L-aminoacide oxydases des enveloppes de Proteus mirabilis : propriétés générales

Abstract

Summary Cell envelopes of Proteus mirabilis are prepared by lysing spheroplasts at a pH value over 10 in the presence of a metal complexing agent. They show a high L-phenylalanine-oxidase activity which amounts to 30–40 p. cent of the oxidase found in the starting material and decreases only slowly during storage in ice. One mole of phenylpyruvate is produced from the aminoacid per half mole of O2 used in the reaction. Other natural or artificial aminoacids are oxidized and belong mostly to two groups : substrates with aliphatic or aromatic non polar side chains and aminoacids with positively charged side chains. Thermal inactivation of the particles shows that these two groups correspond to distinct enzymic systems, both having similar properties. Oxidation of L-aminoacids by Proteus envelopes can be measured with both sensitivity and accuracy by using dichlorophenolindophenol as an electron acceptor in the presence of phenazin methosulfate. Horse cytochrome c is a possible acceptor in this system but ferricyanide is not, contrary to what is observed with the NADH-oxidase associated to the particles. The optimal rate of phenylpyruvate production in the presence of O2 is obtained in the vicinity of pH 7.6. The Km coefficients for various aminoacids range from 2.5 × 10−3 (histidine) to values over 5 × 10−2 M, and are slightly lower for aromatic substrates as compared to aminoacids with bulky aliphatic chains. Substitution at carbon β decreases or suppresses activity. Oxidation of phenylalanine is sensitive to snake venom phospholipase A and is very susceptible to ionic or non ionic detergents. Dependence of the envelope oxidases on membrane phospholipids is further suggested by the Arrhenius plot of reaction velocity as a function of temperature, which is biphasic, the transition being close to 17°C. When O2 is used as the acceptor aminoacid oxidation is blocked by cyanide and decreased by HOQNO. Spectral determination suggests that the whole process is carried on by aminoacid specific flavoproteins and the cytochromes b1, a1 and a2 of the respiratory chain. Detergents apparently exert their action at the flavoprotein level or at the connection between flavoproteins and cytochrome b1.