Reaction of the N-terminal methionine residues in cyanase with diethylpyrocarbonate.
Cyanase is an inducible enzyme in Escherichia coli that catalyzes the reaction of cyanate with bicarbonate to give ammonia and carbon dioxide. The enzyme is a decamer of identical subunits (M(r) = 17,000). Previous studies have shown that modification of either the single cysteine residue or the single histidine residue in each subunit gives an active decameric derivative that dissociates reversibly to inactive dimer derivative, indicating that decameric structure is required for activity and that the SH and imidazole groups are not required for catalytic activity [Anderson, P. M., Korte, J. J., Holcomb, T. A., Cho, Y.-G., Son, C.-M., & Sung, Y.-C. (1994) J. Biol. Chem. 269, 15036-15045]. Here the effects of reaction of the reagent diethylpyrocarbonate (DEPC) with cyanase or mutant cyanases are reported. DEPC reacts stoichiometrically with the histidine residue and at one additional site in each subunit when the enzyme is in the inactive dimer form, preventing reactivation. DEPC reacts stoichiometrically (with the same result on reactivation) at only one site per subunit with the inactive dimer form of cyanase mutants in which the single histidine residue has been replaced by one of several different amino acids by site-directed mutagenesis; the site of the reaction was identified as the amino group of the N-terminal methionine. DEPC does not react with the histidine residue of the active decameric form of wild-type cyanase and does not affect activity of the active decameric form of wild-type or mutant cyanases. Reaction with the N-terminal amino group of methionine apparently prevents reactivation of the mutant enzymes by blocking association to decamer.(ABSTRACT TRUNCATED AT 250 WORDS)
Anderson
P M
PM
Department of Biochemistry and Molecular Biology, University of Minnesota-Duluth 55812.
Korte
J J
JJ
Holcomb
T A
TA
D000595:Amino Acid Sequence; D001665:Binding Sites; D019759:Carbon-Nitrogen Lyases; D004047:Diethyl Pyrocarbonate / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology; D006639:Histidine / Q000737:chemistry; D007700:Kinetics; D008190:Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D046911:Macromolecular Substances; D008715:Methionine / Q000737:chemistry*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D013329:Structure-Activity Relationship
D004220:Disulfides; D046911:Macromolecular Substances; D006639:Histidine; D008715:Methionine; D008190:Lyases; C019953:cyanate hydrolase; D019759:Carbon-Nitrogen Lyases; D004047:Diethyl Pyrocarbonate