Dorweiler Jeanne Sirovatka, Finke Richard G, Matthews Rowena G
Biophysics Research Division, Life Sciences Institute, and Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA.
Biochemistry. 2003 Dec 16;42(49):14653-62. doi: 10.1021/bi035525t.
Cobalamin-dependent methionine synthase (MetH) catalyzes the transfer of methyl groups between methyltetrahydrofolate (CH(3)-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 CH(3)-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 CH(3)-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 CH(3)-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 CH(3)-H(4)folate to cob(I)alamin is 2.8 kcal more favorable than that for methyl transfer to cob(I)inamide. Dimethylbenzimidazole contributes approximately 0.6 kcal/mol of stabilization for the forward reaction and approximately 2.2 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.
钴胺素依赖性甲硫氨酸合酶（MetH）催化甲基在甲基四氢叶酸（CH(3)-H(4)叶酸）和同型半胱氨酸之间的转移，酶结合的钴胺素在甲基转移中起中间体作用。一个包含2至649位残基的MetH片段含有结合并激活CH(3)-H(4)叶酸和同型半胱氨酸的模块，并催化甲基与外源性钴胺素之间的转移。比较含有在较低轴向位置与钴配位的二甲基苯并咪唑核苷酸的钴胺素和缺乏二甲基苯并咪唑核苷酸的钴胺酰胺的反应速率，可以评估二甲基苯并咪唑碱基为结合在MetH(2-649)上的CH(3)-H(4)叶酸与外源性钴胺素（cob(I)alamin）之间的甲基转移提供的稳定程度。当比较钴胺素（cob(I)alamin）或钴胺酰胺（cob(I)inamide）与CH(3)-H(4)叶酸的反应时，观察到的二级速率常数对于钴胺素（cob(I)alamin）快2.7倍；在相反方向上，甲基钴胺酰胺与酶结合的四氢叶酸反应比甲基钴胺素快35倍。这些测量可用于估计二甲基苯并咪唑配体对甲基四氢叶酸与钴胺素（cob(I)alamin）或钴胺酰胺（cob(I)inamide）之间甲基转移的热力学和动力学的影响。从CH(3)-H(4)叶酸向钴胺素（cob(I)alamin）的甲基转移的自由能变化比向钴胺酰胺（cob(I)inamide）的甲基转移更有利2.8千卡。二甲基苯并咪唑对正向反应贡献约0.6千卡/摩尔的稳定作用，对反向反应贡献约2.2千卡/摩尔的去稳定作用。甲基钴胺素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在“碱基在位”和“碱基不在位”状态之间切换[班达里安，V.等人（2003年）美国国家科学院院刊100，8156 - 8163]。目前的结果不支持这种切换在甲基转移催化中起主要作用，并且与配体三联体在甲硫氨酸合酶中的主要作用是在催化过程中控制酶构象分布的假设一致。
