MECHANISM OF ENZYME MEDIATED ACTIVATION OF COENZYME B12

提出了一个研究计划，以确定酶的作用机制 需要5‘-脱氧腺苷钴胺(ADOCb1，辅酶B12)作为辅因子 加快ADOCbl中碳-钴键的均裂速度 9.7到12个数量级。要研究的酶是 核糖核酸三磷酸还原酶(RTPR，EC 1.17.4.2)来自 新近克隆并高效表达的莱氏乳杆菌 在大肠杆菌中。这种酶在ADOCbl依赖的酶中是独一无二的，因为它 催化ADOCbl在无底物的情况下均解，使酶- 诱导均溶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ADOCbl同源溶解作为以下参数的函数 用停流分光光度法测量温度以获得数值 以获取此过程的激活参数。与 ADOCbl非催化均解的活化参数将允许 酶催化反应程度的测定 通过降低热焓和/或通过提高活化熵。 ADOCbl的分子力学计算和核磁共振研究 表明在高温下，ADOCbl显著高于 在构象上比在较低温度下更灵活，在那里 酶是活跃的。因此，人们认为激活 在85-110摄氏度测量的Adobe Cbl参数不适用于 与37℃下的酶诱导均相比较因此， 用初速率法研究ADOCbl在较低温度下的均解动力学 温度将使用非常高的比活度[A2- 在腺苷C2质子上进行了氚反应。 一旦确定了焓和熵催化的程度， 关于催化的一个定量假说，包括以下几个部分 这些元素将被测试：(I)酶诱导的向上弯曲 科林环增加乙酰胺侧链的空间位阻 ADO配体的旋转(导致基态熵和 活化熵的增加)，并且可能是立体的 通过增加科林环之间的接触来伸展Co-C键 氮基和腺苷配体的α-亚甲基氢基，以及(Ii) Co-C键的伸缩和Co-C-C键角的弯曲 活性中心与ADO N7和Ado N7的氢键相互作用 外环氨基，提供热催化作用。这一假设 将试验内容如下：(L)活化动力学研究 RTPR诱导侧边改变的ADOCbl类似物均裂参数的研究 链和腺苷配体结构。(2)~(15)N和~(15)N编辑的~1H核磁共振研究 [U-15N]ADOCbl(发酵产物)与RTPR络合以探测氢气 活性中心与腺苷配基和活性中心的成键作用 侧链酰胺。(3)[A15-13C]ADOCbl的13C核磁共振研究 钴结合碳)与RTPR络合以寻找地面证据 国家Co-C键应变。(4)可可林核磁共振探针的研制 构象，包括使用~(13)C化学位移作为指示 科林环折和NOE约束的分子力学计算 允许测定[U-]络合物中的Corin环构象 13C]ADOCbl(来自发酵)和RTPR。

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.