Phospholipase C from Bacillus cereus. Evidence for essential lysine residues

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Abstract
Phospholipase C [EC 3.1.4.3 from B. cereus] was inactivated by exposure to the 3 amino-group reagents, ethyl acetimidate, 2,4,6-trinitrobenzenesulfonic acid and pyridoxal 5''-phosphate plus reduction. Inactivation by pyrdioxal 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 nm, and 6-N-pyridoxyl-lysine was the only fluorescent component after acid hydrolysis. For complete inactivation 2 mol of pyridoxal 5''-phosphate or 7 mol of 2,4,6-trinitrophenyl were incorporated per mol of enzyme. The 2 apparently essential lysine residues were much more reactive to pyridoxal 5''-phosphate than the other 19 lysine residues in the enzyme. 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 per mol of enzyme, and there was no evidence for 2 especially reactive residues. 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 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. Phospholipase C appears to contain 2 lysine residues that are essential for enzyme activity, but probably not for substrate binding.