Vienozinskis J, Butkus A, Cenas N, Kulys J
Institute of Biochemistry, Lithuanian Academy of Sciences, Vilnius, U.S.S.R.
Biochem J. 1990 Jul 1;269(1):101-5. doi: 10.1042/bj2690101.
The relationship between the NADH:lipoamide reductase and NADH:quinone reductase reactions of pig heart lipoamide dehydrogenase (EC 1.6.4.3) was investigated. At pH 7.0 the catalytic constant of the quinone reductase reaction (kcat.) is 70 s-1 and the rate constant of the active-centre reduction by NADH (kcat./Km) is 9.2 x 10(5) M-1.s-1. These constants are almost an order lower than those for the lipoamide reductase reaction. The maximal quinone reductase activity is observed at pH 6.0-5.5. The use of [4(S)-2H]NADH as substrate decreases kcat./Km for the lipoamide reductase reaction and both kcat. and kcat./Km for the quinone reductase reaction. The kcat./Km values for quinones in this case are decreased 1.85-3.0-fold. NAD+ is a more effective inhibitor in the quinone reductase reaction than in the lipoamide reductase reaction. The pattern of inhibition reflects the shift of the reaction equilibrium. Various forms of the four-electron-reduced enzyme are believed to reduce quinones. Simple and 'hybrid ping-pong' mechanisms of this reaction are discussed. The logarithms of kcat./Km for quinones are hyperbolically dependent on their single-electron reduction potentials (E1(7]. A three-step mechanism for a mixed one-electron and two-electron reduction of quinones by lipoamide dehydrogenase is proposed.
研究了猪心脂酰胺脱氢酶（EC 1.6.4.3）的NADH：脂酰胺还原酶反应与NADH：醌还原酶反应之间的关系。在pH 7.0时，醌还原酶反应的催化常数（kcat.）为70 s-1，NADH对活性中心的还原速率常数（kcat./Km）为9.2×10(5) M-1·s-1。这些常数比脂酰胺还原酶反应的常数低近一个数量级。在pH 6.0 - 5.5时观察到最大的醌还原酶活性。使用[4(S)-2H]NADH作为底物会降低脂酰胺还原酶反应的kcat./Km以及醌还原酶反应的kcat.和kcat./Km。在这种情况下，醌的kcat./Km值降低了1.85 - 3.0倍。NAD+在醌还原酶反应中比在脂酰胺还原酶反应中是更有效的抑制剂。抑制模式反映了反应平衡的移动。据信四电子还原酶的各种形式可还原醌。讨论了该反应的简单和“混合乒乓”机制。醌的kcat./Km的对数与其单电子还原电位（E1(7]呈双曲线相关。提出了脂酰胺脱氢酶对醌进行单电子和双电子混合还原的三步机制。
