Arrangement of substrates at the active site of yeast phosphoglycerate kinase. Effect of sulfate ion.
beta, gamma-Bidentate CrATP, a stable exchange-inert metal-nucleotide analog, was used as a probe of the conformation of the substrates at the active site of yeast phosphoglycerate kinase (PGK). The paramagnetic effects of Cr3+ on the longitudinal relaxation rates (1/T1p) of the 1H, 31P, and 13C nuclei of 3-phospho-D-glycerate (3PGA) were examined to determine the distances between enzyme-bound CrATP and the nuclei of 3PGA in the ternary PGK.CrATP.3PGA complex. Kinetic studies indicated that Ki(CrATP) was dependent on the concentration of 3PGA that was abolished in the presence of activator sulfate ion. Similarly, NMR studies showed that the environment of CrATP bound at the active site was altered by the presence of sulfate ion. The determined metal-to-nucleus distances show the close proximity of the substrates at the active site of PGK, which is consistent with a hinge-bending mechanism of the enzyme for bringing the two substrates together. Additionally, the presence of an activating concentration of sulfate was observed to cause significant changes in both the relative Cr(3+)-to-3PGA distances and the overall conformation of 3PGA when compared to the findings in the absence of sulfate. This is consistent with the active site being in a "less closed" configuration when sulfate is present. Under both conditions, models can be constructed that allow for direct transfer of a phosphoryl group between the bound substrates.
Department of Biochemistry, University of Tennessee, Knoxville 37996-0840.
J D
JD
Gregory
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EH
Serpersu
D000255:Adenosine Triphosphate / Q000737:chemistry; D001665:Binding Sites; D005988:Glyceric Acids / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D009682:Magnetic Resonance Spectroscopy; D008280:Magnetics; D008958:Models, Molecular; D010735:Phosphoglycerate Kinase / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011522:Protons; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D012996:Solutions; D013431:Sulfates / Q000737:chemistry*; D013696:Temperature
D005988:Glyceric Acids; D046911:Macromolecular Substances; D011522:Protons; D012996:Solutions; D013431:Sulfates; C005156:3-phosphoglycerate; D000255:Adenosine Triphosphate; D010735:Phosphoglycerate Kinase