General base catalysis by the phosphatidylcholine-preferring phospholipase C from Bacillus cereus: The role of Glu4 and Asp55

Stephen F. Martin, Paul J. Hergenrother

Research output: Contribution to journalArticlepeer-review


To assess what roles the active site residues Glu4 and Asp55 of the phosphatidylcholine-preferring phospholipase C of Bacillus cereus (PLC(Bc)) might play in binding and catalysis, selected mutants were prepared through site-directed mutagenesis of the plc gene. The mutants were then expressed in Escherichia coli and purified as fusion proteins with the maltose binding protein (MBP). Kinetic analysis showed that mutations at Glu4 had only modest effects on the catalytic activity, whereas those at Asp55 led to proteins whose values for k(cat)K(M) were 104-106 times less than that of the wild- type enzyme. The modest decrease in catalytic activity and the pH-dependent profile of the E4L mutant strongly suggest that glutamic acid at position 4 is not the general base in the PLC(Bc)-catalyzed reaction. Rather, the results support the hypothesis that Glu4 is primarily involved in substrate binding, perhaps by electrostatic stabilization of the positive charge of the choline moiety of the phosphatidylcholine substrate. Examination of X-ray crystallographic data of PLC(Bc) and its various complexes reveals that the carboxylate side chain of Asp55 is positioned such that it could activate a water for nucleophilic attack on the substrate or serve as a ligand for Zn1. However, the involvement of the side chain of Asp55 as an important Zn1 ligand is not consistent with the atomic absorption and thermostability data obtained for the D55L mutant, which are virtually identical with that of the wild-type enzyme. The large reduction in the measured k(cat)/K(M) of the D55E, D55N, and D55L mutants of PLC(Bc) indicates that Asp55 plays a critical role in catalysis and likely serves as the general base in the hydrolysis of phosphatidylcholine by PLC(Bc).

Original languageEnglish (US)
Pages (from-to)5755-5760
Number of pages6
Issue number16
StatePublished - Apr 21 1998
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry


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