LIGAND-INDUCED CONFORMATIONAL-CHANGES IN WILD-TYPE AND MUTANT YEAST PYRUVATE-KINASE

A mutant form of pyruvate kinase in which serine 384 has been mutated to proline has been engineered in the yeast Saccharomyces cerevisiae. Residue 384 is located in a helix in a subunit interface of the tetram eric enzyme, and the mutation was anticipated to alter the conformatio n of the helix and hence destabilize the interface, Previous results i ndicate that the mutant favours the T quaternary conformation over the R conformation, and this is confirmed by the results presented here, Addition of phosphoenolpyruvate (PEP), ADP and fructose-1,6-bisphospha te (Fru-1,6-P-2) singly to the wild-type and mutant enzymes results in a significant quenching of tryptophan fluorescence (12-44%), and for Fru-1,6-P-2, a red shift of 15 nm in the emission maximum, Fluorescenc e titration experiments showed that PEP, ADP and Fru-1,6-P-2 induce co nformations which have similar ligand-binding properties in the wild-t ype and mutant enzymes, However, the Fru-1,6-P-2 induced conformation is demonstrably different from those induced by either ADP or PEP. The enzymes differ in their susceptibility to trypsin digestion and N-eth ylmaleimide inhibition, The thermal stability of the enzyme is unalter ed by the mutation, Far-UV Co spectra show that both enzymes adopt a s imilar overall secondary structure in solution, Taken together, the re sults suggest that the Ser384-Pro mutation causes the enzyme to adopt a different tertiary and/or quaternary structure from the wild-type en zyme and affects the type and extent of the conformational changes ind uced in the enzyme upon ligand binding, A simplified minimal reaction mechanism is proposed in which the R and T states differ in both affin ity and k(cat). Thus, in terms of the models of cooperativity and allo steric interaction, pyruvate kinase is both a K and a V system.