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

Bulletin of the Korean chemical society

Moon-Young Yoon¹  Young-Tae Kim¹  Joungmok Kim¹  Jung-Do Choi¹  Jung-Rim Kim¹ 

关键词: Acetohydroxy acid synthase;    Chemical mechanism;    pH study;    Cofactors;   

DOI  :  

学科分类：化学（综合）

来源: Korean Chemical Society

【 摘 要 】

Acetohydroxy acid synthase (AHAS) is one of several enzymes that require thiamine diphosphate and a divalent cation as essential cofactors. The 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 [Yoon, M.-Y., Hwang, J.-H., Choi, M.-K., Baek, D.-K., Kim, J., Kim, Y.-T., Choi, J.-D. FEBS Letters 555 (2003), 185-191] that the activity is pH-dependent due to changes in Vmax and V/Km. Data were consistent with a mechanism in which substrate was selectively catalyzed by the enzyme with an unprotonated 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 parameters 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 wellconserved 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.

【 授权许可】

Unknown