Involvement of the Arg-Asp-His catalytic triad in enzymatic cleavage of the phosphodiester bond

Biochemistry
R J KubiakK S Bruzik

Abstract

Phosphatidylinositol-specific phospholipase C (PI-PLC) catalyzes the cleavage of the P-O bond in phosphatidylinositol via intramolecular nucleophilic attack of the 2-hydroxyl group of inositol on the phosphorus atom. Our earlier stereochemical and site-directed mutagenesis studies indicated that this reaction proceeds by a mechanism similar to that of RNase A, and that the catalytic site of PI-PLC consists of three major components analogous to those observed in RNase A, the His32 general base, the His82 general acid, and Arg69 acting as a phosphate-activating residue. In addition, His32 is associated with Asp274 in forming a catalytic triad with inositol 2-hydroxyl, and His82 is associated with Asp33 in forming a catalytic diad. The focus of this work is to provide a global view of the mechanism, assess cooperation between various catalytic residues, and determine the origin of enzyme activation by the hydrophobic leaving group. To this end, we have investigated kinetic properties of Arg69, Asp33, and His82 mutants with phosphorothioate substrate analogues which feature leaving groups of varying hydrophobicity and pK(a). Our results indicate that interaction of the nonbridging pro-S oxygen atom of the phosphate group with Arg6...Continue Reading

References

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Jul 18, 2001·Journal of the American Chemical Society·R J Kubiak, K S Bruzik
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Citations

May 17, 2005·European Journal of Medicinal Chemistry·T K VenkatachalamF M Uckun
May 9, 2003·Bioorganic & Medicinal Chemistry·Yinghui LiuWonhwa Cho
Jun 19, 2002·Biochimica Et Biophysica Acta·Daniela CordaChristopher P Berrie
Jun 24, 2003·Biochimica Et Biophysica Acta·Rita MeurisseAnnick Thomas
Jun 6, 2009·Journal of the American Chemical Society·Chuan BaiKarol S Bruzik
Aug 4, 2004·Journal of Bioinformatics and Computational Biology·Chuan Yi TangWei-I Chou
Aug 12, 2009·Cellular and Molecular Life Sciences : CMLS·Daniela CordaStefania Mariggiò
Oct 28, 2006·Biochimica Et Biophysica Acta·Pablo V EscribáOliver Vögler
Feb 6, 2010·Organic & Biomolecular Chemistry·David O Corona-MartínezAnatoly K Yatsimirsky
Mar 13, 2003·Journal of the American Chemical Society·Cornelia MihaiKarol S Bruzik

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