The Cofactors Role on Chemical Mechanism of Recombinant Acetohydroxy Acid Synthase from Tobacco

Abstract

Acetohydroxy acid synthase (AHAS) is one of several enzymes that require thiamine diphosphate and a divalent cation as essential cofactors. Tha active site contains several conserved ionizable groups and all of these appear to be important as judged by the fact that mutation diminishes or abolishes catalytic activity. Recently, we have shown that the activity is pH dependent due to changes in Vmax and V/Km. Data were consistent with a mechanism in which substrate eas selectively catalyzed by the enzyme with an unproyonated base having a pK 6.48, and a protonated group having a pK of 8.25 for catalysis. Here we have in detail studied the pH dependence of the kinetic paramiters of the cofactors(ThDP, FAD, Mg2+) in order to obtain information about the chemical mechanism in the active site. The Vmax of kinetic parameters for all cofactors was pH-dependent on the basic side. The pK of ThDP, FAD, and Mg2+ was 9.5, 9.3, and 10.1 respectively.The V/Km of kinetic parameters for all cofactors was pH-dependent on the acidic and on the basic side. The pK of ThDP, FAD, and Mg2+ was 6.2-6.4 on the acidic side and 9.0-9.1 on the basic side. The well conserved histidine mutant(H392) did not affect the pH-dependence of the kinetic parameters. The data are discussed in terms of the acid-base chemical mechanism.