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 xg and has been purified about 200-fold by means of ammonium sulfate fractionation (between 40 and 55 Olio saturation), gel filtration (Sephadex G-200) and hydroxyl apatite chromatography. The purified enzyme did not react with ~,~-3-hydroxybutyrate, L-lactate, ethanol, L-malate and D,L-isocitrate (final concentration 25 mM). The optimal temperature is 35" for the forward reaction (Ql0 = 1.71 ; energy of activation 8.5 kcal/mole) as well as the reverse reaction (Qlo = 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 a t 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
Zusammenfassung: Die NADP®-abhängige Aldehyd-Dehydrogenase von Acinetobacter calcoaceticus ist nach unseren früheren Befunden ein induzierbares Enzym aus intracytoplasmatischen Inclusionsmembranen. Wir gewannen es als Detergens/Phospholipid-Mischmicelle und demonstrieren in vorliegender Arbeit Ergebnisse zur kinetischen Charakterisierung dieses Enzyms. Die Aldehyd-Dehydrogenase hat ein pH-Optimum um pH 10 und wird durch Aldehydüber-schuß gehemmt. Der A^-Wert für NADP e liegt -unabhängig von der Kettenlänge der Aldehydsubstrate -bei 8.6 x IGT 5 M. Die K m -Werte für die Aldehyde fallen mit steigender Kettenlänge ab (von 1.6 l ( 3 beiButanal auf 1.5 10~6 bei Decanal). Auch die K { -Werte (für die Hemmung durch Aldehydüberschuß) sinken mit wachsender Kettenlänge von 7.5 10~3 (bei Butanal) auf 3.5 10~5M (bei Decanal) ab, genauso wie die Aldehyd konzentrationen, die jeweils die "optimale" Reaktionsgeschwindigkeit bedingen (bei Butanal 5mM, bei Decanal 6 ). Die Anzahl der inhibierenden Aldehydmoleküle pro Enzym-Substrat-Komplex beträgt n= 1. Das Enzym wird durch NADPH als Produkt gehemmt, nicht dagegen durch Fettsäuren. Eine Rückreaktion konnte nicht gemessen werden. Nicht-kompetitive Inhibitoren des Enzyms sind Sn 2 *, Fe 20 , Cu 2e , BOi e , CN 9 , EDTA, o-Phenanthrolin, p-Chlormercuribenzoat, Mercaptoethanol, Phenylmethylsulfonylfluorid und Diisopropylfluorophosphat, nicht dagegen lodacetat. Chloralhydrat ist ein kompetitiver Inhibitor gegen die Aldehyde. Aktivierend wirkte -abhängig von der Kettenlänge der AldehydeButansäure-ethylester.
Kinetics of membrane-bound aldehyde dehydrogenase of Acinetobacter calcoaceticusSummary: In recent investigations we were able to demonstrate that the NADP®-dependent aldehyde dehydrogenase of Acinetobacter calcoaceticus is an inducible enzyme localized in intracytoplasmic membranes limiting alkane inclusions. Long-chain aliphatic hydrocarbons and alkanols are inducers of the enzyme. It was purified by us and now kinetically characterized using the enzyme-micelle form, which contains bacterial phospholipids and a detergent (sodium cholate), too.The pH optimum of aldehyde dehydrogenase was determined to be at pH 10. The enzyme showed substrate inhibition (by aldehyde excess). The K s and K m values of the leading substrate NADP® were found to be 8.6 10~5 and 10.3 10~5M independent of the chain-length of the aldehydes. The K m values of the aldehydes decreased depending on increasing chain-length (butanal: 1.6 10~3, decanal: 1.5 10~6M).The K { values (for inhibition by aldehyde excess)
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