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

Biochemistry
S F Martin, P J Hergenrother

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 s...Continue Reading

Citations

Nov 23, 2006·Applied Microbiology and Biotechnology·Markus A DurbanUwe T Bornscheuer
May 28, 2002·Lipids·Albert González-RouraAmadeu Llebaria
Oct 22, 2003·Journal of Molecular Biology·Graeme C ClarkRichard W Titball
Feb 28, 2007·Archives of Biochemistry and Biophysics·Aaron P BenfieldStephen F Martin
Mar 16, 2016·Biochimica Et Biophysica Acta·Qiongying HuangMary F Roberts
Mar 17, 2021·Biochimica Et Biophysica Acta. Biomembranes·Nejc PetrišičGregor Anderluh
Feb 4, 2010·The Journal of Physical Chemistry. B·Rong-Zhen LiaoFahmi Himo
Jan 11, 2007·Chemical Reviews·Roland K O Sigel, Anna Marie Pyle

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