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 oxalo acetate decarboxylase and pyruvate kinase-like activities. Data from the cr ystalline structure of homologous Escherichia coli PEP carboxykinase sugges t that Arg(333) may be involved in stabilization of enolpyruvate, a postula ted reaction intermediate. In this work, the equivalent Arg(336) from the S , cerevisiae enzyme was changed to Lys or Gin, Kinetic analyses of the vari ed enzymes showed that a positive charge at position 336 is critical for ca talysis of the main reaction, and further suggested different rate limiting steps for the main reaction and the secondary activities. The Arg336Lys al tered enzyme showed increased oxaloacetate decarboxylase activity and devel oped the ability to catalyze pyruvate enolization, These last results suppo rt the proposal that enolpyruvate is an intermediate in the PEP carboxykina se reaction and suggest that in the Arg336Lys PEP carboxykinase a proton do nor group has appeared. (C) 2001 Federation of European Biochemical Societi es. Published by Elsevier Science B.V. All rights reserved.