Interaction of mono- and dianions with cyanase: evidence for apparent half-site binding.
Cyanase is an inducible enzyme in Escherichia coli that catalyzes bicarbonate-dependent hydrolysis of cyanate. The dianions oxalate, oxalacetate, and malonate are slow-binding inhibitors of cyanase, and some monoanions such as azide and chloride also inhibit cyanase activity [Anderson, P. M., & Little, R. M. (1986) Biochemistry 25, 1621-1626]. The purpose of this study was to investigate the interaction of selected dianions and monoanions by kinetic and equilibrium dialysis binding studies in an effort to obtain information about the active site and catalytic mechanism. Measurement of the effectiveness of 30 different dianions as inhibitors of cyanase showed a significant degree of structural and/or isomeric specificity and considerable variation with respect to the slow-binding nature of the inhibition. Oxalate and oxalacetate both show extreme slow-binding inhibition at very low concentrations. Kinetic studies of the rate of inhibition of cyanase by oxalate showed that the reaction is pseudo first order with respect to oxalate concentration and the results are consistent with a pathway in which oxalate forms a complex with the enzyme in a rapid initial reversible step followed by a slow isomerization step leading to a complex with a very low dissociation constant. The rate of inhibition is significantly reduced by the presence of relatively low concentrations of either azide (analogue of cyanate) or bicarbonate. Equilibrium dialysis binding studies showed that the stoichiometry of binding at saturation for oxalate, malonate, chloride, and bicarbonate is about 0.5 mol of ligand bound/mol of subunit for each compound.(ABSTRACT TRUNCATED AT 250 WORDS)
D000619:Aminohydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000838:Anions / Q000494:pharmacology*; D001639:Bicarbonates / Q000494:pharmacology; D001665:Binding Sites; D002250:Carbon Radioisotopes; D019759:Carbon-Nitrogen Lyases; D002712:Chlorides / Q000494:pharmacology; D003956:Dialysis; D004789:Enzyme Activation / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008314:Malonates / Q000494:pharmacology; D010070:Oxalates / Q000494:pharmacology; D019815:Oxalic Acid
D000838:Anions; D001639:Bicarbonates; D002250:Carbon Radioisotopes; D002712:Chlorides; D008314:Malonates; D010070:Oxalates; D019815:Oxalic Acid; C030290:malonic acid; D000619:Aminohydrolases; C019953:cyanate hydrolase; D019759:Carbon-Nitrogen Lyases