General Base Catalysis by the Phosphatidylcholine-Preferring Phospholipase C from Bacillus cereus: The Role of Glu4 and Asp55
- 27 March 1998
- journal article
- research article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 37 (16), 5755-5760
- https://doi.org/10.1021/bi972948k
Abstract
To assess what roles the active site residues Glu4 and Asp55 of the phosphatidylcholine-preferring phospholipase C of Bacillus cereus (PLCBc) 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 kcat/KM were 10(4)-10(6) 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 PLCBc-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 PLCBc 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 kcat/KM of the D55E, D55N, and D55L mutants of PLCBc indicates that Asp55 plays a critical role in catalysis and likely serves as the general base in the hydrolysis of phosphatidylcholine by PLCBc.Keywords
This publication has 10 references indexed in Scilit:
- Determination of the Kinetic Parameters for Phospholipase C (Bacillus cereus) on Different Phospholipid Substrates Using a Chromogenic Assay Based on the Quantitation of Inorganic PhosphateAnalytical Biochemistry, 1997
- Site-directed Mutagenesis of the Catalytic Residues of Bovine Pancreatic Deoxyribonuclease IJournal of Molecular Biology, 1996
- The Effect of a Peptide Helix Macrodipole on the pKa of an Asp Side Chain CarboxylateJournal of the American Chemical Society, 1996
- Crystal Structure of Phospholipase C from Bacillus cereus Complexed with a Substrate AnalogJournal of Molecular Biology, 1993
- The Crystal Structure of Tris-inhibited Phospholipase C from Bacillus cereus at 1·9 Å Resolution: The Nature of the Metal Ion in Site 2Journal of Molecular Biology, 1993
- Crystal structures of phosphate, iodide and iodate-inhibited phospholipase C from Bacillus cereus and structural investigations of the binding of reaction products and a substrate analogueJournal of Molecular Biology, 1992
- Active-site mutagenesis of E. coli alkaline phosphatase: replacement of serine-102 with nonnucleophilic amino acidsThe Journal of Organic Chemistry, 1992
- Kinetics of the Hydrolysis of Monodispersed and Micellar Phosphatidylcholines Catalyzed by a Phospholipase C from Bacillus cereusThe Journal of Biochemistry, 1991
- Apparent phosphate retrieval system in Bacillus cereusJournal of Bacteriology, 1989
- Carboxypeptidase AAccounts of Chemical Research, 1989