2-Aminobenzoyl-CoA Monooxygenase/Reductase. Evidence for Two Distinct Loci Catalyzing Substrate Monooxygenation and Hydrogenation

Langkau, Bernd; Vock, Petra; Massey, Vincent; Fuchs, Georg; Ghisla, Sandro

doi:10.1111/j.1432-1033.1995.0676h.x

Cited by 8 publications

(22 citation statements)

References 18 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In the preceding paper [in this journal (Langkau et al, 1995)], a kinetic scheme was elaborated for enzyme turnover which is consistent with the low NADH oxidase activity of ABzCoA-M/R as compared to the high turnover with NADH and the artificial electron acceptor N-ethylmaleimide (MalNEt).…”

mentioning

confidence: 77%

“…The enzyme was purified according to . Activity of ABzCoA-M/R was assayed by following the disappearance of NADH at 340 nm using either ABzCoA or MalNEt (Langkau et al, 1995) as substrates. H,O, was determined enzymically according to Lockridge et al (1972).…”

Section: Methodsmentioning

confidence: 99%

“…All experiments were performed at 4°C in 50 mM potassium phosphate pH 7.4, containing 10 mM EDTA in the cases where the enzyme was reduced photochemi- cally prior to the experiment. Anaerobiosis and photoreduction of ABzCoA-M/R was achieved as described previously (Langkau et al, 1995), but using special tonometers by which the solutions were transferred to the stopped-flow syringes. Unless otherwise noted, all concentrations mentioned in the context of stopped flow experiments are those of the reagent after mixing, i.e.…”

Section: Methodsmentioning

confidence: 99%

“…In the work described in this paper steady-state, pre-steady-state and post-steady-state reactions of ABzCoA-M/R have been studied using the stopped-flow technique. Based on these results and on those of the preceding paper (Langkau et al, 1995), a more detailed mechanistic scheme is proposed, in which the reactions occumng on the monooxygenating site (E') take place independently from those at the hydrogenating site (E") and converge in forming the final products. Terms and values for k, , , K,(NADH), K,(ABzCoA) and K,(Oz) have been derived.…”

mentioning

confidence: 91%

“…on the relative concentrations of reactants and on incubation times . ABzCoA-MIR is most unusual in that it catalyzes two mechanistically different reactions and appears to have two separate active centers, built up by two identical protein chains and forming a dimer with two FAD molecules (Langkau et al, 1995). Only one molecule ABzCoA or analogous CoA ester is boundtwo enzyme FAD molecules, indicating the presence of only one binding siteldimer.…”

mentioning

confidence: 99%

See 4 more Smart Citations

Kinetic and Mechanistic Studies on the Reactions of 2-Aminobenzoyl-CoA Monooxygenase/Reductase

Langkau¹,

Ghisla²

1995

Eur J Biochem

Self Cite

View full text Add to dashboard Cite

The kinetic mechanism of the flavoprotein 2-aminobenzoyl-CoA monooxygenase lreductase with its natural substrates 2-aminobenzoyl-CoA, NADH and 0, 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 m i x ' 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 FADH, 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-') 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 k,, 2-80 s-I. 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-', 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 (kbs = 0.05 s?). This third 'post-catalytic' process is not relevant for catalysis. The primary product 2-amino-5-oxocyclohex-I -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 0, to form H,O,. 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. Biochern. 185,. A kinetic mechanism is proposed based on the concept that aminobenzoyl-CoA monooxygenaselreductase has two active sites which catalyze independently monooxygenation and hydrogenation of substrate or intermediate.

show abstract

mentioning

confidence: 77%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 91%

mentioning

confidence: 99%

See 3 more Smart Citations