Spectrophotometric pH titrations and nitration with tetranitromethane of the tyrosyl residues in yeast phosphoglycerate kinase.
Spectrophotometric pH titrations of phosphoglycerate kinase (EC 2.7.2.3) reveal seven tyrosyl residues. In the native state one tyrosyl residue has pKapp equal to 9.3, another has pKapp of about 12.9, and five have pKapp values close to 11.0. Titration above pH 10 causes concomitant reduction of the catalytic activity. Reactivation of the enzyme occurs during storage at pH 7.8. In 6 M guanidine - HCl seven tyrosyl residues with pKapp values equal to 10.0 appear. Nitration of three tyrosyl residues occurs easily when tetranitromethane is used in excess. Four tyrosyl residues appear to be masked or buried. The tyrosyl residue having pKapp equal to 9.3 can be selectively nitrated. Simultaneously the enzyme loses 40% of its catalytic activity. No change in the Km value for one or the other of the two substrates, MgATP or 3-phospho-D-glycerate, was observed in the mononitrated enzyme. On the other hand MgATP protects the tyrosyl residue from nitration whereas 3-phospho-D-glycerate at corresponding condition appears harmless. These results suggest the low ionizing tyrosyl residue to be situated close to the binding site of MgATP, possibly in a pocket just behind. Circular dichroism measurements indicated that minor successive changes occur in the secondary structure, mainly the beta-structure, when the enzyme is being nitrated. It is reasonable to think that these structural changes, possible in combination with steric hindrance, are responsible for the decrease in catalytic activity. Dimerization of the enzyme occurs if the single thiol group is not masked before the tetranitromethane treatment.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D001665:Binding Sites; D002942:Circular Dichroism; D006863:Hydrogen-Ion Concentration; D006902:Hydroxymercuribenzoates / Q000494:pharmacology; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008697:Methane / Q000031:analogs & derivatives*; D010735:Phosphoglycerate Kinase / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013774:Tetranitromethane / Q000494:pharmacology*; D014443:Tyrosine / Q000032:analysis
D006902:Hydroxymercuribenzoates; D014443:Tyrosine; D000255:Adenosine Triphosphate; D010735:Phosphoglycerate Kinase; D008274:Magnesium; D013774:Tetranitromethane; D008697:Methane