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., and 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at only one site per subunit with the inactive dimer form of cyanase mutants in which the single histidine residue has been replaced by one of several different amino acids by site directed mutagenesis; the site of the reaction was identified as the amino group of the N-terminal methionine.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 cyapases. Reaction with the N-terminal amino group of methionine apparently prevents reactivation of the mutant enzymes by blocking association to decamer. However, some reactivation of the wild-type cyanase occurs when the free histidine is regenerated by treatment with hydroxylamine; a possible explanation is that the presence of the histidine residue overcomes the effect of the reaction of DEPC with the N-terminal amino group, which is consistent with previous observations that histidine plays a significant role in stabilizing the decamer and facilitating decamer formation. The rate of reaction of the dimer of cyanase with DEPC is increased 2-3-fold by the presence of 50 mM bicarbonate, suggesting that the substrate bicarbonate can bind to the inactive dimer, but that the binding constant is much higher than for the active decamer.