NICKEL-SPECIFIC, SLOW-BINDING INHIBITION OF CARBON-MONOXIDE DEHYDROGENASE FROM RHODOSPIRILLUM-RUBRUM BY CYANIDE
NICKEL-SPECIFIC, SLOW-BINDING INHIBITION OF CARBON-MONOXIDE DEHYDROGENASE FROM RHODOSPIRILLUM-RUBRUM BY CYANIDE
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DOI：
10.1021/bi00438a011
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发表时间：
1989-06-13
期刊：
影响因子：
2.9
通讯作者：
LUDDEN, PW
中科院分区：
文献类型：
--
作者：
ENSIGN, SA;HYMAN, MR;LUDDEN, PW
在存在和不存在 CO 和电子受体的情况下研究了氰化物对红色红螺菌纯化一氧化碳脱氢酶的抑制作用。抑制是一个时间依赖性过程，在两组条件下均表现出伪一级动力学。两种底物均存在时，抑制的真正二级速率常数为 72.2 M-1 s-1，对于与氰化物一起孵育的还原酶和氧化酶，分别为 48.9 和 79.5 M-1 s-1。与 100 μM KCN 孵育 25 分钟后，CO 部分保护酶免受抑制。计算出氰化物和CO与酶的结合的解离常数为8.46μM (KCN)和4.70μM (CO)。除去未结合的氰化物后，在 CO 气氛下，氰化物抑制是完全可逆的。 N2 无法逆转抑制作用。缺镍 (apo) CO 脱氢酶被 NiCl2 激活的能力不受事先与氰化物孵育的影响。添加 NiCl2 不会逆转氰化物对 Holo-CO 脱氢酶的抑制作用。通过凝胶过滤色谱法，14CN-仍然与全酶结合，但不与脱辅基酶结合。这些发现表明氰化物是一种缓慢结合、活性位点定向、镍特异性、可逆的 CO 脱氢酶抑制剂。我们提出，氰化物通过成为 CO 类似物并通过酶结合镍结合来抑制 CO 脱氢酶。
The inhibition of purified carbon monoxide dehydrogenase from Rhodospirillum rubrum by cyanide was investigated in both the presence and absence of CO and electron acceptor. The inhibition was a time-dependent process exhibiting pseudo-first-order kinetics under both sets of conditions. The true second-order rate constants for inhibition were 72.2 M-1 s-1 with both substrates present and 48.9 and 79.5 M-1 s-1, respectively, for the reduced and oxidized enzymes incubated with cyanide. CO partially protected the enzyme against inhibition after 25-min incubation with 100 .mu.M KCN. Dissociation constants of 8.46 .mu.M (KCN) and 4.70 .mu.M (CO) were calculated for the binding of cyanide and CO to the enzyme. Cyanide inhibition was fully reversible under an atmosphere of CO after removal of unbound cyanide. N2 was unable to reverse the inhibition. The competence of nickel-deficient (apo) CO dehydrogenase to undergo activation by NiCl2 was unaffected by prior incubation with cyanide. Cyanide inhibition of holo-CO dehydrogenase was not reversed by addition of NiCl2. 14CN- remained associated with holoenzyme but not with apoenzyme through gel filtration chromatography. These findings suggest that cyanide is a slow-binding, active-site-directed, nickel-specific, reversible inhibitor of CO dehydrogenase. We propose that cyanide inhibits CO dehydrogenase by being an analogue of CO and by binding through enzyme-bound nickel.