Substrate specificity of copper-containing plant amine oxidases

The steady-state kinetic parameters of the amine oxidases purified from Lathyrus cicera (LCAO) and Pisum sativum (PSAO) seedling were measured on a series of common substrates, previously tested on bovine serum amine oxidase (BSAO). LCAO, as PSAO, was substantially more reactive than BSAO with aliphatic diamines and histamine. The kcat and kcat/Km for putrescine were four and six order of magnitude higher, respectively. Differences were smaller with some aromatic monoamines. The plot of kcat versus hydrogen ions concentration produced bell-shaped curves, the maximum of which was substrate dependent, shifting from neutral pH with putrescine to alkaline pH with phenylethylamine and benzylamine. The latter substrates made the site more hydrophobic and increased the pKa of both enzyme-substrate and enzyme-product adducts. The plot of kcat/Km versus hydrogen ion concentration produced approximately parallel bell-shaped curves. Similar pKa couples were obtained from the latter curves, in agreement with the assignment as free enzyme and free substrate pKa. The limited pH dependence of kinetic parameters suggests a predominance of hydrophobic interactions.