ANALYSIS OF AN INVARIANT COFACTOR-PROTEIN INTERACTION IN THIAMIN DIPHOSPHATE-DEPENDENT ENZYMES BY SITE-DIRECTED MUTAGENESIS - GLUTAMIC-ACID-418 IN TRANSKETOLASE IS ESSENTIAL FOR CATALYSIS

A homologous expression system and a purification protocol for pure, h ighly active recombinant yeast transketolase have been developed. The invariant transketolase residue Glu(418), which forms a hydrogen bond to the N-1' nitrogen atom of the pyrimidine ring of the cofactor thiam in diphosphate has been replaced by glutamine and alanine. Crystallogr aphic analyses of the mutants show that these amino acid substitutions do not induce structural changes beyond the site of mutation. In both cases, the cofactor binds in a manner identical to the wild-type enzy me. Significant differences in the CD spectra of the mutant transketol ases compared with the spectrum of wild-type enzyme indicate differenc es in the electron distribution of the aminopyrimidine ring of the cof actor. The E418Q mutant shows 2% and the E418A mutant shows about 0.1% of the catalytic activity of wild-type enzyme. The affinities of the mutant enzymes for thiamin diphosphate are comparable with wild-type t ransketolase, The hydrogen bond between the coenzyme and the side chai n of Glu(418) is thus not required for coenzyme binding but essential for catalytic activity. The results demonstrate the functional importa nce of this interaction and support the molecular model for cofactor d eprotonation, the first step in enzymatic thiamin catalysis.