Tan A K, Ramsay R R
Department of Biochemistry and Biophysics, University of California, San Francisco 94143.
Biochemistry. 1993 Mar 9;32(9):2137-43. doi: 10.1021/bi00060a003.
Monoamine oxidases A and B have identical flavin sites but different, although overlapping, amine substrate specificity. Reoxidation of ternary complexes containing substrate is much faster than of free enzyme, and the enhancement is greater in the A form than the B form. The oxidative half-reaction was studied with a variety of substrates to elucidate the specificity of the effect and to probe the different influences of substrate on the flavin reoxidation in the two forms of the enzyme. The second-order rate constant for the reoxidation was highest with monoamine oxidase A when kynuramine was the ligand (508 x 10(3) M-1 s-1) compared to 4 x 10(3) M-1 s-1 in its absence. MPTP (166 x 10(3) M-1 s-1) also enhanced reoxidation well, but indole substrates stimulated only poorly (e.g., tryptamine, 29 x 10(3) M-1 s-1; serotonin, 50 x 10(3) M-1 s-1). For the A form, the reduction of the flavin was rate-limiting in all cases. For the B form, reoxidation was rate-limiting for beta-phenylethylamine and contributed to the determination of the overall rate with several substrates. The ratio of the enhanced rate of oxidation to the rate of reduction correlated with the redox state of the enzyme in turnover experiments. All the observations are consistent with alternate paths of reoxidation, via either free enzyme or a reduced enzyme-substrate complex. The flux through each path is determined by the relative dissociation constants and rate constants.
单胺氧化酶A和B具有相同的黄素位点，但胺底物特异性不同，尽管存在重叠。含有底物的三元复合物的再氧化比游离酶快得多，且A形式的增强作用比B形式更大。用多种底物研究了氧化半反应，以阐明该效应的特异性，并探究底物对两种酶形式中黄素再氧化的不同影响。当犬尿胺作为配体时，单胺氧化酶A的再氧化二级速率常数最高（508×10³ M⁻¹ s⁻¹），相比之下，无配体时为4×10³ M⁻¹ s⁻¹。MPTP（166×10³ M⁻¹ s⁻¹）也能很好地增强再氧化，但吲哚底物的刺激作用较差（例如，色胺为29×10³ M⁻¹ s⁻¹；5-羟色胺为50×10³ M⁻¹ s⁻¹）。对于A形式，在所有情况下黄素的还原都是限速步骤。对于B形式，β-苯乙胺的再氧化是限速步骤，并且对几种底物的总速率测定有影响。在周转实验中，氧化增强速率与还原速率的比值与酶的氧化还原状态相关。所有观察结果都与通过游离酶或还原型酶-底物复合物的交替再氧化途径一致。通过每条途径的通量由相对解离常数和速率常数决定。
