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Yeast phosphoglycerate kinase (yPGK) is a monomeric two domain protein used as folding model representative of large proteins. We inserted short unstructured sequences (four Gly or four Thr) into the connections between secondary structure elements and studied the consequences of these insertions on the folding process and stability of yPGK. All the mutated proteins can refold efficiently. The effect per residue on stability is larger for the first inserted residue. Insertion in two long betaalpha loops (at residue positions 71 and 129) is more destabilizing than an insertion in a short alphabeta loop (at residue position 89) located on the opposite side of the N-terminal domain. The effect on stability is mainly due to a large increase of the unfolding rate rather than a decrease of the folding rate. This suggests that these connections between secondary structure elements do not play an active role in directing the folding process. Insertion into the short alphabeta loop (position 89) has limited effects on stability and results in the detection of a kinetic phase not previously seen with the wild-type protein, suggesting that insertions in this particular loop do qualitatively affect the folding process without a large effect on folding efficiency. For the two long betaalpha loops (positions 71 and 129) located in the inner surface of the N-terminal domain, the effects on stability are possibly associated with decoupling of the two domains as observed by differential scanning calorimetry during thermal unfolding.

Horse muscle phosphoglycerate kinase, like other mammalian phosphoglycerate kinases, contains seven cysteine residues of which two react rapidly with 5,5'-dithio-bis(2-nitrobenzoate) (Nbs2) following second-order kinetics (k = 640 M-1.s-1). Selective cyanylation of the fast-reacting cysteines, followed by chemical cleavage and subsequent sodium dodecyl sulfate/polyacrylamide gel electrophoresis analysis of the resulting polypeptides, suggested that these cysteines are at positions 378 and 379. Cysteine residues were introduced into yeast phosphoglycerate kinase by site-directed mutagenesis. Mutant enzymes, each containing only one cysteine residue at position 364, 376, or 377, were constructed from a mutant devoid of cysteine (Cys97----Ala). In the last two mutants, the cysteines were at positions corresponding to Cys378 and Cys379, respectively, in the horse muscle enzyme. The chemical reactivity of the cysteine groups in these latter two yeast mutant enzymes was similar to that of the fast-reacting cysteines in the horse muscle enzyme. Furthermore, they were similarly modified upon substrate binding. All these data demonstrate unambiguously that the fast-reacting cysteines in the horse muscle enzyme are Cys378 and Cys379.

The reversibility of the unfolding-refolding transition of horse muscle phosphoglycerate kinase, induced by guanidine hydrochloride (Gdn X HCl), was studied using the regain of enzyme activity as a probe of the native structure. An irreversibility in the reactivation process was detected when the protein was incubated in a critical concentration of denaturant (0.7 +/- 0.1 M Gdn X HCl). This apparent irreversibility was observed for the unfolding process (N----D) as well as for the refolding process (D----N). The formation of the trough followed biphasic kinetics at 23 degrees C, the first phase obeying a first-order reaction corresponded to an isomerization of an intermediate; the second phase, protein-concentration-dependent, was suppressed by lowering the temperature to 4 degrees C. The structural properties of the inactive species were studied; all the beta structures were recovered, but about 29% of the helical structures remained unfolded, and two SH groups were buried. Simulated kinetics were compared with the experimental results and were used to extend the minimum folding scheme previously proposed from equilibrium and kinetic studies [Betton et al. (1984) Biochemistry 23, 6654-6661; Betton et al. (1985) Biochemistry 24, 4570-4577]. The intermediates trapped under these conditions were structured but devoid of catalytic activity. Taking into account the structural properties of these species, the nature of the interactions involved in their formation and stabilization is discussed.