Purification and properties of carnitine dehydratase from Escherichia coli--a new enzyme of carnitine metabolization.
Carnitine dehydratase from Escherichia coli 044 K74 is an inducible enzyme detectable in cells grown anaerobically in the presence of L(-)-carnitine or crotonobetaine. It has been purified 500-fold to electrophoretic homogeneity by chromatography on phenyl-Sepharose, hydroxyapatite, DEAE-Sepharose, second phenyl-Sepharose and finally gel filtration on a Sephadex G-100 column. During the purification procedure a low-molecular-weight effector essential for enzyme activity was separated from the enzyme. The addition of this still unknown effector caused reactivation of the apoenzyme. The relative molecular mass of the apoenzyme has been estimated to be 85,000. It seems to be composed of two identical subunits with a relative molecular mass of 45,000. The purified and reactivated enzyme has been further characterized with respect to pH and temperature optimum (7.8 and 37-42 degrees C), equilibrium constant (Keq = 1.5 +/- 0.2) and substrate specifity. The enzyme is inhibited by thiol reagents. The Km value for crotonobetaine is 1.2.10(-2) M. gamma-Butyrobetaine, D(+)-carnitine and choline are competitive inhibitors of crotonobetaine hydration.
Jung
H
H
Department of Biochemistry, Karl Marx University Leipzig, G.D.R.
Jung
K
K
Kleber
H P
HP
D000217:Acyltransferases; D000693:Anaerobiosis; D001051:Apoenzymes / Q000032:analysis; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D002331:Carnitine / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D006836:Hydro-Lyases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D011489:Protein Denaturation; D013379:Substrate Specificity; D013696:Temperature
D001051:Apoenzymes; D001426:Bacterial Proteins; D000217:Acyltransferases; C060278:crotonobetainyl-CoA carnitine coenzyme A transferase; D006836:Hydro-Lyases; D002331:Carnitine