MECHANISTIC STUDY OF B12-DEPENDENT METHIONINE SYNTHASE

蛋氨酸合成酶(METH)利用甲基四氢叶酸衍生的甲基，催化同型半胱氨酸转化为蛋氨酸。B12作为中间甲基载体。甲基基因突变的患者表现为严重的神经功能障碍、巨幼细胞性贫血、同型半胱氨酸尿症或低蛋氨酸血症。冰毒是已在哺乳动物中发现的两种依赖B12的酶之一。由于人类冰毒与来自大肠杆菌的酶具有实质性的序列相似性，因此对细菌酶的研究可能会为人类酶的催化机制提供有价值的见解。对大肠杆菌Meth的B12结合区的晶体结构表明，在酶结合的B12中，二甲基苯并咪唑被His759的咪唑取代。His759与附近的Asp757和Ser810形成了配体三联体，并被认为在催化过程中发挥了作用。这项建议的目标是量化和剖析配体三联体的作用。通过将辅因子与蛋白质结合而实现的速率增强将被量化。为了将配体三联体对构象变化的贡献与对化学变化的贡献分开，将分别检测野生型酶和配体三联体变体的单独过表达片段的活性。这些研究可能有助于阐明配体三联体在冰毒和一类新兴的B12依赖酶中的作用，这些酶更喜欢辅因子的碱基配位。

Methionine synthase (MetH) catalyzes the conversion of homocysteine to methionine, using a methyl group that is derived from methyltetrahydrofolate. B12 serves as an intermediate methyl group carrier. Patients with mutations in the MetH gene display severe neurological dysfunction, megaloblastic anemia, homocystinuria, or hypomethioninemia. MetH is one of two B12 dependent enzymes that have been identified in mammals. Since human MetH shows substantial sequence identity to the enzyme from Escherichia coli, studies with the bacterial enzyme may provide valuable insights into the catalytic mechanism of the human enzyme. The crystal structure of the B12 binding domain of the E. coli MetH revealed that in the enzyme bound B12, the dimethylbenzimidazole is replaced by the imidazole of His759. The His759 and the nearby Asp757 and Ser810 form a ligand triad and have been suggested to play a role during catalysis. The goal of this proposal is to quantitate and to dissect the role of the ligand triad. The rate enhancements achieved by binding of the cofactor to the protein will be quantitated. To separate the contribution of the ligand triad to conformational changes from those to the chemistry, separately overexpressed fragments of the wild type enzyme and ligand triad variants will be examined for activity. These studies may help clarify the utility of the ligand triad in MetH and in an emerging class of B12 dependent enzymes that prefer base-off coordination of the cofactor.