ROLE OF THE C-TERMINAL HELIX IN THE FOLDING AND STABILITY OF YEAST PHOSPHOGLYCERATE KINASE

In Order to determine the role of the C-terminal helix in the folding and stability of yeast phosphoglycerate kinase, a mutant deleted of th e 12 C-terminal residues (PGK Delta 404-415) was constructed. This mut ant folds in a conformation very similar to that of the wild-type prot ein, but exhibits a very low activity (0.1% of that of the wild-type e nzyme). The main structural effect of the deletion of the C-terminal h elix is an increase in flexibility of the whole protein and a decrease in stability by about 5 kcal/mol. The structural properties of the tr uncated protein are very similar, at least qualitatively, to those in the isolated domains. The accessibility of the thiol group of Cys 97 i s identical to that in the isolated N-domain. The large solvent effect on the tryptophan fluorescence in the native protein at very low conc entration of denaturant reveals an increase of flexibility of the C-do main, similar to that observed on the isolated C-domain. NMR measureme nts show that the pH dependence of His C2H and C4H chemical shifts in the truncated protein perfectly matches those of the isolated domains. The addition of the missing peptide provokes a 40-fold increase in en zyme activity at saturation. A dissociation constant of 80 mu M was de termined. This peptide, which displays a random structure in solution, folds in a helical structure in the region 405-410 as assessed by TRN OESY. All these results show that the C-terminal part of yeast phospho glycerate kinase is not necessary for most of the initial folding step s but acts to lock the C-domain on the N-domain, thus ensuring the exp ression of full enzyme activity. Without this sequence, the protein ha s the sum of the properties of the two isolated domains.