MECHANISM OF ENZYME MEDIATED ACTIVATION OF COENZYME B12

提出了一项研究计划，以确定酶的作用机制， 需要5 '-脱氧腺苷钴胺素（5'-deoxy-adenosylcobalamin，辅酶B12）作为辅因子 通过以下方式加速H2O Cbl的碳-钴键的均裂速率： 9.7到12个数量级。要研究的酶是 核糖核苷酸三磷酸还原酶（RTPR，EC 1.17.4.2）来自 最近被克隆和过表达的莱氏乳杆菌 在大肠杆菌这种酶在依赖于β-Cbl的酶中是独特的，因为它 在没有底物的情况下催化β-Cbl的均裂，因此酶- 诱导的均裂可以在没有周转的情况下进行研究。动力学 将研究RTPR诱导的CSCb 1均裂的作用， 通过停流分光光度法测定温度，以获得数值 这个过程的激活参数。相比于 用于未催化的H2 Cbl均裂的活化参数将允许 确定酶催化反应的程度 通过降低焓和/或通过提高活化熵。 分子力学计算和核磁共振研究 这表明在高温下， 构象柔性比它在较低温度下， 酶是活跃的。因此，据信， 在85-110摄氏度下测量的CO_2 Cbl的参数不适用于 与37 ℃下酶诱导的均裂比较。 用初速率法研究了PbCl_2在较低温度下的均裂动力学 温度将使用非常高的比活度[A2-A4]来开发。 在腺苷C2质子处氚化的[3 H]-[3 H]-[3 H]-[3 H] Cbl。 一旦确定了热力学和熵催化的程度， 催化作用的一种定量假设，包括以下内容： 将测试以下元件：（i）酶促诱导的 咕啉环以增加乙酰胺侧链的空间限制 Ado配体的旋转（导致基态熵的降低， 活化熵的增加）并且可能在空间上 通过增加咕啉环之间的接触来拉伸Co-C键 所述Ado配体的氮和α亚甲基氢，和（ii） 拉伸的Co-C键和弯曲的Co-C-C键角， 活性位点和Ado N7之间的氢键相互作用， 环外氨基，提供双碳催化。这一假设 将测试如下：（l）活化的动力学研究 RTPR诱导的具有改变的侧边的HCCbl类似物的均裂的参数 链和Ado配体结构。(2)15 N和15 N-编辑的1H NMR研究 [U-15 N] NH 4Cbl（来自发酵）与RTPR络合以探测氢 活性位点与Ado配体以及与 侧链酰胺。(3)[A15- 13 C] NH 4Cbl的13 C NMR研究（标记在图10中） 钴结合碳）与RTPR络合，以寻找 说明Co-C键应变。(4)咕啉核磁共振探针的研制 构象，包括使用13 C化学位移作为构象的指示剂， 咕啉环折叠和NOE约束的分子力学计算， 允许测定[U-]络合物中的咕啉环构象 13 C]将Cbl（来自发酵）与RTPR-起。

A program of study is proposed to determine the mechanism by which enzymes requiring 5'-deoxy-adenosylcobalamin (AdoCbl, coenzyme B12) as a cofactor accelerate the rate of homolysis of the carbon-cobalt bond of AdoCbl by 9.7 to 12 orders of magnitude. The enzyme to be studied is the ribonucleotide triphosphate reductase (RTPR, EC 1.17.4.2) from Lactobacillus leichmannii which has recently been cloned and overexpressed in E. coli. This enzyme is unique among AdoCbl-dependent enzymes since it catalyzes the homolysis of AdoCbl without substrate, so that enzyme- induced homolysis can be studied in the absence of turnover. The kinetics of RTPR-induced AdoCbl homolysis will be studied as a function of temperature by stopped flow spectrophotometry in order to obtain values for the activation parameters for this process. Comparison to the activation parameters for the uncatalyzed homolysis of AdoCbl will permit a determination of the extent to which the enzyme catalyzes the reaction by lowering the enthalpy and/or by raising the entropy of activation. Molecular mechanics calculations and NMR studies of AdoCbl strongly suggest that at high temperatures, AdoCbl is substantially more conformationally flexible than it is at lower temperatures where the enzyme is active. As a result, it is believed that the activation parameters for AdoCbl measured at 85-110 degrees C are not appropriate for comparison to enzyme-induced homolysis at 37 degreesC Consequently, an initial rate method for studying the homolysis kinetics of AdoCbl at lower temperatures will be developed using very high specific activity [A2- 3H]AdoCbl tritiated at the adenosine C2 proton. Once the extent of enthalpic and entropic catalysis has been determined, a quantitative hypothesis for catalysis consisting of the following elements will be tested: (i) enzymatically induced upward flexing of the corrin ring to increase the steric restriction of acetamide side chain rotation by the Ado ligand (causing a decrease in ground state entropy and an increase in the entropy of activation) and possibly sterically stretching the Co-C bond by increased contact between the corrin ring nitrogens and the alpha methylene hydrogens of the Ado ligand, and (ii) stretching of the Co-C bond and bending of the Co-C-C bond angle by hydrogen bonding interactions between the active site and the Ado N7 and exocyclic amino groups, providing enthalpic catalysis. This hypothesis will be tested as follows: (l) Kinetic studies of the activation parameters for RTPR-induced homolysis of AdoCbl analogs with altered side chain and Ado ligand structure. (2) 15N and 15N-edited 1H NMR studies of [U-15N]AdoCbl (from fermentation) complexed to RTPR to probe hydrogen bonding interactions of the active site with the Ado ligand and with the side chain amides. (3) 13C NMR studies of [A15-13C]AdoCbl (labeled at the cobalt-bound carbon) complexed to RTPR to look for evidence of ground state Co-C bond strain. (4) Development of NMR probes of corrin conformation, including the use of 13C chemical shifts as an indicator of corrin ring fold and NOE constrained molecular mechanics calculations, to permit determination of corrin ring conformation in complexes of [U- 13C]AdoCbl (from fermentation) with RTPR.