Tritium isotope effects in adenosylcobalamin-dependent methylmalonyl-CoA mutase
Tritium isotope effects in adenosylcobalamin-dependent methylmalonyl-CoA mutase
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DOI：
10.1021/bi961250o
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发表时间：
1996-09-10
期刊：
影响因子：
2.9
通讯作者：
Leadlay, PF
中科院分区：
文献类型：
--
作者：
Meier, TW;Thoma, NH;Leadlay, PF
来自谢氏丙酸杆菌的甲基丙二酰辅酶A是腺苷钴胺素依赖性酶，其催化甲基丙二酰辅酶A和琥珀酰辅酶A的可逆异构化。测定了氚在酶促反应过程中从5 ′-[H-3]腺苷钴胺素中损失的速率以及氚在底物和产物中出现的相对速率。在添加甲基丙二酰辅酶A的全酶的溶液中，氚被完全释放的辅因子在约500毫秒内。没有发现氚结合到酶或释放到水中。在甲基丙二酰-CoA和琥珀酰-CoA中发现放射性，其恒定比例为1：3，在反应的前300 ms期间没有变化。在将琥珀酰辅酶A加入全酶溶液中后，氚以基本相同的速率释放，并且在甲基丙二酰辅酶A和琥珀酰辅酶A中发现放射性以1：3的相同恒定比率。使用C-14标记的甲基丙二酰-CoA作为底物测量的氚同位素对酶催化的氢转移的影响为k（H）/k（T）= 4.9。该低值表明，氢转移仅是部分速率限制，并且至少一个随后的缓慢步骤，例如产物释放，对总反应速度有实质性贡献。氚的相同的分区，无论使用的基板，表明基板自由基到产品自由基的重排是不限速的。非常低的氚同位素效应和所有的氚被发现与CoA酯或辅因子结合的事实使得蛋白质自由基不太可能是甲基丙二酰-CoA突变酶催化的重排中的中间体。
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'-[H-3]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 C-14-labeled methylmalonyl-CoA as substrate, was k(H)/k(T) = 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.