Chemical modification of yeast 3-phosphoglycerate kinase.
Sulfhydryl reagents, as well as mild hydrogen peroxide oxidation, do not inhibit the activity of yeast phosphoglycerate kinase, indicating that the single thiol group and 3 methionine residues present in the enzyme are not essential for activity. Nitration of phosphoglycerate kinase by tetranitromethane inhibits the enzyme by reaction with a single tyrosine residue. Substrates provide partial protection against inactivation by nitration. Circular dichroism spectra indicate that no conformational changes occur upon nitration. However, perturbation of the microenvironment surrounding the aromatic amino acid residues, particularly tyrosine, was observed. The same perturbation was observed on addition of the substrate 3-phosphoglycerate kinase to native phosphoglycerate kinase. The role of lysine in the action of yeast phosphoglycerate kinase has been studied by modification with O-methylisourea, 2-methoxy-5-nitrotropone, and pyridoxal phosphate. Guanidination shows that there are lysines essential for phosphoglycerate kinase; extrapolation to zero activity indicates that there are three essential lysines as judged by nitrotroponylation and three essential lysines when the enzyme is reacted with pyridoxal phosphate. Substrates afford partial protection and extrapolation to total protection indicates that up to three lysines are protected by MgITP and one lysine by 3-phosphoglycerate. Spectrofluorescence and optical rotatory dispersion measurements show that there is no detectable conformational change for the guanidinated phosphoglycerate kinase and that there are slight changes in the spectra suggesting that there may be slight conformational changes for the nitrotroponylated and the pyridoxal phosphate-modified enzymes.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D002942:Circular Dichroism; D007700:Kinetics; D008239:Lysine / Q000032:analysis; D008298:Maleates; D008970:Molecular Weight; D009574:Nitro Compounds; D009905:Optical Rotatory Dispersion; D010446:Peptide Fragments / Q000032:analysis; D010735:Phosphoglycerate Kinase / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011732:Pyridoxal Phosphate; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D014357:Trypsin; D014443:Tyrosine / Q000032:analysis; D014508:Urea / Q000031:analogs & derivatives
D000596:Amino Acids; D008298:Maleates; D009574:Nitro Compounds; D010446:Peptide Fragments; D014443:Tyrosine; D011732:Pyridoxal Phosphate; D014508:Urea; D010735:Phosphoglycerate Kinase; D014357:Trypsin; D008239:Lysine