Site-directed mutagenesis in basic amino acid residues of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase.
Mutant Arg76Gln and Lys290Gln Saccharomyces cerevisiae phosphoenolpyruvate carboxykinases have been prepared and analyzed. No alteration in the apparent kinetic constants were detected for the Arg76Gln mutant enzyme, while the Lys290Gln mutant showed a 12-fold decrease in V(max)/K(m)ADP. These results indicate that Arg76 is not involved in CO2 binding, but support the hypothesis that the binding of this substrate induces a conformational change that protects the region around Arg76 from trypsin action [Herrera et al. (1993) J. Protein Chem. 12, 413-418]. These findings also indicate that Lys290, a highly reactive residue against pyrydoxal phosphate [Bazaes et al. (1995), FEBS Lett. 360, 207-210], does not perform an essential function for the enzyme activity.
Chávez
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Departamento de Ciencias Quimicas, Facultad de Quimica y Biologia, Universidad de Santiago de Chile.
Krautwurst
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Cardemil
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D001120:Arginine; D001665:Binding Sites; D003001:Cloning, Molecular; D005973:Glutamine; D007700:Kinetics; D008239:Lysine; D016297:Mutagenesis, Site-Directed; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D017354:Point Mutation; D011487:Protein Conformation; D011732:Pyridoxal Phosphate / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D014357:Trypsin
D011994:Recombinant Proteins; D005973:Glutamine; D011732:Pyridoxal Phosphate; D001120:Arginine; D014357:Trypsin; D010729:Phosphoenolpyruvate Carboxykinase (GTP); D008239:Lysine