The roles of ATP4- and Mg2+ in control steps of phosphoglycerate kinase.
1H-NMR measurements were made of solutions of yeast phosphoglycerate kinase containing the nucleotide substrate, ATP, and Mg2+ in varying concentrations in order to investigate the affect that the metal ion has on the mode of ATP binding to the enzyme. From the change in the chemical shifts of the 'basic-patch' histidine resonances (His62, His167 and His170) and the nucleotide C8H, C2H and C1'H resonances it is apparent that there are at least two ATP-binding sites on the enzyme. Downfield shifts observed for the above histidine resonances at low nucleotide/enzyme molar ratios indicates that the primary binding site involves electrostatic interactions between the nucleotide triphosphate chain and the basic-patch region of the N-terminal domain. The secondary binding site is shown to involve predominantly hydrophobic interactions between the adenosine moiety and the protein. Evidence from previous two-dimensional NMR experiments [Fairbrother et al. (1990) Eur. J. Biochem. 190, 161-169] suggests that the secondary site is equivalent to the crystallographically observed catalytic site. The affinity of the catalytic site is increased relative to the primary electrostatic site with increasing Mg2+ concentration. The possible importance of these observations in the regulation of this enzyme in vivo are discussed.
Fairbrother
W J
WJ
Inorganic Chemistry Laboratory, University of Oxford, England.
Graham
H C
HC
Williams
R J
RJ
D000255:Adenosine Triphosphate / Q000494:pharmacology* / Q000502:physiology; D001665:Binding Sites / Q000187:drug effects / Q000502:physiology; D004563:Electrochemistry; D008274:Magnesium / Q000494:pharmacology*; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D010735:Phosphoglycerate Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013431:Sulfates / Q000494:pharmacology
D013431:Sulfates; D000255:Adenosine Triphosphate; D010735:Phosphoglycerate Kinase; D008274:Magnesium