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J. Biol. Chem., Vol. 279, Issue 19, 20490-20500, May 7, 2004

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Cross-linking Phosphatidylinositol-specific Phospholipase C Traps Two Activating Phosphatidylcholine Molecules on the Enzyme^*

Xin Zhang, Hania Wehbi, and Mary F. Roberts

From the Merkert Chemistry Center, Boston College, Chestnut Hill, Massachusetts 02467

Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PI-PLC), a bacterial model for the catalytic domain of mammalian PI-PLC enzymes, was cross-linked by 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride to probe for the aggregation and/or conformational changes of PI-PLC when bound to activating phosphatidylcholine (PC) interfaces. Dimers and higher order multimers (up to 31% of the total protein when cross-linked at pH 7) were observed when the enzyme was cross-linked in the presence of PC vesicles. Aggregates were also detected with PI-PLC bound to diheptanoyl-PC (diC₇PC) micelles, although the fraction of cross-linked multimers (19% at pH 7) was lower than when the enzyme was cross-linked in the presence of vesicles. PI-PLC cross-linked in the presence of a diC₇PC interface exhibited an enhanced specific activity for PI cleavage. The extent of this cross-linking-enhanced activation was reduced in PI-PLC mutants lacking either tryptophan in the rim (W47A and W242A) of this ()₈-barrel protein. The higher activity of the native protein cross-linked in the presence of diC₇PC correlated with an increased affinity of the protein for two diC₇PC molecules as detected by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry. In contrast to wild type protein, W47A and W242A had only a single diC₇PC tightly associated when cross-linked in the presence of that activator molecule. These results indicate that (i) each rim tryptophan residue is involved in binding a PC molecule at interfaces, (ii) the affinity of the enzyme for an activating PC molecule is enhanced when the protein is bound to a surface, and (iii) this conformation of the enzyme with at least two PC bound that is stabilized by chemical cross-linking interacts more effectively with activating interfaces, leading to higher observed specific activities for the phosphotransferase reaction.

Received for publication, January 29, 2004 , and in revised form, February 27, 2004.

^* This work was supported by National Institutes of Health Grant GM 60418 (to M. F. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 617-552-3616; Fax: 617-552-2705; E-mail: mary.roberts{at}bc.edu.

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