Characterisation of yeast phosphoglycerate kinase modified by mutagenesis at residue 21.
Site-directed mutagenesis has been used to produce mutant forms of yeast phosphoglycerate kinase in which the conserved active-site residue, Arg21, has been replaced by a methionine or a lysine. Kinetic results obtained using these mutant enzymes show that their Km for both 3-phospho-D-glycerate and ATP are significantly different from those recorded for the wild-type enzyme. The Vmax for the lysine mutant is reduced by a factor of two from that of the wild-type enzyme whereas the Vmax for the methionine mutant is reduced more than sevenfold. A very clean electron-density-difference map shows little, if any, evidence of a structural change associated with the C-terminal domain, although resonances in the NMR spectra associated with the ATP-binding site (C-terminal domain) are also affected by the mutation as one might expect from the kinetic results. The NMR data show that binding at both the 3-phospho-D-glycerate and the non-productive ATP-binding site (associated with the N-terminal domain) are affected in the mutant in a way which is different to that associated with the wild-type enzyme. These results, taken together with the X-ray and kinetic data, indicate that the non-productive ATP-binding site and the activating anion-binding site are both associated with the basic patch region of yeast phosphoglycerate kinase.
Walker
P A
PA
Department of Biochemistry, School of Medical Sciences, The University, Bristol, England.
Joâo
H C
HC
Littlechild
J A
JA
Williams
R J
RJ
Watson
H C
HC
D000595:Amino Acid Sequence; D000818:Animals; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010735:Phosphoglycerate Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D014961:X-Ray Diffraction
D010735:Phosphoglycerate Kinase