NDLI: Determination of pKa values of the histidine side chains of phosphatidylinositol-specific phospholipase C from Bacillus cereus by NMR spectroscopy and site-directed mutagenesis.

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Determination of pKa values of the histidine side chains of phosphatidylinositol-specific phospholipase C from Bacillus cereus by NMR spectroscopy and site-directed mutagenesis.

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Ryan, M. Dahlquist, F. W. Liu, T. Griffith, O. H.
Description	Country affiliation: United States Author Affiliation: Liu T ( Institute of Molecular Biology, University of Oregon, Eugene 97403, USA.)
Abstract	Two active site histidine residues have been implicated in the catalysis of phosphatidylinositol-specific phospholipase C (PI-PLC). In this report, we present the first study of the pKa values of histidines of a PI-PLC. All six histidines of Bacillus cereus PI-PLC were studied by 2D NMR spectroscopy and site-directed mutagenesis. The protein was selectively labeled with 13C epsilon 1-histidine. A series of 1H-13C HSQC NMR spectra were acquired over a pH range of 4.0-9.0. Five of the six histidines have been individually substituted with alanine to aid the resonance assignments in the NMR spectra. Overall, the remaining histidines in the mutants show little chemical shift changes in the 1H-13C HSQC spectra, indicating that the alanine substitution has no effect on the tertiary structure of the protein. H32A and H82A mutants are inactive enzymes, while H92A and H61A are fully active, and H81A retains about 15% of the wild-type activity. The active site histidines, His32 and His82, display pKa values of 7.6 and 6.9, respectively. His92 and His227 exhibit pKa values of 5.4 and 6.9. His61 and His81 do not titrate over the pH range studied. These values are consistent with the crystal structure data, which shows that His92 and His227 are on the surface of the protein, whereas His61 and His81 are buried. The pKa value of 6.9 corroborates the hypothesis of His82 acting as a general acid in the catalysis. His32 is essential to enzyme activity, but its putative role as the general base is in question due to its relatively high pKa.
ISSN	09618368
e-ISSN	1469896X
Journal	Protein Science
Issue Number	9
Volume Number	6
Language	English
Publisher	Wiley-Blackwell (on behalf of The Protein Society)
Publisher Date	1997-09-01
Publisher Place	United States
Access Restriction	Open
Subject Keyword	Bacillus Cereus Enzymology Histidine Chemistry Magnetic Resonance Spectroscopy Mutagenesis, Site-directed Type C Phospholipases Binding Sites Hydrogen-ion Concentration Models, Molecular Molecular Structure Phosphatidylinositol Diacylglycerol-lyase Phosphoinositide Phospholipase C Genetics Metabolism Research Support, U.s. Gov't, P.h.s. Discipline Biochemistry
Content Type	Text
Resource Type	Article
Subject	Medicine Molecular Biology Biochemistry

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