Conversion of non-allosteric pyruvate kinase isozyme into an allosteric enzyme by a single amino acid substitution.

Pyruvate kinase M1, a non-allosteric isozyme, was converted into an allosteric enzyme by replacement of an amino acid in the intersubunit contact. The substitution of Ala-398 with Arg resulted in the pronounced allosteric enzyme. The Hill coefficient and the substrate concentration giving one-half of Vmax for the mutant with respect to phosphoenolpyruvate were 2.7 and 0.41 mM, respectively, whereas those values for the wild type were 1.0 and 0.049 mM. This mutation, however, gave rise to only minor effects on the apparent values of Km for ADP and on Vmax. Furthermore, in contrast to the wild-type enzyme, the mutant was activated by fructose 1, 6-bisphosphate. The Hill coefficient of the mutant was no longer increased by the allosteric inhibitor, L-phenylalanine, indicating that the equilibrium for the unligated enzyme is largely shifted toward the T-state. These results suggest that Ala-398 is one of the most critical residues allowing the enzyme to prefer the R-state and that allosteric regulation of pyruvate kinase involves amino acid residues in the intersubunit contact.