The strongly conserved lysine 256 of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase is essential for phosphoryl transfer

Lysine 256, a conserved amino acid of Saccharomyces cerevisiae phosphoenolpyruvate (PEP) carboxykinase located in the consensus kinase la 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 K-m for MnADP but about a 20 000-fold decrease in V-max 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 mu M for wild-type S. cerevisiae PEP carboxykinase, and the corresponding values for the Lys256Arg, Lys256Gln, and Lys256Ala mutants were 2.0 +/- 0.6 mu M, 17 +/- 2 mu M, and 20 +/- 6 mu M, respectively. These results collectively show that a positively charged residue is required for proper binding of MnATP and that Lys(256) plays an essential role in transition state stabilization during phosphoryl transfer for S. cerevisiae PEP carboxykinase.