Phospholipase C from Bacillus cereus. Evidence for essential lysine residues.
1. Phospholipase C was inactivated by exposure to the three amino-group reagents, ethyl acetamidate, 2,4,6-trinitrobenzensulphonic acid and pyridoxal 5'-phosphate plus reduction. 2. Inactivation by pyridoxal 5'-phosphate showed the characteristics of Schiff's base formation with the enzyme. The pyridoxal 5'-phosphate-treated enzyme after reduction had an absorbance maximum at 325 mm and 6-N-pyridoxyl-lysine was the only fluorescent component after acid hydrolysis. 3. For complete inactivation, 2 mol of pyridoxal 5'-phosphate or 7 mol of 2,4,6-trinitrophenyl were incorporated/mol of enzyme. 4. The two apparently essential lysine residues were much more reactive to pyridoxal 5'-phosphate than the other 19 lysine residues in the enzyme. 5. Binding of phospholipase C to a substrate-based affinity gel caused marked protection against inactivation by pyridoxal 5'-phosphate. For complete inactivation of the gel-bound enzyme, 5 mol of pyridoxal 5'-phosphate were incorporated/mol of enzyme and there was no evidence of two especially reactive lysine residues. 6. On application of pyridoxal 5'-phosphate-treated enzyme (remaining activity 30% of original) to a column of the affinity gel, some material bound and some did not. The latter contained very little enzyme activity and was heavily incorporated with reagent (9.06 mol/mol of enzyme). The former had a specific activity of 34% of that of the control and contained 1.29 mol of reagent/mol of enzyme. 7. Thus phospholipase C appears to contain two lysine residues that are essential for enzyme activity, but probably not for substrate binding.
D001409:Bacillus cereus / Q000201:enzymology*; D002846:Chromatography, Affinity; D007094:Imides; D008239:Lysine / Q000032:analysis; D010740:Phospholipases / Q000032:analysis* / Q000037:antagonists & inhibitors; D011732:Pyridoxal Phosphate; D014302:Trinitrobenzenesulfonic Acid
D007094:Imides; D011732:Pyridoxal Phosphate; D014302:Trinitrobenzenesulfonic Acid; D010740:Phospholipases; D008239:Lysine