Purification and properties of carnitine dehydrogenase from Pseudomonas aeruginosa

Abstract

Carnitine dehydrogenase from Pseudomonas aeruginosa A7244 catalyzes the oxidation of carnitine to 3‐dehydrocarnitine and is specific for l‐carnitine and NAD+. The enzyme is formed by induction. l‐carnitine and 3‐dehydrocarnitine are inducers. Both substances can be utilized by the bacteria under aerobic conditions as sole source of carbon and nitrogen. After treatment of bacteria with lysozyme carnitine dehydrogenase is found in the supernatant of 90000 ×g and has been purified about 200‐fold by means of ammonium sulfate fractionation (between 40 and 55% saturation), gel filtration (Sephadex G‐200) and hydroxyl apatite chromatography. The purified enzyme did not react with d,l‐3‐hydroxybutyrate, l‐lactate, ethanol, l‐malate and d,l‐isocitrate (final concentration 25 mM). The optimal temperature is 35° for the forward reaction (Q10= 1.71; energy of activation 8.5 kcal/mole) as well as the reverse reaction (Q10= 1.99; energy of activation 8.2 kcal/mole). The oxidation reaction with carnitine as substrate exhibited optimum activity between pH 9.0 and 9.5. The reduction reaction with 3‐dehydrocarnitine and NADH as substrates showed optimum activity at pH 7.0. The apparent Michaelis constants, Km, for the substrates carnitine and NAD+ and for the products of the reaction 3‐dehydrocarnitine and NADH were determined. The pK/pH‐ and – log [V/Km]/pH‐profiles of the forward reaction allow the assumption of thiol groups in the active center of the enzyme. Analogues of l‐carnitine (d‐carnitine, glycine betaine and choline) are competitive inhibitors of l‐carnitine oxidation. The equilibrium constant of the reaction of carnitine dehydrogenase was determined and found to be 1.3 ×10−11 (standard error: ± 0.5 ± 10−11) at 30°. Possibilities of an optical test to determine l‐carnitine and 3‐dehydrocarnitine using carnitine dehydrogenase are discussed.