Kinetic and mechanistic studies on the reactions of 2-aminobenzoyl-CoA monooxygenase/reductase.

The kinetic mechanism of the flavoprotein 2-aminobenzoyl-CoA monooxygenase/reductase with its natural substrates 2-aminobenzoyl-CoA, NADH and O2 has been investigated using the stopped-flow technique. Initial rate measurements indicate the formation of a ternary complex between oxidized enzyme and the two substrates 2-aminobenzoyl-CoA and NADH, a turnover number of approximately 40 min-1 was found at pH 7.4 and 4 degrees C. 2-Aminobenzoyl-CoA binds to oxidized enzyme to form a complex which is in a approximately 1:1 equilibrium with a second, spectrophotometrically distinguishable one. Binding of 2-amino benzoyl-CoA to reduced enzyme is, in contrast, a simple second-order process. Reduction of oxidized enzyme, both uncomplexed and in complex with 2-aminobenzoyl-CoA, by NADH is strongly biphasic. The first fast phase yields enzyme in which 50% of the total FAD is reduced to the FADH2 state. This rate is not affected by the presence of 2-aminobenzoyl-CoA. In contrast, 2-aminobenzoyl-CoA enhances approximately 100-fold the second phase, the reduction of the residual 50% FAD. This second phase of reduction (kobs = 2.0 s-1) is partially rate-limiting in catalysis. The oxygen reaction of uncomplexed, reduced enzyme is also biphasic and no oxygenated intermediate was detected. Reoxidation of substrate-complexed, reduced enzyme involves three spectroscopically distinguishable species. The first observable intermediate is highly fluorescent suggesting that it consists largely of flavin-4a-hydroxide. Thus, insertion of oxygen into 2-aminobenzoyl-CoA is essentially complete at this point and has a kobs > or = 80 s-1. The subsequent phase is accompanied by formation of the main product, 2-amino-5-oxocyclohex-1-enecarboxyl CoA. This step consists in a hydrogenation of the primary, oxygenated and non-aromatic CoA intermediate; it has a rate approximately 1.3 s-1, which is thus the second rate-limiting step in catalysis. As a side reaction of the oxidized enzyme and at low NADH concentrations the initially formed product disappears at a very slow rate (kobs approximately 0.05 s-1). This third 'post-catalytic' process is not relevant for catalysis. The primary product 2-amino-5-oxocyclohex-1-enecarboxyl-CoA is dehydrogenated by the oxidized enzyme to yield the aromatic 2-amino-5-hydroxybenzoyl-CoA as secondary product. The reduced enzyme formed in this process is reoxidized by O2 to form H2O2.(ABSTRACT TRUNCATED AT 400 WORDS)