Relevance of Arg457 for the nucleotide affinity of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase.
Phosphoenolpyruvate carboxykinases catalyze one of the first steps in the biosynthesis of glucose and depending on the enzyme origin, preferentially use adenine or guanine nucleotides as substrates. The Saccharomyces cerevisiae enzyme has a marked preference for ADP (or ATP) over other nucleotides. Homology models of the enzyme in complex with ADP or ATP show that the guanidinium group of Arg457 is close to the adenine base, suggesting that this group might be involved in the stabilization of the nucleotide substrate. To evaluate this we have performed the mutation Arg457Met, replacing the positively charged guanidinium group by a neutral residue. The mutated enzyme retained the structural characteristics of the wild-type protein. Fluorescence titration experiments showed that mutation causes a loss of 1.7 kcal mol(-1) in the binding affinity of the enzyme for ADPMn. Similarly, kinetic analyses of the mutated enzyme showed 50-fold increase in K(m) for ADPMn, with minor alterations in the other kinetic parameters. These results show that Arg457 is an important factor for nucleotide binding by S. cerevisiae PEP carboxykinase.
Tobar
Iván
I
Facultad de Química y Biología, Universidad de Santiago de Chile, Casilla 40, Santiago 33, Chile.
González-Nilo
Fernando D
FD
Jabalquinto
Ana M
AM
Cardemil
Emilio
E
D001120:Arginine / Q000378:metabolism*; D049109:Cell Proliferation; D005453:Fluorescence; D015966:Gene Expression Regulation, Fungal; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009711:Nucleotides / Q000378:metabolism*; D019764:Phosphoenolpyruvate Carboxykinase (ATP) / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017354:Point Mutation; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000201:enzymology* / Q000235:genetics
D009711:Nucleotides; D001120:Arginine; D019764:Phosphoenolpyruvate Carboxykinase (ATP)