title: 31P NMR studies of enzyme-bound substrate complexes of yeast 3-phosphoglycerate kinase. 1. Effects of sulfate and pH. Mg(II) affinity at the two ATP sites.
abstract: 31P NMR measurements were made (at 121.5 MHz and 5 degrees C) on enzyme-bound substrate complexes of 3-phosphoglycerate kinase in order to address three questions pertaining to (i) the integrity of the enzyme-substrate complexes with Mg(II) in the presence of sulfate concentrations typical of those used for crystallization in X-ray studies, (ii) the relative affinities of Mg(II) to ATP bound at the two sites on the enzyme, and (iii) the pH behavior of the different phosphate groups in the enzyme complexes. 31P chemical shift and spin-spin coupling constant changes showed that at concentrations of 0.5 M and higher, sulfate ion interferes with Mg(II) chelation to ATP and ADP free in solution as well as in their enzyme-bound complexes. The effect on enzyme complexes is stronger for the E.MgATP complex than for the E.MgADP complex. Sulfate ion (50 mM) also causes a approximately 0.5 ppm upfield chemical shift of the 31P resonance of enzyme-bound 3-P-glycerate even in the absence of ATP or Mg(II). A quantitative estimate of the dispartate affinities of Mg(II) to ATP bound at the two sites on the enzyme was made on the basis of computer simulation of changes in the line shape of beta-P (ATP) resonance and of changes in 31P chemical shift of the corresponding gamma-P (ATP) in the E.ATP complex with increasing [Mg(II)]. The concentrations of the relevant species that contribute to these 31P NMR signals were computed by assuming independent binding at the two sites.(ABSTRACT TRUNCATED AT 250 WORDS)
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites; D006019:Glycolysis; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D010735:Phosphoglycerate Kinase / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013431:Sulfates / Q000494:pharmacology
chemical_list: D013431:Sulfates; D000255:Adenosine Triphosphate; D010735:Phosphoglycerate Kinase; D008274:Magnesium