Methylation of carbon monoxide dehydrogenase from Clostridium thermoaceticum and mechanism of acetyl coenzyme A synthesis
Methylation of carbon monoxide dehydrogenase from Clostridium thermoaceticum and mechanism of acetyl coenzyme A synthesis
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DOI：
10.1021/ja963597k
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发表时间：
1997-04-30
影响因子：
15
通讯作者：
Lindahl, PA
中科院分区：
文献类型：
--
作者：
Barondeau, DP;Lindahl, PA
对来自热醋酸梭菌的一氧化碳脱氢酶进行了甲基化，使所有结合的甲基都能与一氧化碳和辅酶A(或OH-)反应生成乙酰辅酶A(或乙酸酯)。甲基不能结合缺乏A-簇不稳定镍的酶，但在Ni2+水溶液中孵育后可以结合这些样品，这一过程已知重新插入不稳定镍并重新激活酶。结合甲基对1，10-菲咯啉去除活性镍的能力有抑制作用，其结合量与活性镍的量大致相关。这是乙酰辅酶A合成中使用的甲基与不稳定的镍结合的有力证据。有证据表明，定义A-簇的自旋耦合金属以外的氧化还原位(称为D位)必须在甲基化发生之前被还原。甲基和乙酰基中间体似乎都是EPR沉默的。乙酰基中间体与OH-反应缓慢生成乙酸酯，与COAS(-)快速反应生成乙酰辅酶A。当A-簇还原并与CO结合的酶(S=1/2 A(红)-CO状态)被甲基化时，得到的乙酰基中间体也是EPR沉默的，表明底物添加顺序对得到的乙酰基中间体的EPR沉默没有影响。D位似乎是n=2的氧化还原剂，其作用是在甲基化时还原氧化的A-簇，并在产物乙酰辅酶A解离时氧化A-簇。D在两种氧化状态下都是EPR沉默的，并且不是酶中任何已知的金属簇。D可能是与不稳定的镍配位的一对特殊的半胱氨酸，可以在异常低的电位下被氧化成半胱氨酸(类似于-530 mV vs NHE)。不含D或其功能等价物的催化机理，或使用A-团簇的约化S=1/2 CO结合形式作为中间体的催化机理，与目前的数据不一致。结合本研究的结果，提出了一种新的催化机理。
Carbon monoxide dehydrogenase from Clostridium thermoaceticum was methylated such that all bound methyl groups could subsequently react with CO and coenzyme A (or OH-) to yield acetyl-coenzyme A (acetyl-CoA) (or acetate). Methyl groups could not bind enzyme lacking the labile Ni of the A-cluster, but could bind such samples after incubation in aqueous Ni2+, a process known to reinsert the labile Ni and reactivate the enzyme. Bound methyl groups inhibited the ability of 1,10-phenanthroline to remove the labile Ni, and the amount bound approximately correlated with the amount of labile Ni. This is strong evidence that the methyl group used in acetyl-CoA synthesis binds the labile Ni. Evidence is presented that a redox site (called the D site) other than the spin-coupled metals that define the A-cluster must be reduced before methylation can occur. Both methyl and acetyl intermediates appear to be EPR-silent. The acetyl intermediate reacted slowly with OH- to yield acetate and rapidly with CoAS(-) to yield acetyl-CoA. When enzyme in a state with the A-cluster reduced and bound with CO (the S = 1/2 A(red)-CO state) was methylated, the resulting acetyl intermediate was also EPR-silent, indicating that the order of substrate addition had no effect on the EPR silence of the resulting acetyl intermediate. The D site appears to be an n = 2 redox agent that functions to reduce the oxidized A-cluster upon methylation and to oxidize the A-cluster as the product acetyl-CoA dissociates. D is EPR-silent in both of its oxidation states and is not any of the known metal clusters in the enzyme. D may be a special pair of cysteines coordinated to the labile Ni that can be oxidized to cystine at unusually low potentials (similar to -530 mV vs NHE). Catalytic mechanisms that do not include D or its functional equivalent, or that employ the reduced S = 1/2 CO-bound form of the A-cluster as an intermediate, are inconsistent with the present data. A new catalytic mechanism incorporating the results of this study is proposed.