60 YEARS OF THIAMIN DIPHOSPHATE BIOCHEMISTRY

The mechanism of ThDP enzymes originates in the anionic (ylid) structu re of the coenzyme, On the other hand, no ylid species (as permanently existing structure) could be detected by C-13(2)-NMR studies with PDC (yeast), when the cofactor binds to the active site. Therefore, the r ate of ylid formation as the first step of the catalytic mechanism dis tinguishes decisively the power (k(cat)) of all ThDP enzymes. H-2/H-1- exchange experiments with PDC, TK, PDH and POX have shown that within the active center of ThDP enzymes (under native pH conditions!) the am inopyrimidine part generates the essential ylid structure by enhancing the dissociation rate (acidity) of the C-2-H bond up to PG orders of magnitude. Moreover, it could be proved that the mechanism of substrat e activation of PDC (yeast) is also connected directly with the C-2-H activation by the aminopyrimidine part. Experiments with analogs of Th DP or modified apoenzymes (via site-directed mutagenesis) have shown t hat this mechanism requires as essential elements a hydrogen bond betw een the pyrimidine N-1' atom and a conserved Glu side chain of the dif ferent apoenzymes as well as the (evolutionary conserved) V-conformati on. The latter positions the 4'-amino group in direct (functional) con tact to the C-2-H bond, A proposal is discussed, how the 4'-positioned amino group in cooperation with the N-1' atom could increase the C-2- H dissociation rate. (C) 1998 Elsevier Science B.V. All rights reserve d.