An NMR study of anion binding to yeast phosphoglycerate kinase.
Anion binding to yeast phosphoglycerate kinase has been investigated using 1H-NMR spectroscopy. The use of anionic paramagnetic probes. [Cr(CN)6]3- and [Fe(CN)6]3-, has enabled the location of the primary anion binding site in the 'basic-patch' region of the amino-terminal domain. The anions interact most closely with Arg-65 and Arg-168. The binding of these and a variety of other anions to this site is directly competitive with the binding of the substrate, 3-phosphoglycerate. Binding of 3-phosphoglycerate and 1.3-bisphosphoglycerate is, however, stronger than expected on the basis of anionic charge and causes conformational changes in the protein not seen with any of the other simple spherical anions investigated. This must be part, at least, of the substrate specificity. Evidence for a secondary anion binding site involving the side chains of surface lysine residues is also presented. It is suggested that the primary anion site is responsible for the observed activation by anions at low concentrations while the secondary site leads to inhibition at higher anion concentrations. The kinetics fit these deductions and a scheme for kinase activity is presented.
Inorganic Chemistry Laboratory, University of Oxford, England.
W J
WJ
Fairbrother
H C
HC
Graham
R J
RJ
Williams
D000838:Anions; D001665:Binding Sites; D002840:Chromates / Q000494:pharmacology; D003035:Cobalt / Q000494:pharmacology; D003486:Cyanides / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005292:Ferricyanides / Q000494:pharmacology; D005988:Glyceric Acids / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D010735:Phosphoglycerate Kinase / Q000037:antagonists & inhibitors*; D011487:Protein Conformation / Q000187:drug effects; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013379:Substrate Specificity; D013431:Sulfates
D000838:Anions; D002840:Chromates; D003486:Cyanides; D005292:Ferricyanides; D005988:Glyceric Acids; D013431:Sulfates; C007931:hexacyanoferrate III; C021038:hexacyanochromate; C027033:hexacyanocobaltate (III); D003035:Cobalt; C005156:3-phosphoglycerate; D010735:Phosphoglycerate Kinase