THE MECHANISM OF THE QUINONE REDUCTASE REACTION OF PIG-HEART LIPOAMIDE DEHYDROGENASE

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 (k(cat.)) is 70 s-1 and the rate constant of the active-centre reduction by NADH (k(cat.)/K(m)) is 9.2 x 105 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 k(cat.)/K(m) for the lipoamide reductase reaction and both k(cat.) and k(cat.)/K(m) for the quinone reductase reaction. The k(cat.)/K(m) 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 k(cat.)/K(m) for quinones are hyperbolically dependent on their single-electron reduction potentials (E71). A three-step mechanism for a mixed one-electron and two-electron reduction of quinones by lipoamide dehydrogenase is proposed.