15] Cofactor designing in functional analysis of thiamin diphosphate enzymes

Abstract

Publisher Summary Thiamin diphosphate (ThDP) has evolved as a cofactor of enzymes catalyzing the splitting and resynthesis of C–C bonds. It reacts exclusively with substrates of the general structure R–CO–X, where X as a cationic leaving group (CO 2 or R–CHO) is replaced by another positively charged residue (Y). ThDP enzymes are pyruvate decarboxylase (PDC) with Y = H - , transketolases (TK) and acetolactate synthase (ALS), with carbonyl compounds as Y, or the α -oxoacid dehydrogenase complexes, such as pyruvate dehydrogenase complex (PDH), with a cyclic disulfide. In all ThDP enzymes the first substrate contains, besides the activated carbonyl group and the cationic leaving group X, an alkyl residue R, which defines the substrate specificity of the ThDP enzyme. The second substrate Y characterizes the enzyme species. This chapter presents the method of cofactor designing in functional analysis of thiamin diphosphate enzymes. Methods have been developed that describe the binding properties of inactive analogs, helping to elucidate which groups are involved in binding and the specific influence of molecular parameters on the binding mechanism of the cofactors.