Purification and properties of a novel type of malonate decarboxylase from Acinetobacter calcoaceticus.
A novel type of malonate decarboxylase which is induced and located in periplasm of Acinetobacter calcoaceticus grown on malonate as sole carbon and energy source was purified to homogeneity. The purified 185-kDa decarboxylase was found to be an oligomer composed of three different polypeptides in a ratio of 2:1:1, designated alpha (65 kDa), beta (32 kDa), and gamma (25 kDa). Optimum pH was 6.8, while pI was 6.39. The enzyme was highly specific for malonate with Km 1.4 mM. This enzyme contained a biotin prosthetic group on alpha subunit which does not seem to be directly related to malonate decarboxylation. The purified enzyme showed almost no activity; activity was restored, however, by treatment with acetic anhydride or malonyl-CoA, which formed an acetyl-enzyme. The 14C-acylated enzyme was isolated by a gel filtration from the reaction mixtures containing [2-14C]malonyl-CoA and the enzyme or [2-14C]malonate, malonyl-CoA, and the enzyme. When treated by thiol-specific reagents, such as N-ethylmaleimide, 5,5'-dinitro-bis(2-nitrobenzoic acid), and 2-nitro-5-thiocyanobenzoic acid, the purified enzyme showed no increase in activity after the acetic anhydride treatment. The thiol-specific reagents failed to inhibit, however, the catalytically active acetyl-enzyme, suggesting that the site of acylation is the thiol group. Further evidence was provided when sodium borohydride inactivated the acetyl-enzyme. These results suggest that malonate decarboxylation by this enzyme may proceed by a catalytic cycle in which the acetyl group on the active enzyme is displaced by malonate, which binds covalently to a thiol group on the enzyme and is subsequently decarboxylated.
Kim
Y S
YS
Department of Biochemistry, College of Science, Yonsei University, Seoul, Korea.
Byun
H S
HS
D016954:Acinetobacter calcoaceticus / Q000201:enzymology*; D000215:Acylation; D000595:Amino Acid Sequence; D001710:Biotin / Q000737:chemistry; D002262:Carboxy-Lyases / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism; D002384:Catalysis; D002847:Chromatography, Agarose; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003593:Cytoplasm / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004790:Enzyme Induction; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011487:Protein Conformation; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000737:chemistry
D013438:Sulfhydryl Compounds; D001710:Biotin; D002262:Carboxy-Lyases; C071852:malonate decarboxylase