A SUBUNIT INTERFACE MUTANT OF YEAST PYRUVATE-KINASE REQUIRES THE ALLOSTERIC ACTIVATOR FRUCTOSE-1,6-BISPHOSPHATE FOR ACTIVITY

A variant form of yeast pyruvate kinase (EC 2.7.1.40) with Ser-384 mut ated to proline has been engineered in order to study the allosteric p roperties of this enzyme. Both the mutant and wildtype enzymes were ov erexpressed in a strain of yeast in which the genomic copy of the pyru vate kinase gene had been disrupted by an insertion of the Ura3 gene. Both enzymes were purified to homogeneity and their kinetic properties characterized. The wild-type enzyme displays sigmoid kinetics with re spect to phosphoenolpyruvate (PEP) concentration, and is activated by the allosteric effector fructose 1,6-bisphosphate with concomitant red uction in co-operativity. In contrast, the mutant was found to be depe ndent on the presence of the effector for catalytic activity and was i nactive in its absence. The fully activated mutant enzyme had a k(cat. ) 1.6 times greater than that of the wild-type enzyme. The mutation in troduced into the enzyme is in an intersubunit contact which is known to be critical for the allosteric properties of the enzyme, and is far removed from the active site. The major effect of the mutation seems to be to stabilize the low-affinity T state of the apoenzyme, although k(cat.) is also affected. The S-0.5 for PEP and S-0.5 for ADP of the wild-type enzyme were 0.22 +/- 0.004 and 0.15 +/- 0.01 mM respectively (means +/- S.E.M.). In the activated mutant enzyme, these kinetic par ameters increased to 0.67 +/- 0.03 and 0.43 +/- 0.03 mM respectively. The cooperativity between ADP-binding sites was altered in the mutant enzyme, with the Hill coefficient (h) for ADP increasing to 1.65 +/- 0 .07 in the presence of the effector, compared with a value of 1.01 +/- 0.07 for the wild-type enzyme under the same conditions. CD spectrosc opy revealed the secondary structure of the mutant enzyme to be little different from that of the wild-type enzyme, indicating that the two enzymes have similar secondary structures in solution. Precise tertiar y and quaternary structures such as intersubunit and interdomain inter actions may be modified. An improved purification procedure has been d evised that allows large quantities of enzyme to be rapidly prepared.