Mutation Arg336 to Lys in Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase originates an enzyme with increased oxaloacetate decarboxylase activity.
Saccharomyces cerevisiae phosphoenolpyruvate (PEP) carboxykinase catalyzes one of the first reactions in the biosynthesis of carbohydrates. Apart from the physiologically important reaction, the enzyme also presents low oxaloacetate decarboxylase and pyruvate kinase-like activities. Data from the crystalline structure of homologous Escherichia coli PEP carboxykinase suggest that Arg(333) may be involved in stabilization of enolpyruvate, a postulated reaction intermediate. In this work, the equivalent Arg(336) from the S. cerevisiae enzyme was changed to Lys or Gln. Kinetic analyses of the varied enzymes showed that a positive charge at position 336 is critical for catalysis of the main reaction, and further suggested different rate limiting steps for the main reaction and the secondary activities. The Arg336Lys altered enzyme showed increased oxaloacetate decarboxylase activity and developed the ability to catalyze pyruvate enolization. These last results support the proposal that enolpyruvate is an intermediate in the PEP carboxykinase reaction and suggest that in the Arg336Lys PEP carboxykinase a proton donor group has appeared.
Departamento de Ciencias Quimicas, Facultad de Quimica y Biologia, Universidad de Santiago de Chile.
L
L
Llanos
R
R
Briones
A
A
Yévenes
F D
FD
González-Nilo
P A
PA
Frey
E
E
Cardemil
D001120:Arginine / Q000737:chemistry*; D001665:Binding Sites; D002262:Carboxy-Lyases / Q000378:metabolism; D002942:Circular Dichroism; D004926:Escherichia coli / Q000201:enzymology; D005973:Glutamine / Q000737:chemistry; D007700:Kinetics; D008239:Lysine / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D019764:Phosphoenolpyruvate Carboxykinase (ATP) / Q000737:chemistry*; D011485:Protein Binding; D011489:Protein Denaturation; D011522:Protons; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D013997:Time Factors; D014508:Urea / Q000494:pharmacology
D011522:Protons; D005973:Glutamine; D014508:Urea; D001120:Arginine; D002262:Carboxy-Lyases; C021916:oxaloacetate decarboxylase; D019764:Phosphoenolpyruvate Carboxykinase (ATP); D008239:Lysine