Spectroscopic studies of the reaction between bovine serum amine oxidase (copper-containing) and some hydrazides and hydrazines

Abstract

The carbonyl cofactor of bovine serum amine oxidase, recently identified as pyrroloquinoline quinone [Ameyama, Hayashi, Matsushita, Shinagawa & Adachi (1984) Agric. Biol. Chem. 48, 561-565; Lobenstein-Verbeek, Jongejan, Frank & Duine (1984) FEBS Lett. 170, 305-309], reacts stoichiometrically and irreversibly with hydrazides of phenylacetic acid and of benzoic acid. With the phenylacetic hydrazides a reversible intermediate step was detected by competition with substrate, carbonylic reagents or phenylhydrazine, a typical inhibitor of the enzyme. All hydrazides form an intense broad band with maximum absorbance in a narrow wavelength range (350-360 nm), irrespective of the acyl group, suggesting that the transition is located on the organic cofactor. A different situation is found with some phenylhydrazines, where extended conjugation can occur between the cofactor and the phenyl pi-electron system via the azo group, as shown by the lower energy and higher intensity of the transition. In this case the transition is sensitive to substituents in the phenyl ring. The c.d. spectrum of the adducts is influenced by the type of hydrazide (derived from phenylacetic acid or benzoic acid), by pH and by NN-diethyldithiocarbamate binding to copper, probably as a result of shifts of equilibria between hydrazone-azo tautomers.

Crystal Structure of Amine Oxidase from Bovine Serum

“…In fact, the end of the arm of one subunit is positioned at the mouth of the active-site channel of the other subunit, suggesting the possibility of interaction between the two active sites. To this regard, in the case of BSAO half-of-thesites reactivity has been reported, 49,50 suggesting the possibility of a negative cooperativity.…”

“…44 However, functional and mechanistic studies on BSAO have yielded limited structural information. 19,[21][22][23]32,[45][46][47][48][49][50][51] As such, our current understanding of this enzyme, including its substrate specificity in terms of structural features, is largely speculative.…”

Benzylhydrazine as a pseudo-substrate of bovine serum amine oxidase

“…Hydrazines are known to react in 1: 1 stoichiometry with BSAO, forming either the hydrazone or the azo derivative (Morpurgo et al, 1988). The species absorbing at 405 nm which was formed within mixing time both in air and under anaerobic conditions (Fig.…”

“…The copper contribution to the optical absorption is very low, and the intense absorption band at 480 nm (e = 3800 M-l cm-') is due to the organic cofactor (Suzuki et al, 1983). The band, which disappears under reducing conditions, was suggested (Morpurgo et al, 1988) to be an n -+ or* transition peculiar to polycyclic quinones (Murrell, 1963).…”

“…The present paper reports a study of the reactions of BSAO with benzylhydrazine (BHy). Like other carbonyl reagents BHy forms a coloured adduct with the enzyme (Morpurgo et al, 1988). In most cases the adduct is stable, but with some hydrazines, including BHy, it decomposes on prolonged aerobic incubation, with eventual recovery of the enzymic activity (Hucko-Haas & Reed, 1970).…”