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

Abstract

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 ≈40 min−1 was found at pH 7.4 and 4°C. 2-Aminobenzoyl-CoA binds to oxidized enzyme to form a complex which is in a ≈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 ≈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≥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 ≈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 ≈ 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. This explains the formation of different products depending on the actual concentration of NADH in the catalytic system, which has been reported previously [Buder, R., Ziegler, K., Fuchs, G., Langkau, B. & Ghisla, S. (1989) Eur. J. Biochem. 185, 637 643]. A kinetic mechanism is proposed based on the concept that aminobenzoyl-CoA monooxygenase/reductase has two active sites which catalyze independently monooxygenation and hydrogenation of substrate or intermediate.