2-AMINOBENZOYL-COA MONOOXYGENASE REDUCTASE - EVIDENCE FOR 2 DISTINCT LOCI CATALYZING SUBSTRATE MONOOXYGENATION AND HYDROGENATION/

2-Aminobenzoyl-CoA monooxygenase/reductase catalyzes both monooxygenat ion and hydrogenation of anthraniloyl-CoA. Its reactivity with 11 subs trate analogs has been investigated. Only 2-aminobenzoyl-CoA (anthrani loyl-CoA) in its normal and deuterated (5-H-2) form is a full substrat e, and only traces of 2-hydroxybenzoyl-CoA (salicyloyl-CoA) are probab ly monooxygenated but not hydrogenated. The purified enzyme is a homod imer and has been resolved preparatively into three major species by a nion-exchrtnge chromatography on Mono Q. All three species have the sa me specific activity when reconstituted to full content of FAD, they d iffer, however, substantially in their mode of binding FAD. The oxidiz ed or fully reduced enzyme forms bind tightly 0.5 mol/mol of the subst rate 2-aminobenzoyl-CoA (K-d = 1-2 mu M). The enzyme can be depleted o f approximate to 50% of its FAD, which corresponds to essentially comp lete removal from one of the two binding sites, reflecting a large dif ference in the affinity for FAD. From this it is deduced that the two sites are not equivalent. Removal of FAD from one binding site leads t o loss of the hydrogenation capacity of the enzyme, while monooxygenat ion catalysis is retained. The FAD cofactors of the two binding sites differ drastically in their reactivities towards NADH, oxygen and N-et hylmaleimide. Exchange of reducing equivalents between the two FAD cof actors at the respective binding sites is very slow and irrelevant com pared to the rates of catalysis. It is concluded that the enzyme, whic h has been proposed to consist of two identical polypeptide chains [Al tenschmidt, U., Bokranz, M. and Fuchs, G. (1992) Eur J. Biochem 207, 7 15-722], contains two active centers which differ substantially in the ir catalytic activity. One center belongs to the class of monooxygenas es, the other one to the (de)hydrogenases. This must result from subst antially different interaction of the same flavin cofactors with prote in functional groups and is, to our knowledge, unprecedented in flavop rotein enzymology.