Meier T W, Thomä N H, Leadlay P F
Department of Biochemistry, University of Cambridge, United Kingdom.
Biochemistry. 1996 Sep 10;35(36):11791-6. doi: 10.1021/bi961250o.
Methylmalonyl-CoA mutase from Propionibacterium shermanii is an adenosylcobalamin-dependent enzyme which catalyzes the reversible isomerization of methylmalonyl-CoA and succinyl-CoA. The rate of tritium loss from 5'-[3H]adenosylcobalamin during the enzymic reaction and the relative rates of tritium appearance in substrate and product were examined. Upon the addition of methylmalonyl-CoA to a solution of holoenzyme, tritium was completely released from the cofactor within about 500 ms. No tritium was found either bound to the enzyme or released into the water. The radioactivity was found in methylmalonyl-CoA and succinyl-CoA in a constant ratio of 1 to 3, which did not change during the first 300 ms of the reaction. Upon the addition of succinyl-CoA to a solution of holoenzyme, tritium was released at essentially the same rate, and the radioactivity was found in methylmalonyl-CoA and succinyl-CoA in the identical constant ratio of 1 to 3. The tritium isotope effect on the enzyme-catalyzed hydrogen transfer, measured using 14C-labeled methylmalonyl-CoA as substrate, was kH/kT = 4.9. This low value shows that hydrogen transfer is only partly rate limiting and that at least one subsequent slow step, such as product release, contributes substantially to the overall reaction velocity. The identical partitioning of tritium, regardless of the substrate used, shows that the rearrangement of the substrate radical into the product radical is not rate limiting. The very low tritium isotope effect and the fact that all the tritium is found bound either to the CoA esters or to the cofactor make it very unlikely that a protein radical is an intermediate in the methylmalonyl-CoA mutase-catalyzed rearrangement.
来自谢氏丙酸杆菌的甲基丙二酰辅酶A变位酶是一种依赖腺苷钴胺素的酶，可催化甲基丙二酰辅酶A和琥珀酰辅酶A的可逆异构化反应。研究了酶促反应过程中5'-[3H]腺苷钴胺素的氚损失速率以及底物和产物中氚出现的相对速率。向全酶溶液中加入甲基丙二酰辅酶A后，约500毫秒内辅酶中的氚完全释放。未发现氚与酶结合或释放到水中。在甲基丙二酰辅酶A和琥珀酰辅酶A中发现的放射性呈1比3的恒定比例，在反应的前300毫秒内该比例不变。向全酶溶液中加入琥珀酰辅酶A后，氚以基本相同的速率释放，且在甲基丙二酰辅酶A和琥珀酰辅酶A中发现的放射性比例同样为恒定的1比3。以14C标记的甲基丙二酰辅酶A为底物，测得的酶催化氢转移的氚同位素效应为kH/kT = 4.9。这个低值表明氢转移只是部分限速步骤，至少有一个后续的慢步骤，如产物释放，对整体反应速度有很大贡献。无论使用何种底物，氚的分配相同，这表明底物自由基重排为产物自由基不是限速步骤。极低的氚同位素效应以及所有氚都只与辅酶A酯或辅酶结合这一事实，使得蛋白质自由基极不可能是甲基丙二酰辅酶A变位酶催化重排反应的中间体。
