Kinetische Untersuchungen zum Reaktionsmechanismus der Carnitindehydrogenase aus Pseudomonas aeruginosa

Abstract

Carnitine dehydrogenase from Pseudomonas aeruginosa catalyzes the reversible reduction of 3-dehydrocarnitine to carnitine. The enzyme is specific for NADH and 3-dehydrocarnitne in the reduction and for NAD+ and l-carnitine in the oxidation reaction. Carnitine dehydrogenase differs from other dehydrogenases in from other dehydrogenases in the polar structure of both of its substrates, carnitine and 3-dehydrocarnitine. So differences in the enzyme-substrate binding and in the reaction mechanism might be expected. The enzyme used in our experiments was of a high specific activity (600 μmoles NAD+/min/mg protein), purified by means of ammonium sulphate fractionation, gel filtration and DEAE cellulose and hydroxyl apatite chromatography. The dependence of initial velocity on the substrate concentration was investigated in the oxidation and reduction reacations. The results are presented in the form of double reciprocal plots. The slopes and intercepts of these primary plots were replotted versus reciprocal substrate concentrations yielding secondary plots. Slopes and intercepts of these secondary plots correspond to the kinetic parameters ϕ0, ϕ1, ϕ2, ϕ12 (reduction reaction) and ϕ0, ϕ′1, ϕ′2, and ϕ′12, (oxidation reaction). The quotients obtained from these values agree with the conditions for a special case of ordered Bi Bi mechanism, the so-called Theorell-Chance mechanism. The limiting Michaelis constants for both substrates in the reduction and oxidation reactions, the dissociation constants for the binary complexes of the enzyme with NADH or NAD+, and the equilibrium constants of the overall reacrtion are presented. The product inhibition patterns also correspond to those predicted by the Theorell-Chance mechanism.