THE STRONGLY CONSERVED LYSINE-256 OF SACCHAROMYCES-CEREVISIAE PHOSPHOENOLPYRUVATE CARBOXYKINASE IS ESSENTIAL FOR PHOSPHORYL TRANSFER

Citation
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
Citations number
55
Categorie Soggetti
Biology
Journal title
Volume
37
Issue
18
Year of publication
1998
Pages
6295 - 6302
Database
ISI
SICI code
Abstract
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.