抑制剂OAA在存在两种阳离子的情况下都会增加苹果酸结合位点之间的相互作用，在存在Mn2+时观察到正协同性增加，而在存在Mg2+时负协同性增加。动力学数据可以用一个模型来解释，该模型涉及酶的顺序配体诱导构象变化，导致明显的正协同行为和负协同行为的混合。还考虑了涉及不同类别的非相互作用结合位点或不同酶形式的其他解释。金属辅因子Mg2+和Mn2+似乎稳定了酶的两种不同构象状态，这两种状态对不同的底物和效应物浓度的反应不同。用纯酶进行的蛋白水解消化研究进一步证实了在存在两种阳离子的情况下酶的构象状态发生了改变。这些结果与先前报道的烟酰胺腺嘌呤二核苷酸（NAD+）依赖性苹果酸酶和NAD+依赖性异柠檬酸脱氢酶的结果惊人地相似，支持了金属辅因子作为调节实体发挥作用的观点。
