Allosteric regulation of pyruvate kinase M2 isozyme involves a cysteine residue in the intersubunit contact.
Pyruvate kinase M2 isozyme mutants with amino acid substitutions in the subunit interface were prepared and characterized. The substitutions were made in the allosteric M2 isozyme by the corresponding residues of the nonallosteric M1 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.
Ikeda
Y
Y
Department of Biochemistry, Fukui Medical University, 23-3 Shimoaizuki, Matsuoka, Fukui, 910-1193 Japan.
Noguchi
T
T
D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008741:Methyl Methanesulfonate / Q000031:analogs & derivatives / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011770:Pyruvate Kinase / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017386:Sequence Homology, Amino Acid; D018411:Spodoptera
D007527:Isoenzymes; C014674:methyl methanethiosulfonate; D008741:Methyl Methanesulfonate; D011770:Pyruvate Kinase; D003545:Cysteine