Mechanism of Enzyme Mediated Activation of Coenzyme B12

描述（申请人摘要）：提出一项研究计划，以确定 需要5 ′-脱氧-腺苷钴胺素的酶（CYP1Cb1， 辅酶B12）加速了碳-钴键的均裂速率， 将Cbl的数量级降低9到12个数量级。要研究的酶是 核糖核苷三磷酸还原酶（RTPR），来自莱氏乳杆菌。 首席研究员实验室最近的工作表明， 通过RTPR催化Cb1 Co-C键均裂发生大的（14 kcal mol-1）的活化焓降低，但熵不变。 活化，相对于非酶，热Co-C键均裂。三 假设的催化剂的双金属均裂将是 研究了：（1）Co-C键均裂过渡态的稳定性 通过氢原子从基本活性位点半胱氨酸的部分转移， Cys408;（2）活性中心相互作用引起的基态Co-C键畸变 （3）机械化学触发，其中 酶促压缩的轴向Co-N键的Ct3Cbl导致接地 状态不稳定的Co-C键，或电子稳定的 过渡态的增加Co-Nax轨道重叠。以下实验 目的是调查这些可能性：（1）研究 图10示出了被用作药物的CXCbl结构类似物的结构-活性关系， 用于RTPR的部分活性辅酶，包括X射线晶体学， 核磁共振限制的分子模拟和RTPR诱导的Co-C动力学研究 （2）RTPR络合作用对~（13）C 富13 C的C_3Cb_1的NMR化学位移和1H-13 C偶合常数 钴结合的碳，并对15N NMR化学位移的15N Cbl的 在每个Ado氮中和在每个Ado氮中单独富集15N， 轴向核苷酸的配位氮;（3）用 低空间体积的非天然下轴核苷酸（如苯并咪唑 和咪唑）比天然的轴向核苷酸，包括X射线 晶体学，NMR限制的分子建模，和完整的动力学 RTPR诱导的和 Co-C键的非酶热均裂;（4）Co-C键的非酶热均裂模型研究 含有分子内巯基官能团的RTPR的活性位点连接到 用不同长度的系绳将Cbl固定住。此外， RTPR的活性物质实际上是二聚体，将通过测定 蛋白质浓度对观察到的结合常数的影响 在一些实施方案中，所述抗体包含抗CDCbl和变构效应物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.