Jarrett J T, Drennan C L, Amaratunga M, Scholten J D, Ludwig M L, Matthews R G
Biophysics Research Division, University of Michigan, Ann Arbor 48109, USA.
Bioorg Med Chem. 1996 Aug;4(8):1237-46. doi: 10.1016/0968-0896(96)00119-8.
Methionine synthase from Escherichia coli is a B12-dependent enzyme that utilizes a methylcobalamin prosthetic group. In the catalytic cycle, the methyl group of methylcobalamin is transferred to homocysteine, generating methionine and cob(I)-alamin, and cob(I)alamin is then remethylated by a methyl group from methyltetrahydrofolate. Methionine synthase occasionally undergoes side reactions that produce the inactive cob(II)alamin form of the enzyme. One such reaction is photolytic homolysis of the methylcobalamin C-Co bond. Binding to the methionine synthase apoenzyme protects the methylcobalamin cofactor against photolysis, decreasing the rate of this reaction by approximately 50-fold. The X-ray structure of the cobalamin-binding region of methionine synthase suggests how the protein might protect the methylcobalamin cofactor in the resting enzyme. In particular, the upper face (methyl or beta face) of the cobalamin cofactor is in contact with several hydrophobic residues provided by an alpha-helical domain, and these residues could slow photolysis by caging the methyl radical and favoring recombination of the CH3./cob(II)alamin radical pair. We have introduced mutations at three positions in the cap domain; phenylalanine 708, phenylalanine 714, and leucine 715 have each been replaced by alanine. Calculations based on the wild-type structure predict that two of these three mutations (Phe708Ala and Leu715Ala) will increase solvent accessibility to the methylcobalamin cofactor, and in fact these mutations result in dramatic increases in the rate of photolysis. The third mutation, Phe714Ala, is not predicted to increase the accessibility of the cofactor and has only a modest effect on the photolysis rate of the enzyme. These results confirm that the alpha-helical domain covers the cofactor in the resting methylcobalamin enzyme and that residues from this domain can protect the enzyme against photolysis. Further, we show that binding the substrate methyltetrahydrofolate to the wild-type enzyme results in a saturable increase in the rate of photolysis, suggesting that substrate binding induces a conformational change in the protein that increases the accessibility of the methylcobalamin cofactor.
来自大肠杆菌的甲硫氨酸合酶是一种依赖维生素B12的酶，它利用甲基钴胺素作为辅基。在催化循环中，甲基钴胺素的甲基被转移至同型半胱氨酸，生成甲硫氨酸和钴胺素（I），然后钴胺素（I）被来自甲基四氢叶酸的甲基重新甲基化。甲硫氨酸合酶偶尔会发生副反应，产生无活性的钴胺素（II）形式的酶。其中一种反应是甲基钴胺素的碳-钴键发生光解均裂。与甲硫氨酸合酶脱辅酶结合可保护甲基钴胺素辅因子免受光解，使该反应速率降低约50倍。甲硫氨酸合酶钴胺素结合区域的X射线结构表明了蛋白质在静止酶中可能如何保护甲基钴胺素辅因子。特别是，钴胺素辅因子的上表面（甲基或β面）与一个α-螺旋结构域提供的几个疏水残基接触，这些残基可能通过捕获甲基自由基并促进CH3./钴胺素（II）自由基对的重组来减缓光解。我们在帽结构域的三个位置引入了突变；苯丙氨酸708、苯丙氨酸714和亮氨酸715分别被丙氨酸取代。基于野生型结构的计算预测，这三个突变中的两个（苯丙氨酸708丙氨酸和亮氨酸715丙氨酸）将增加甲基钴胺素辅因子的溶剂可及性，事实上这些突变导致光解速率显著增加。第三个突变，苯丙氨酸714丙氨酸，预计不会增加辅因子的可及性，对酶的光解速率只有适度影响。这些结果证实，α-螺旋结构域在静止的甲基钴胺素酶中覆盖了辅因子，并且该结构域的残基可以保护酶免受光解。此外，我们表明，将底物甲基四氢叶酸与野生型酶结合会导致光解速率出现饱和性增加，这表明底物结合会诱导蛋白质发生构象变化，从而增加甲基钴胺素辅因子的可及性。
