pH dependence of the reaction catalyzed by yeast Mg-enolase.
The pH dependence of the chemical shifts of the 31P resonances of enzyme-bound substrates 2-phosphoglycerate (PGA) and phosphoenolpyruvate (PEP) were measured to obtain further insight into the catalytic mechanism of yeast enolase. The 31P resonances of PGA and PEP bound to the enolase-Mg complex are individually observed by NMR. The Keq,internal = 1.5 favoring PEP was measured. A pH dependence of the 31P chemical shifts gives pKa values of 5.82 and 6.16 for bound PGA and PEP, respectively, indicating that both ligands bind predominantly with their phosphate groups as the dianionic species and their ionization has been altered. The phosphoryl group of PGA has been suggested as playing a role in catalysis [Nowak, T., Mildvan, A. S., and Kenyon, G. L. (1973) Biochemistry 12, 1690-1701]. The pH dependence of the kinetic parameters for Mg-enolase shows a single break in the plot of pKm, PGA vs pH at pH 6.27 with a pH independence above pH 7. This is consistent with the trianion of PGA preferably binding to the enzyme. The kcat profile gives pKA values of 5.94 and 8.35, and kcat/Km profiles give pKA values of 5.85, 6.25, and 8.39. Activation studies with Mg2+ show a pH independence for the activator constant (Ka), but a pH-dependent inhibition at higher concentrations of Mg2+. The log kcat and kcat/Ka profiles from Mg2+ activation give pKA values of about 5.9 and 8.4. These results confirm the importance of residues with pKA values of about 5.9 and 8.4 (His and Lys residues?) but do not support a function for the phosphoryl group of the substrate. The pH dependence of the Ki,Mg2+ gives pKA fits of 5. 95, 7.13, and 8.35. Data from cation inhibition suggest that the phosphate of the substrate and a His residue on enolase may bind the inhibitory Mg2+.
Vinarov
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DA
Department of Chemistry and Biochemistry, University of Notre Dame, Indiana 46556, USA.
Nowak
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D002021:Buffers; D002384:Catalysis; D002413:Cations, Divalent; D005988:Glyceric Acids / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D010710:Phosphates / Q000378:metabolism; D010751:Phosphopyruvate Hydratase / Q000378:metabolism*; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity
D002021:Buffers; D002413:Cations, Divalent; D005988:Glyceric Acids; D010710:Phosphates; C008885:2-phosphoglycerate; D010751:Phosphopyruvate Hydratase; D008274:Magnesium