Studies on phosphoglyceromutase from chicken breast muscle: chemical modification of lysyl residues.
To examine the role of lysyl residues in the activity of the enzyme, phosphoglyceromutase (PGM) from chicken breast muscle was chemically modified with trinitrobenzenesulfonate (TNBS) and pyridoxal 5'-phosphate. Trinitrophenylation resulted in modification of about nine lysines per mole of PGM with almost complete activity loss. Substrate (3-PGA) offered some protection to TNBS inactivation but cofactor (2,3-DPGA) did not. Reduction of the Schiff's base complex between pyridoxal 5'-phosphate and PGM gave irreversible inactivation of the enzyme. Inactivation was due to incorporation of 1 mol of pyridoxal 5'-phosphate per mole of PGM dimer through the epsilon-amino group of a lysyl residue. The effect of pyridoxal 5'-phosphate was specific for intact native enzyme and reaction with only one lysine per dimer was not due to induced conformational changes nor to dissociation of the reacted enzyme. 3-PGA prevented much of the reaction with pyridoxal 5'-phosphate with preservation of 70% of the activity and was a competitive inhibitor of the active site directed reagent. Cofactor (2,3-DPGA) acting noncompetitively, reduced the rate at which inactivation occurred with pyridoxal 5'-phosphate. Incorporation of 2,3-[32P]DPGA into PGM irreversibly inactivated with pyridoxal 5'-phosphate and NaBH4 was incomplete indicating hindrance to phosphorylation in the modified enzyme. The results indicate that a lysyl residue is located at or near the active site of PGM and that it is probably involved in the binding of 3-PGA.
D000818:Animals; D001665:Binding Sites; D002645:Chickens; D002942:Circular Dichroism; D007700:Kinetics; D008239:Lysine; D009132:Muscles / Q000201:enzymology*; D009578:Nitrobenzenes / Q000494:pharmacology*; D010736:Phosphoglycerate Mutase / Q000378:metabolism*; D010770:Phosphotransferases / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011732:Pyridoxal Phosphate / Q000494:pharmacology*; D013056:Spectrophotometry, Ultraviolet; D014302:Trinitrobenzenesulfonic Acid / Q000494:pharmacology*
D009578:Nitrobenzenes; D011732:Pyridoxal Phosphate; D014302:Trinitrobenzenesulfonic Acid; D010770:Phosphotransferases; D010736:Phosphoglycerate Mutase; D008239:Lysine