Phosphatidylinositol-specific phospholipase C: kinetic and stereochemical evidence for an interaction between arginine-69 and the phosphate group of phosphatidylinositol

Biochemistry
R J HondalM D Tsai

Abstract

A new substrate analogue, (2R)-1,2-dipalmitoyloxypropanethiophospho-1-D-myo-inositol (DPsPI), has been used in a new, continuous assay for phosphatidylinositol-specific phospholipase C (PI-PLC). DPsPI is superior to other substrate analogs that have been used for assaying PI-PLC since it is synthesized as a pure diastereomer and maintains both acyl chains of the natural substrate, dipalmitoylphosphatidylinositol (DPPI). The assay that has been developed using this new analogue has allowed us to elucidate detailed kinetic data so far lacking in the field. In addition, several mutants of PI-PLC were constructed and assayed. The results show that Arg-69 is essential for catalysis, since mutations at this position led to a 10(3)- 10(4)-fold decrease in activity with respect that of to the wild-type (WT) enzyme. An alanine mutant of Asp-67, a residue also found at the active site, displays activity similar to that of WT. We have also used nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy to analyze the structural integrity and conformational stability of the mutants. The results show that the overall global conformation of the enzyme is not perturbed by the mutants. The 15N-1H HSQC NMR spectrum of WT PI-PLC i...Continue Reading

References

Nov 1, 1992·Journal of Biomolecular NMR·M PiottoV Sklenár
Apr 1, 1991·FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology·E A DennisY A Hannun
Jan 1, 1991·Methods in Enzymology·W Cho, F J Kézdy
Jan 1, 1987·Annual Review of Biochemistry·M J Berridge

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Citations

Aug 3, 2002·Nature Reviews. Genetics·Catharine H Rankin
Jul 18, 2001·Journal of the American Chemical Society·R J Kubiak, K S Bruzik
Mar 13, 2003·Journal of the American Chemical Society·Cornelia MihaiKarol S Bruzik

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