Phosphatidylinositol-Specific Phospholipase C: Kinetic and Stereochemical Evidence for an Interaction between Arginine-69 and the Phosphate Group of Phosphatidylinositol

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• 作者：Robert J. Hondal ; Suzette R. Riddle ; Alexander V. Kravchuk ; Zhong Zhao ; Hua Liao ; Karol S. Bruzik ; and Ming-Daw Tsai
• 刊名：Biochemistry
• 出版年：1997
• 出版时间：June 3, 1997
• 年：1997
• 卷：36
• 期：22
• 页码：6633 - 6642
• 全文大小：254K
• 年卷期：v.36,no.22(June 3, 1997)
• ISSN：1520-4995

文摘

A new substrate analogue,(2R)-1,2-dipalmitoyloxypropanethiophospho-1-D-myo-inositol(DPsPI), has been used in a new, continuous assay forphosphatidylinositol-specific phospholipase C(PI-PLC). DPsPI is superior to other substrate analogs that havebeen used for assaying PI-PLC since itis synthesized as a pure diastereomer and maintains both acyl chains ofthe natural substrate,dipalmitoylphosphatidylinositol (DPPI). The assay that has beendeveloped using this new analogue hasallowed us to elucidate detailed kinetic data so far lacking in thefield. In addition, several mutants ofPI-PLC were constructed and assayed. The results show that Arg-69is essential for catalysis, sincemutations at this position led to a10³-10⁴-fold decrease in activity withrespect that of to the wild-type(WT) enzyme. An alanine mutant of Asp-67, a residue also found atthe active site, displays activitysimilar to that of WT. We have also used nuclear magneticresonance (NMR) and circular dichroism(CD) spectroscopy to analyze the structural integrity andconformational stability of the mutants. Theresults show that the overall global conformation of the enzyme is notperturbed by the mutants. The¹⁵N-¹H HSQC NMR spectrum of WT PI-PLC is alsoreported at 600 MHz. The stereoselectivity of thereaction toward the stereoisomers of another analogue,1,2-dipalmitoyl-sn-glycero-3-thiophospho-1-myo-inositol (DPPsI), was used to probe whether Arg-69 interacts with thephosphate moiety of the substrate.We have calculated that the WT enzyme shows a stereoselectivityratio of 160000:1 in favor of theR_pisomer versus the S_p isomer. The R69Kmutant displayed a significant 10⁴-fold relaxationofstereoselectivity. Our data support the role of Arg-69 instabilizing the negative charge on thepentacoordinate phosphate in the transition state duringcatalysis.