H. Krautwurst et al., THE STRONGLY CONSERVED LYSINE-256 OF SACCHAROMYCES-CEREVISIAE PHOSPHOENOLPYRUVATE CARBOXYKINASE IS ESSENTIAL FOR PHOSPHORYL TRANSFER, Biochemistry, 37(18), 1998, pp. 6295-6302
Lysine 256, a conserved amino acid of Saccharomyces cerevisiae phospho
enolpyruvate (PEP) carboxykinase located in the consensus kinase la se
quence of the enzyme, was changed to alanine, arginine, or glutamine b
y site-directed mutagenesis. These substitutions did not result in gro
ss changes in the protein structure, as indicated by circular dichrois
m, tryptophan fluorescence spectroscopy, and gel-exclusion chromatogra
phy. The three variant enzymes showed almost unaltered K-m for MnADP b
ut about a 20 000-fold decrease in V-max for the PEP carboxylation rea
ction, as compared to wild-type PEP carboxykinase. The variant enzymes
presented oxaloacetate decarboxylase activity at levels similar to th
ose of the native protein; however, they lacked pyruvate kinase-like a
ctivity. 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 mutan
ts were 2.0 +/- 0.6 mu M, 17 +/- 2 mu M, and 20 +/- 6 mu M, respective
ly. These results collectively show that a positively charged residue
is required for proper binding of MnATP and that Lys(256) plays an ess
ential role in transition state stabilization during phosphoryl transf
er for S. cerevisiae PEP carboxykinase.