Identification of a new reaction intermediate in the oxidation of methylamine dehydrogenase by amicyanin

The two-electron oxidation of tryptophan tryptophylquinone (TTQ) in substra te-reduced methylamine dehydrogenase (MADH) by amicyanin is known to procee d via an N-semiquinone intermediate in which the substrate-derived amino gr oup remains covalently attached to TTQ [Bishop, G. R,, and Davidson, V. L. (1996) Biochemistry 35, 8948-8954]. A new method for the stoichiometric for mation of the N-semiquinone in vitro has allowed the study of the oxidation of the N-semiquinone by amicyanin in greater detail than was previously po ssible. Conversion of N-semiquinone TTQ to the quinone requires two biochem ical events, electron transfer to amicyanin and release of ammonia from TTQ , Using rapid scanning stopped-flow spectroscopy, it is shown that this occ urs by a sequential mechanism in which oxidation to an imine (N-quinone) pr ecedes hydrolysis by water and ammonia release. Under certain reaction cond itions, the N-quinone intermediate accumulates prior to the relatively slow hydrolysis step. Correlation of these transient kinetic data with steady-s tate kinetic data indicates that the slow hydrolysis of the N-quinone by wa ter does not occur in the steady state. In the presence of excess substrate , the next methylamine molecule initiates a nucleophilic attack of the N-qu inone TTQ, causing release of ammonia that is concomitant with the formatio n of the next enzyme-substrate cofactor adduct. In light of these results, the usually accepted steady-state reaction mechanism of MADH is revised and clarified to indicate that reactions of the quinone form of TTQ are side r eactions of the normal catalytic pathway. The relevance of these conclusion s to the reaction mechanisms of other enzymes with carbonyl cofactors, the reactions of which proceed via Schiff base intermediates, is also discussed .