General base catalysis by the phosphatidylcholine-preferring phospholipase C from Bacillus cereus: the role of Glu4 and Asp55.
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.
Martin
S F
SF
Department of Chemistry and Biochemistry, The University of Texas at Austin 78712, USA. sfmartin@mail.utexas.edu
Hergenrother
P J
PJ
D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism*; D001409:Bacillus cereus / Q000201:enzymology*; D002384:Catalysis; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D010713:Phosphatidylcholines / Q000378:metabolism*; D013379:Substrate Specificity / Q000235:genetics; D010738:Type C Phospholipases / Q000235:genetics / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism
C092445:1,2-dihexanoyl-sn-glycero-3-phosphorylcholine; D008024:Ligands; D010713:Phosphatidylcholines; D001224:Aspartic Acid; D018698:Glutamic Acid; D010738:Type C Phospholipases; D015032:Zinc