EFFECTS OF METAL-IONS, THIAMINE DIPHOSPHATE ANALOGS AND SUBUNIT INTERACTIONS ON THE RECONSTITUTION BEHAVIOR OF PYRUVATE DECARBOXYLASE FROM BREWERS-YEAST

The reconstitution of pyruvate decarboxylase starts with reversible bi nding of thiamine diphosphate and Mg2+-ions to the apoenzyme, followed by a rate-limiting conformational change to the catalytically active holoenzyme. Investigations with diphosphoesters of 4-methyl-5-(2-hydro xyethyl) thiazolium derivatives have shown that the diphosphate residu e of thiamine diphosphate is the most important part of the coenzyme r esponsible for the first reversible binding step. Methylation of the N 1'-atom of the pyrimidine ring of thiamine diphosphate or 4'-oxythiami ne diphosphate prevents the coenzyme from binding stably to the apoenz yme, so that the methylated coenzyme displays no coenzyme activity. In contrast, thiamine diphosphate analogues with bulky residues on the n eighbouring C2'-atom of the pyrimidine ring form active holoenzyme com plexes. This result shows the essential role of the N1'-atom of thiami ne diphosphate in stable cofactor binding. The cofactor binding rate t o the dimeric and tetrameric apoenzymes indicates that the cofactor is located in the contact regions of the subunits in the tetrameric enzy me