THE REACTION-MECHANISM OF COPPER AMINE OXIDASE - DETECTION OF INTERMEDIATES BY THE USE OF SUBSTRATES AND INHIBITORS

Intermediate states in the catalytic mechanism of lentil copper amine oxidase have been investigated by ESR and optical spectroscopy, Using highly purified apo- and holoenzyme in combination with a poor substra te and a range of inhibitors, under both aerobic and anaerobic conditi ons, the single steps of the reaction mechanism can be slowed down or 'frozen' completely. In this way, a sequence of six intermediate speci es in the catalytic cycle has been established. Oxidative deamination of p-(dimethylamino)benzylamine is 5 x 10(5) times slower than for put rescine; the rate-limiting step is shown to be release of the aldehyde product. This process is not affected in the apoenzyme, but subsequen t intramolecular electron transfer to form the characteristic free rad ical intermediate is completely blocked, and the apoenzyme is trapped as an aminoresorcinol species. Classic hydrazine and hydrazide inhibit ors bind to the 6-hydroxydopa cofactor in the same way as active subst rates, but rearrangements lead to formation of stable intermediate add ucts at the step preceding release of aldehyde. The semicarbazide-6-hy droxydopa adduct is shown to bind simultaneously to Cu(II), providing the first direct evidence for localization of 6-hydroxydopa close to t he copper site.