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 dioxid e. The enzyme is a decamer of identical subunits (M(r) = 17 000). Prev ious studies have shown that modification of either the single cystein e residue or the single histidine residue in each subunit gives an act ive decameric derivative that dissociates reversibly to inactive dimer derivative, indicating that decameric structure is required for activ ity and that the SH and imidazole groups are not required for catalyti c 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 stoichiome trically with the histidine residue and at one additional site in each subunit when the enzyme is in the inactive dimer form, preventing rea ctivation. DEPC reacts stoichiometrically (with the same result on rea ctivation) at only one site per subunit with the inactive dimer form o f cyanase mutants in which the single histidine residue has been repla ced by one of several different amino acids by site directed mutagenes is; 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 pre vents reactivation of the mutant enzymes by blocking association to de camer. 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 ove rcomes the effect of the reaction of DEPC with the N-terminal amino gr oup, which is consistent with previous observations that histidine pla ys a significant role in stabilizing the decamer and facilitating deca mer 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 th at the binding constant is much higher than for the active decamer.