PARTICIPATION OF COB(I)ALAMIN IN THE REACTION CATALYZED BY METHIONINE SYNTHASE FROM ESCHERICHIA-COLI - A STEADY-STATE AND RAPID REACTION KINETIC-ANALYSIS
PARTICIPATION OF COB(I)ALAMIN IN THE REACTION CATALYZED BY METHIONINE SYNTHASE FROM ESCHERICHIA-COLI - A STEADY-STATE AND RAPID REACTION KINETIC-ANALYSIS
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DOI：
10.1021/bi00502a013
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发表时间：
1990-12-18
期刊：
影响因子：
2.9
通讯作者：
MATTHEWS, RG
中科院分区：
文献类型：
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作者：
BANERJEE, RV;FRASCA, V;MATTHEWS, RG
通过稳态和稳态前动力学分析，研究了大肠杆菌K12钴胺素依赖性蛋氨酸合酶催化反应的动力学机理。甲硫氨酸合酶催化的反应涉及甲基从甲基四氢叶酸转移到高半胱氨酸以生成四氢叶酸和甲硫氨酸。假设的反应机制调用甲基基团的初始转移到酶，以产生酶结合的甲钴胺和四氢叶酸。酶结合的甲钴胺素然后将其甲基捐赠给同型半胱氨酸，以产生甲硫氨酸和钴（I）维生素。在这项研究中解决的关键问题是：（1）反应是否涉及一个顺序或乒乓机制？(2)酶结合的辅酶I是一种动力学活性中间体吗？(3)如果反应确实涉及顺序机制，那么底物结合的“游离”酶的性质是什么;即，辅基是cob（I）alamin还是methycobalamin状态？稳态和快速反应研究均在25 ℃下进行。C在厌氧条件下。在稳态条件下的初始速度分析表明，一个家庭的平行线建议乒乓机制或有序的顺序机制。稳态产物抑制研究提供了证据的有序的顺序机制，其中第一个底物结合是甲基四氢叶酸和最后一个产品被释放是四氢叶酸。然后进行预稳态动力学研究以确定各种反应的速率常数。酶结合的辅酶（I）丙氨酸被证明与甲基四氢叶酸反应非常迅速（观察到的速率常数为250 s-1，而最大速度条件下的周转数为19 s-1）。酶结合的辅酶（I）alamin也被证明形成迅速（140 s-1）时，同型半胱氨酸与甲基化酶混合，从而建立其动力学相关的中间体。一个最小的动力学机制，容纳的稳态数据和测得的速率常数被用来模拟系统的动力学行为。根据辅基在“游离”酶中是处于cob（I）alamin还是甲钴胺状态，模拟做出了非常不同的预测，并且仅当使用含有结合的甲钴胺的酶作为起始酶时，才产生与真实的数据的极好拟合。从这些研究中得出结论，甲基钴胺素含甲硫氨酸合酶是酶的“游离”形式，底物以有序的顺序方式与之结合，并且酶结合的辅酶（I）丙氨酸是动力学活性中间体。
The kinetic mechanism of the reaction catalyzed by cobalamin-dependent methionine synthase from Escherichia coli K12 has been investigated by both steady-state and pre-steady-state kinetic analyses. The reaction catalyzed by methionine synthase involves the transfer of a methyl group from methyltetrahydrofolate to homocysteine to generate tetrahydrofolate and methionine. The postulated reaction mechanism invokes an initial transfer of the methyl group to the enzyme to generate enzyme-bound methylcobalamin and tetrahydrofolate. Enzyme-bound methylcobalamin then donates its methyl group to homocysteine to generate methionine and cob(I)alamin. The key questions that were addressed in this study were the following: (1) Does the reaction involve a sequential or ping-pong mechanism? (2) Is enzyme-bound cob(I)alamin a kinetically competent intermediate? (3) If the reaction does involve a sequential mechanism, what is the nature of the "free" enzyme to which the substrates bind; i.e., is the prosthetic group in the cob(I)alamin or methylcobalamin state? Both the steady-state and rapid reaction studies were conducted at 25.degree. C under anaerobic conditions. Initial velocity analysis under steady-state conditions revealed a family of parallel lines suggesting either a ping-pong mechanism or an ordered sequential mechanism. Steady-state product inhibition studies provided evidence for an ordered sequential mechanism in which the first substrate to bind is methyltetrahydrofolate and the last product to be released is tetrahydrofolate. Pre-steady-state kinetic studies were then conducted to determine the rate constants for the various reactions. Enzyme-bound cob(I)alamin was shown to react very rapidly with methyltetrahydrofolate (with an observed rate constant of 250 s-1 versus a turnover number under maximal velocity conditions of 19 s-1). Enzyme-bound cob(I)alamin was also shown to form rapidly (140 s-1) when homocysteine was mixed with methylated enzyme, thus establishing its kinetic relevance as an intermediate. A minimal kinetic mechanism that accommodates the steady-state data and the measured rate constants was employed to simulate the kinetic behavior of the system. The simulations made very different predictions depending on whether the prosthetic group was in the cob(I)alamin or methylcobalamin state in "free" enzyme and yielded an excellent fit to the real data only when enzyme containing bound methylcobalamin was employed as the starting enzyme. From these studies it was concluded that methylcobalamin-containing methionine synthase is the "free" form of the enzyme, to which the substrates bind in an ordered sequential fashion, and that enzyme-bound cob(I)alamin is a kinetically competent intermediate.