Adenosylcobalamin-dependent glutamate mutase: examination of substrate and coenzyme binding in an engineered fusion protein possessing simplified subunit structure and kinetic properties.
Adenosylcobalamin-dependent glutamate mutase: examination of substrate and coenzyme binding in an engineered fusion protein possessing simplified subunit structure and kinetic properties.
复制标题
腺苷钴胺依赖性谷氨酸变位酶：检查具有简化亚基结构和动力学特性的工程融合蛋白中的底物和辅酶结合。
DOI：
10.1021/bi971374g
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发表时间：
1997
期刊：
影响因子：
2.9
通讯作者：
Marsh,EN
中科院分区：
文献类型：
--
作者：
Chen,HP;Marsh,EN
谷氨酸酯酶由两个弱缔合亚基E和S组成，它们联合收割机形成辅酶结合位点。活性全酶在一个动力学复杂的过程中组装，其中腺苷钴胺素（adenosylcobalamin，CBL）的化学计量和表观Kd都取决于两个亚基的相对浓度，因为这是酶的比活性。为了便于对这种酶的机理和结构研究，我们通过11个氨基酸（Gly-Gln）5-Gly接头片段将S亚基遗传融合到E亚基的C-末端。这种蛋白质GlmES以化学计量方式结合HbCbl，并且对HbCbl的亲和力和周转数都不依赖于蛋白质浓度。测定了底物和辅酶的kcat和Km以及氘同位素对Vmax和Vmax/Km的影响。与野生型相比，对β-Cbl的亲和力不变，但对于l-谷氨酸向（2S，3S）-3-甲基天冬氨酸的转化，l-谷氨酸的kcat和Km均降低约三分之一，并且同位素效应降低，表明产物释放更具限速性。为了验证关于辅酶活化的假设，我们研究了腺苷钴胺素、甲钴胺和辅酶（II）维生素B1与酶的结合。这两种物质的结合亲和力基本相同（2 μM），这表明与预期相反，蛋白质与腺苷部分之间的相互作用不会削弱基态的钴碳键。
Glutamate mutase is comprised of two weakly associating subunits, E and S, that combine to form the coenzyme binding site. The active holoenzyme assembles in a kinetically complex process in which both the stoichiometry and apparentKdfor adenosylcobalamin (AdoCbl) are dependent upon the relative concentrations of the two subunits, as is the enzyme's specific activity. To facilitate mechanistic and structural studies on this enzyme we have genetically fused the S subunit to the C-terminus of the E subunit through an 11 amino acid (Gly-Gln)5-Gly linker segment. This protein, GlmES, binds AdoCbl stoichiometrically and neither the affinity for AdoCbl nor the turnover number depends upon protein concentration. ThekcatandKmfor both substrate and coenzyme, together with the deuterium isotope effects onVmaxandVmax/Km, have been determined for the GlmES-catalyzed reaction proceeding in both directions. Compared with wild type, the affinity for AdoCbl is unchanged, but for the conversion ofl-glutamate to (2S,3S)-3-methylaspartate bothkcatandKmforl-glutamate are decreased by about a third and the isotope effects are reduced, suggesting product release to be more rate-limiting. To test hypotheses concerning the activation of the coenzyme, we examined the binding of adenosylcobalamin, methylcobalamin, and cob(II)alamin to the enzyme. Each of these is bound with essentially the same affinity (2 μM), suggesting that, contrary to expectations, interactions between the protein and the adenosyl moiety do not serve to weaken the cobalt−carbon bond in the ground state.