Allosteric regulation of pyruvate kinase M2 isozyme involves a cysteine residue in the intersubunit contact

Pyruvate kinase M-2 isozyme mutants with amino acid substitutions in the subunit interface were prepared and characterized. The substitutions were made in the allosteric M-2 isozyme by the corresponding residues of the nonallosteric M-1 isozyme to identify the residue involved in the allosteric effects. The replacement of Cys-423 by Leu led to substantial loss of both homotropic and heterotropic allosteric effects while the substitutions at Phe-389, Arg-398, Ala-401, Pro-402, Thr-408, and Ile-427 did not. The altered kinetic properties of the Cys-423-substituted mutant resulted from the shift of the allosteric transition toward the active R-state since the mutant exhibits the allosteric properties in the presence of an allosteric inhibitor, L-phenylalanine, The inverse correlation between the hydrophobicity of residue 423 and the extent of stabilization of the R-state was found by analysis of mutants with un-ionizable amino acids at position 423, Furthermore, the modification of Cys-423 with methyl methanethiosulfonate led to a shift of the allosteric transition toward the R-state, probably the result of increased hydrophobicity of the residue. These results suggest that Cys-423 is involved in the allosteric regulation of the enzyme through hydrophobic interactions.