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Protein Science, Vol 6, Issue 9 1937-1944, Copyright © 1997 by Cold Spring Harbor Laboratory Press

ARTICLE

Determination of pK(a) values of the histidine side chains of phosphatidylinositol-specific phospholipase C from Bacillus cereus by NMR spectroscopy and site-directed mutagenesis

T. LIU, M. RYAN, F. W. DAHLQUIST and O. H. GRIFFITH
Institute of Molecular Biology and Department of Chemistry, University of Oregon, Eugene, Oregon 97403

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 pK(a) 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 (13)C({epsilon}1)-histidine. A series of (1)H-(13)C 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 (1)H-(13)C 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 pK(a) values of 7.6 and 6.9, respectively. His92 and His227 exhibit pK(a) 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 pK(a) 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 pK(a).
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