THE COENZYME A-SYNTHESIZING PROTEIN COMPLEX AND ITS PROPOSED ROLE IN COA BIOSYNTHESIS IN BAKERS-YEAST

An improved procedure is described for the recovery and purification o f the coenzyme A-synthesizing protein complex (CoA-SPC) of Saccharomyc es cerevisiae (bakers' yeast). The molecular mass of the CoA-SPC, dete rmined prior to and following its purification, is estimated by Sephac ryl S-300 size exclusion chromatography to be between 375 000-400 000. Two previously unreported catalytic activities attributed to CoA-SPC have been identified. One of these is CoA-hydrolase activity which cat alyzes the hydrolysis of CoA to form 3',5'-ADP and 4'-phosphopantethei ne, and the other is dephospho-CoA-pyrophosphorylase activity which ca talyzes a reaction between 4'-phosphopantetheine and ATP to form depho spho-CoA. The dephospho-CoA then reacts with ATP, catalyzed by the: de phospho-CoA-kinase, to reform CoA. This sequence of reactions, referre d to as the CoA/4'-phosphopantetheine cycle, provides a mechanism by w hich the 4'-phosphopantetheine can be recycled to form CoA. Each turn of the cycle utilizes two mol of ATP and produces one mol of ADP, one mol of PPi, and one mol of 3',5'-ADP. Other than the hydrolysis of CoA by CoA-SPC, the 4'-phosphopantetheine for the cycle apparently could be supplied by alternate sources. One alternate source may be the conv entional pathway of CoA biosynthesis. Intact CoA-SPC has been separate d into two segments, One segment is designated apo-CoA-SPC and the oth er segment is referred to as the 10 000-15 000 M-r subunit. The 5'-ADP -4'-pantothenic acid-synthetase, 5'-ADP-4'-pantothenylcysteine-synthet ase, 5'-ADP-4'-pantothenylcysteine-decarboxylase, and CoA-hydrolase ac tivities reside in the apo-CoA-SPC segment of CoA-SPC. Whereas the dep hospho-CoA-kinase and the dephospho-CoA-pyrophosphorylase activities r eside in the 10 000-15 000 M-r subunit. Thus, the 10 000-15 000 M-r su bunit mimics the bifunctional enzyme complex that catalyzes the final two steps in the conventional pathway of CoA biosynthesis.