Kinetic Studies on a 4‐Methoxybenzoate O‐Demethylase from Pseudomonas putida

Abstract

A direct, sensitive and reliable photometric assay procedure for monitoring the activity of non-specific 4-methoxybenzoate O-demethylases of microorganisms is described. The assay is based on the O-demethylation of 3-nitro-4-methoxybenzoate to the yellow-colored product 3-nitro-4-hydroxybenzoate. Using this assay and by monitoring the oxidation rate of reduced pyridine nucleotides, the kinetic properties of a purified, reconstituted enzyme system composed of 4-methoxybenzoate monooxygenase [EC 1.14.99.15] (O-demethylating) and a reductase from P. putida were investigated. The Km value of the monooxygenase of this enzyme system towards different substrates (i.e., tight couplers, uncouplers and partial uncouplers) rises from the low value of 0.07 .mu.M for the tight couplers to .apprx. 55 .mu.M for the uncouplers. The effect of possible inhibitors and metal ions on the reconstituted enzyme system was investigated. The inhibition pattern was almost identical to that found for the purified reductase, only bathophenanthrolinedisulfonate showing a greater inhibition of the reconstituted enzyme system. The affinity of the reductase towards NADH was .apprx. 200-fold greater than that towards NADPH. The affinity of this reductase to NADH depended on the nature of the electron acceptor and was > 10 times higher when the monooxygenase.cntdot.substrate complex was used as the electron acceptor, than when cytochrome c or 2,6-dichloroindophenol was used. These differences are discussed on the basis of enzyme-enzyme interactions between the reductase and the monooxygenase.