Kinetic mechanism of the hydrogen-oxidizing hydrogenase from soybean nodule bacteroids.
Kinetic mechanism of the hydrogen-oxidizing hydrogenase from soybean nodule bacteroids.
复制标题
大豆根瘤类菌氧化氢化酶的动力学机制。
DOI：
10.1021/bi00511a025
复制
发表时间：
1981
期刊：
影响因子：
2.9
通讯作者：
R. Burris
中科院分区：
文献类型：
--
作者：
D. Arp;R. Burris
用高纯度的酶研究了大豆根瘤类菌单向H2氧化氢化酶的动力学机制。 H2 的 Km 测量值在 0.97 至 2.6 microM 之间变化，亚甲基蓝的 Km 测量值在 6 至 17 microM 之间变化。以 H2 和亚甲基蓝为底物，初速度模式是相交的。高水平的亚甲蓝具有抑制作用 (KI =2.4 mM)：这种抑制作用与 H2 具有竞争性。 CO 是 H2 的竞争性抑制剂 (KI = 157 microM)，与亚甲蓝没有竞争性。 O2 使酶失活（t 1/2 大约 1 小时），但在短时间暴露于低浓度时也是氢化酶活性的可逆抑制剂。 O2 的抑制作用与 H2 没有竞争性，与亚甲基蓝也没有竞争性。氢化酶不受C2H2抑制；在 C2H2 下预孵育会使酶失活。减少的甲基和苄基紫精可降低氢化酶产生 H2 的速率。还原型甲基紫精的 Km 为 11 microM。 H2 是 H2 演化的有效抑制剂：与还原的甲基紫精相比，该抑制是非竞争性的。氢化酶将催化 D2 和 H2O 之间反应形成 HD 和 H2 的低速率交换。我们提出了一种酶的双位点乒乓机制，其中 H2 在一个位点可逆激活，电子载体在第二个位点相互作用。
The kinetic mechanism of the unidirectional H2-oxidizing hydrogenase from soybean nodule bacteroids has been investigated with highly purified enzyme. Measurements of the Km for H2 vary from 0.97 to 2.6 microM, and the Km for methylene blue varies from 6 to 17 microM. With H2 and methylene blue as substrates, the initial velocity patterns are intersecting. High levels of methylene blue are inhibitory (KI =2.4 mM): the inhibition is competitive vs. H2. CO is a competitive inhibitor of H2 (KI = 157 microM) and noncompetitive vs. methylene blue. O2 inactivates the enzyme (t 1/2 approximately 1 h) but also is a reversible inhibitor of hydrogenase activity upon short exposure to low concentrations. Inhibition by O2 is uncompetitive vs. H2 and noncompetitive vs. methylene blue. Hydrogenase was not inhibited by C2H2; preincubation under C2H2 inactivates the enzyme. Reduced methyl and benzyl viologens support low rates of H2 evolution by the hydrogenase. The Km for reduced methyl viologen is 11 microM. H2 is a potent inhibitor of H2 evolution: the inhibition is noncompetitive vs. reduced methyl viologen. The hydrogenase will catalyze a low rate of exchange in the reaction between D2 and H2O to form both HD and H2. We propose a two-site ping-pong mechanism for the enzyme in which H2 is reversibly activated at one site and e- carriers interact at the second site.