Malonate decarboxylase of Malonomonas rubra, a novel type of biotin-containing acetyl enzyme.
Cell suspensions or crude extracts of Malonomonas rubra grown anaerobically on malonate catalyze the decarboxylation of this substrate at a rate of 1.7-2.5 mumol.min-1.mg protein-1 which is consistent with the malonate degradation rate during growth. After fractionation of the cell extract by ultracentrifugation, neither the soluble nor the particulate fraction alone catalyzed the decarboxylation of malonate, but on recombination of the two fractions 87% of the activity of the unfractionated extract was restored. The decarboxylation pathway did not involve the intermediate formation of malonyl-CoA, but decarboxylation proceeded directly with free malonate. The catalytic activity of the enzyme was completely abolished on incubation with hydroxylamine or NaSCN. Approximately 50-65% of the original decarboxylase activity was restored by incubation of the extract with ATP in the presence of acetate, and the extent of reactivation increased after incubation with dithioerythritol. Reactivation of the enzyme was also obtained by chemical acetylation with acetic anhydride. These results indicate modification of the decarboxylase by deacetylation leading to inactivation and by acetylation of the inactivated enzyme specimens leading to reactivation. It is suggested that the catalytic mechanism involves exchange of the enzyme-bound acetyl residues by malonyl residues and subsequent decarboxylation releasing CO2 and regenerating the acetyl-enzyme. The decarboxylase was inhibited by avidin but not by an avidin-biotin complex indicating that biotin is involved in catalysis. A single biotin-containing 120-kDa polypeptide was present in the extract and is a likely component of malonate decarboxylase.
Hilbi
H
H
Mikrobiologisches Institut, Eidgenössische Technische Hochschule, ETH-Zentrum, Zürich, Switzerland.
Dehning
I
I
Schink
B
B
Dimroth
P
P
D001360:Avidin / Q000494:pharmacology; D001421:Bacteria, Anaerobic / Q000201:enzymology* / Q000254:growth & development; D001710:Biotin / Q000032:analysis / Q000378:metabolism*; D015153:Blotting, Western; D002262:Carboxy-Lyases / Q000302:isolation & purification / Q000378:metabolism*; D002458:Cell Fractionation; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D013347:Subcellular Fractions / Q000201:enzymology
D001360:Avidin; D001710:Biotin; D002262:Carboxy-Lyases; C071852:malonate decarboxylase