The strongly conserved lysine 256 of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase is essential for phosphoryl transfer.
Lysine 256, a conserved amino acid of Saccharomycescerevisiae phosphoenolpyruvate (PEP) carboxykinase located in the consensus kinase 1a sequence of the enzyme, was changed to alanine, arginine, or glutamine by site-directed mutagenesis. These substitutions did not result in gross changes in the protein structure, as indicated by circular dichroism, tryptophan fluorescence spectroscopy, and gel-exclusion chromatography. The three variant enzymes showed almost unaltered Km for MnADP but about a 20 000-fold decrease in Vmax for the PEP carboxylation reaction, as compared to wild-type PEP carboxykinase. The variant enzymes presented oxaloacetate decarboxylase activity at levels similar to those of the native protein; however, they lacked pyruvate kinase-like activity. The dissociation constant for the enzyme-MnATP complex was 1.3 +/- 0.3 microM for wild-type S. cerevisiae PEP carboxykinase, and the corresponding values for the Lys256Arg, Lys256Gln, and Lys256Ala mutants were 2.0 +/- 0.6 microM, 17 +/- 2 microM, and 20 +/- 6 microM, respectively. These results collectively show that a positively charged residue is required for proper binding of MnATP and that Lys256 plays an essential role in transition state stabilization during phosphoryl transfer for S. cerevisiae PEP carboxykinase.
Krautwurst
H
H
Departamento de Ciencias Químicas, Facultad de Química y Biología, Universidad de Santiago de Chile, Casilla 40, Santiago 33, Chile.
Bazaes
S
S
González
F D
FD
Jabalquinto
A M
AM
Frey
P A
PA
Cardemil
E
E
D000255:Adenosine Triphosphate / Q000378:metabolism; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D007700:Kinetics; D008239:Lysine / Q000032:analysis / Q000378:metabolism*; D008958:Models, Molecular; D019764:Phosphoenolpyruvate Carboxykinase (ATP) / Q000737:chemistry / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D000255:Adenosine Triphosphate; D019764:Phosphoenolpyruvate Carboxykinase (ATP); D008239:Lysine