Raybuck S A, Bastian N R, Orme-Johnson W H, Walsh C T
Department of Chemistry, Massachusetts Institute of Technology, Cambridge 02139.
Biochemistry. 1988 Oct 4;27(20):7698-702. doi: 10.1021/bi00420a019.
CO dehydrogenase from Clostridium thermoaceticum is a nickel-containing enzyme that catalyzes both the reversible conversion of CO2 to CO (for incorporation into the carbonyl group of acetate) and the synthesis of acetyl-CoA from methyl corrinoid, CO, and CoASH. The latter activity is conveniently assayed by monitoring the exchange of [1-14C]acetyl-CoA (carbonyl group) with 12CO. Kinetic parameters for the highly oxygen sensitive exchange activity have been determined: Km (acetyl-CoA) = 600 microM; Vmax = 440 min-1. In addition, coenzyme A analogues have been tested as inhibitors of the exchange to probe the active site of the enzyme; each has no effect on the CO2 in equilibrium CO activity of CO dehydrogenase. Coenzyme A, the substrate for acetate biosynthesis, is a potent competitive inhibitor, KI = 7 microM. Comparison of this value with that for desulfo-CoA (KI = 6000 microM) suggests that a key mode of binding is through the sulfur atom, possibly to a metal site on the enzyme. The relatively high affinity of the enzyme for CoASH relative to acetyl-CoA is consistent with its proposed operation in the acetogenic direction. The differential sensitivity to oxygen and storage of the two activities of CO dehydrogenase as well as the contrasting effect of coenzyme A inhibitors suggests that acetate assemblage occurs at a site distinct from that for CO dehydrogenation.
来自热醋梭菌的一氧化碳脱氢酶是一种含镍酶，它既能催化二氧化碳可逆转化为一氧化碳（用于并入乙酸的羰基），又能催化从甲基类咕啉、一氧化碳和辅酶A合成乙酰辅酶A。后一种活性可通过监测[1-¹⁴C]乙酰辅酶A（羰基）与¹²CO的交换来方便地测定。已经确定了对高氧敏感的交换活性的动力学参数：Km（乙酰辅酶A）=600微摩尔；Vmax = 440分钟⁻¹。此外，还测试了辅酶A类似物作为该交换反应的抑制剂，以探测该酶的活性位点；每种类似物对一氧化碳脱氢酶的平衡一氧化碳活性中的二氧化碳均无影响。辅酶A是乙酸生物合成的底物，是一种有效的竞争性抑制剂，KI = 7微摩尔。将该值与脱硫辅酶A的值（KI = 6000微摩尔）进行比较表明，一种关键的结合方式是通过硫原子，可能是与酶上的一个金属位点结合。该酶对辅酶A相对于乙酰辅酶A具有较高的亲和力，这与其在产乙酸方向上的假定作用一致。一氧化碳脱氢酶的两种活性对氧气和储存的敏感性差异以及辅酶A抑制剂的不同作用表明，乙酸组装发生在与一氧化碳脱氢不同的位点。
