Characterization of the structure and properties of the His 62-->Ala and Arg 38-->Ala mutants of yeast phosphoglycerate kinase: an investigation of the catalytic and activatory sites by site-directed mutagenesis and NMR.
The role of two "basic patch" residues, Arg-38 and His-62, in the catalytic function and anion-dependent activation of yeast 3-phosphoglycerate kinase (PGK) was investigated by site-directed mutagenesis. Steady-state kinetics and NMR experiments were conducted to characterize the functional properties and structural integrity of the R38A and H62A mutants. The results of these studies, in combination with earlier mutagenesis experiments, suggest that Arg-38 is the only catalytically essential residue among the conserved histidines and arginines of the basic patch. It appears that, similar to the remaining basic patch residues, His-62 is important for anion-dependent activation but not for enzyme activity. Cumulative evidence from this study and from previous mutagenesis experiments suggests that the basic patch region is in effect an extended anion binding site that encompasses both the catalytic and the general anion-binding site. It is proposed that substitution of any one of the basic patch residues results in an increased localization of the catalytic site. Substrate and product may still bind to this site, but a simultaneous binding of activatory anions, required for activation, has been impaired. NMR experiments suggest that the conformational changes observed upon binding of 3-PG to wild-type PGK are necessary for anion- and substrate-dependent activation.
Sherman
M A
MA
Beckman Research Institute of the City of Hope, Division of Biology, Duarte, California 91010.
Fairbrother
W J
WJ
Mas
M T
MT
D000409:Alanine; D000595:Amino Acid Sequence; D001120:Arginine; D001483:Base Sequence; D004789:Enzyme Activation; D006639:Histidine; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010735:Phosphoglycerate Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D009838:Oligodeoxyribonucleotides; D006639:Histidine; D001120:Arginine; D010735:Phosphoglycerate Kinase; D000409:Alanine