Thomä N H, Meier T W, Evans P R, Leadlay P F
Department of Biochemistry, Cambridge Centre for Molecular Recognition, University of Cambridge, United Kingdom.
Biochemistry. 1998 Oct 13;37(41):14386-93. doi: 10.1021/bi981375o.
The adenosylcobalamin-dependent methylmalonyl-CoA mutase catalyzes the reversible rearrangement of methylmalonyl-CoA into succinyl-CoA by a free-radical mechanism. The recently solved X-ray crystal structure of methylmalonyl-CoA mutase from Propionibacterium shermanii has shown that tyrosine 89 is an active-site residue involved in substrate binding. The role of tyrosine 89, a conserved residue among methylmalonyl-CoA mutases, has been investigated by using site-directed mutagenesis to replace this residue with phenylalanine. The crystal structure of the Tyr89Phe mutant was determined to 2.2 A resolution and was found to be essentially superimposable on that of wild-type. Mutant and wild-type enzyme have very similar KM values, but kcat for the Tyr89Phe mutant is 580-fold lower than for wild-type. The rate of release of tritium from 5'-[3H]adenosylcobalamin during the enzymatic reaction and its rate of appearance in substrate and product were measured. The tritium released was found to partition unequally between methylmalonyl-CoA and succinyl-CoA, in a ratio of 40:60 when the reaction was initiated by addition of methylmalonyl-CoA and in a ratio of 10:90 when the reaction was initiated by addition of succinyl-CoA. The overall release of tritium was four times faster when succinyl-CoA was used as substrate. The tritium isotope effect on the enzyme catalyzed hydrogen transfer, measured with methylmalonyl-CoA as a substrate, was kH/kT = 30, which is within the expected range for a full primary kinetic tritium isotope effect. The different partitioning of tritium, dependent upon which substrate was used, and the normal value for the kinetic tritium isotope effect contrast markedly with the behavior of wild-type mutase. It appears that the loss of a single interaction involving the hydroxyl group of tyrosine 89 both affects the stability of radical intermediates and decreases the rate of interconversion of the substrate- and product-derived radicals.
腺苷钴胺素依赖性甲基丙二酰辅酶A变位酶通过自由基机制催化甲基丙二酰辅酶A可逆重排为琥珀酰辅酶A。最近解析的来自谢氏丙酸杆菌的甲基丙二酰辅酶A变位酶的X射线晶体结构表明，酪氨酸89是参与底物结合的活性位点残基。通过定点诱变将该残基替换为苯丙氨酸，对甲基丙二酰辅酶A变位酶中保守的酪氨酸89的作用进行了研究。Tyr89Phe突变体的晶体结构在2.2埃分辨率下测定，发现其与野生型基本重叠。突变体和野生型酶具有非常相似的KM值，但Tyr89Phe突变体的kcat比野生型低580倍。测定了酶促反应过程中5'-[3H]腺苷钴胺素中氚的释放速率及其在底物和产物中的出现速率。发现释放的氚在甲基丙二酰辅酶A和琥珀酰辅酶A之间分配不均，当反应由添加甲基丙二酰辅酶A引发时，比例为40:60，当反应由添加琥珀酰辅酶A引发时，比例为10:90。当使用琥珀酰辅酶A作为底物时，氚的总体释放速度快四倍。以甲基丙二酰辅酶A为底物测定的氚同位素对酶催化氢转移的影响为kH/kT = 30，这在完全一级动力学氚同位素效应的预期范围内。氚的不同分配取决于使用的底物，以及动力学氚同位素效应的正常值与野生型变位酶的行为形成明显对比。似乎涉及酪氨酸89羟基的单一相互作用的丧失既影响自由基中间体的稳定性，又降低了底物衍生自由基和产物衍生自由基的相互转化速率。
