Cobalamin-dependent methionine synthase: Probing the role of the axial base in catalysis of methyl transfer between methyltetrahydrofolate and exogenous Cob(I)alamin or Cob(I)inamide
Cobalamin-dependent methionine synthase: Probing the role of the axial base in catalysis of methyl transfer between methyltetrahydrofolate and exogenous Cob(I)alamin or Cob(I)inamide
复制标题
DOI：
10.1021/bi035525t
复制
发表时间：
2003-12-16
期刊：
影响因子：
2.9
通讯作者：
Matthews, RG
中科院分区：
文献类型：
--
作者：
Dorweiler, JS;Finke, RG;Matthews, RG
钴胺素依赖性甲硫氨酸合酶（MetH）催化甲基四氢叶酸（CH 3-H（4）叶酸）和高半胱氨酸之间的甲基转移，酶结合的钴胺素充当甲基转移的中间体。包含残基2-649的MetH片段含有结合和活化CH 3-H（4）叶酸和高半胱氨酸并催化甲基转移到外源钴胺素和从外源钴胺素转移的模块。通过比较钴胺素（其含有与下轴位置的钴配位的二甲基苯并咪唑核苷酸）和钴氨酰胺（其缺乏二甲基苯并咪唑核苷酸）的反应速率，可以评估二甲基苯并咪唑碱基为与MetH（2-649）结合的CH 3-H（4）叶酸和外源性cob（I）alamin之间的甲基转移提供的稳定程度。当比较cob（I）alamin或cob（I）inamide与CH 3-H（4）folate的反应时，观察到cob（I）alamin的二级速率常数快2.7倍;在相反方向，甲基cobinamide与酶结合的四氢叶酸的反应比甲基钴胺素快35倍。这些测量结果可用于估计二甲基苯并咪唑配体对甲基四氢叶酸与co（I）丙氨酸或co（I）酰胺之间的甲基转移的热力学和动力学的影响。甲基从CH_3-H（4）叶酸转移到co（I）丙氨酸的自由能比甲基转移到co（I）酰胺的自由能高2.8kcal。二甲基苯并咪唑对正向反应的稳定作用约为0.6kcal/mol，对逆反应的不稳定作用约为2.2kcal/mol。甲钴胺与全长甲硫氨酸合酶的结合伴随着配体取代，并且已经证明辅因子的“碱基开启”和“碱基关闭”状态之间的转换[Bandarian，V.，等人（2003）Proc.Natl. Acad. Sci. U.S.A. 100，8156-8163]。目前的结果不赞成这样的切换在催化甲基转移的主要作用，并与甲硫氨酸合酶中的配体三联体的主要作用是在催化过程中控制酶构象的分布的假设是一致的。
Cobalamin-dependent methionine synthase (MetH) catalyzes the transfer of methyl groups between methyltetrahydrofolate (CH3-H(4)folate) and homocysteine, with the enzyme-bound cobalamin serving as an intermediary in the methyl transfers. An MetH fragment comprising residues 2-649 contains modules that bind and activate CH3-H(4)folate and homocysteine and catalyze methyl transfers to and from exogenous cobalamin. Comparison of the rates of reaction of cobalamin, which contains a dimethylbenzimidazole nucleotide coordinated to the cobalt in the lower axial position, and cobinamide, which lacks the dimethylbenzimidazole nucleotide, allows assessment of the degree of stabilization the dimethylbenzimidazole base provides for methyl transfer between CH3-H(4)folate bound to MetH(2-649) and exogenous cob(I)alamin. When the reactions of cob(I)alamin or cob(I)inamide with CH3-H(4)folate are compared, the observed second-order rate constants are 2.7-fold faster for cob(I)alamin; in the reverse direction, methylcobinamide reacts 35-fold faster than methylcobalamin with enzyme-bound tetrahydrofolate. These measurements can be used to estimate the influence of the dimethylbenzimidazole ligand on both the thermodynamics and kinetics of methyl transfer between methyltetrahydrofolate and cob(I)alamin or cob(I)inamide. The free energy change for methyl transfer from CH3-H(4)folate to cob(I)alamin is 2.8 kcal more favorable than that for methyl transfer to cob(I)inamide. Dimethylbenzimidazole contributes similar to0.6 kcal/mol of stabilization for the forward reaction and similar to2.2 kcal/mol of destabilization for the reverse reaction. Binding of methylcobalamin to full-length methionine synthase is accompanied by ligand substitution, and switching between "base-on" and "base-off" states of the cofactor has been demonstrated [Bandarian, V., et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 8156-8163]. The present results disfavor a major role for such switching in catalysis of methyl transfer, and are consistent with the hypothesis that the primary role of the ligand triad in methionine synthase is controlling the distribution of enzyme conformations during catalysis.