Mechanism of Enzyme Mediated Activation of Coenzyme B12

描述(申请人的摘要)：提出一个学习计划，以确定 需要5‘-脱氧腺苷钴胺的酶(ADOCb1， 辅酶B12)加快了碳钴键的均裂速度 AdoCbl增加了9到12个数量级。要研究的酶是 莱氏乳杆菌核糖核苷三磷酸还原酶(RTPR)。 首席调查员实验室最近的研究表明， 用RTPR催化ADOCbl Co-C键均裂反应发生大的(14kcal) 摩尔~(-1)降低活化热，但不改变表观活化能 相对于非酶的、热的Co-C键均解而言的活化。三 ADOCbl均解热催化的假设为 研究：(1)Co-C键均裂过渡态的稳定性 通过从基本活性部位半胱氨酸部分转移氢原子， Cys408；(2)活性中心相互作用引起的基态Co-C键变形 与ADOCbl的ADO配体的RTPR；(3)机械力化学触发，其中 ADOCbl的轴向Co-N键的酶压缩导致要么接地 Co-C键的状态不稳定，或电子稳定 由于Co-Nax轨道重叠增加而形成的过渡态。下面的实验 旨在调查这些可能性：(1)研究 ADOCbl结构类似物的构效关系 RTPR的部分活性辅酶，包括X射线结晶学， 核磁共振约束的分子模拟和RTPR诱导的Co-C反应动力学研究 键均裂作用；(2)络合对~(13)C反应的影响 富含~(13)C的ADOCbl的核磁共振化学位移和~1H-~(13)C耦合常数 钴结合碳和ADOCbl的~(15)N核磁共振化学位移 在每个ADO氮素中分别富含15N，在 轴核苷酸的配位氮；(3)ADOCbl的研究 低空间体积的非天然低轴核苷酸(如苯并咪唑 和咪唑)比天然的轴核苷酸，包括X射线 结晶学、核磁共振约束的分子模拟和完全动力学 研究(包括温度依赖性)RTPR诱导的和 Co-C键的非酶热均解；(4)模型研究 含有分子内硫醇官能团的RTPR的活性部位 由不同长度的系绳系住。此外，有可能 RTPR的活性物种实际上是一种二聚体，将通过确定 蛋白质浓度对观察到的结合常数的影响 ADOCbl和变构效应器dGTP。

DESCRIPTION (Applicant's abstract): A program of study is proposed to determine the mechanism by which an enzyme requiring 5'-deoxy-adenosylcobalamin (AdoCbl, coenzyme B12) accelerates the rate of homolysis of the carbon-cobalt bond of AdoCbl by 9 to 12 orders of magnitude. The enzyme to be studied is the ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii. Recent work from the principal investigator's laboratory has shown that catalysis of AdoCbl Co-C bond homolysis by RTPR occurs with a large (14 kcal mol-1) decrease in the enthalpy of activation but no change in the entropy of activation, relative to non-enzymatic, thermal Co-C bond homolysis. Three hypotheses for the enthalpic catalysis of AdoCbl homolysis will be investigated: (1) Stabilization of the transition state for Co-C bond homolysis by partial transfer of a hydrogen atom from the essential active site cysteine, Cys408; (2) Ground state Co-C bond distortion by interaction of the active site of RTPR with the Ado ligand of AdoCbl; (3) Mechanochemical triggering, in which enzymatic compression of the axial Co-N bond of AdoCbl leads either to ground state destabilization of the Co-C bond, or to electronic stabilization of the transition state by increased Co-Nax orbital overlap. The following experiments are designed to investigate these possibilities: (1) Studies of structure-activity relationships of AdoCbl structural analogs which are partially active coenzymes for RTPR, including X-ray crystallography, NMR-restrained molecular modeling, and kinetic studies of the RTPR-induced Co-C bond homolysis; (2) Studies of the effect of complexation to RTPR on the 13C NMR chemical shift and 1H- 13C coupling constants of AdoCbl enriched in 13C in the cobalt-bound carbon, and on the 15N NMR chemical shift of AdoCbl's individually enriched in 15N in each of the Ado nitrogens and in the coordinating nitrogen of the axial nucleotide; (3) Studies of AdoCbl's with unnatural lower axial nucleotides of lower steric bulk (such as benzimidazole and imidazole) than the natural axial nucleotide, including X-ray crystallography, NMR-restrained molecular modeling, and complete kinetic studies (including temperature dependence) of the RTPR-induced and non-enzymatic thermal homolysis of the Co-C bond; and (4) Studies of models of the active site of RTPR containing an intramolecular thiol function attached to AdoCbl by tethers of varying lengths. In addition, the possibility that the active species of RTPR is actually a dimer will be investigated by determining the effect of protein concentration on the observed binding constants for AdoCbl and the allosteric effector dGTP.