Role of Tryptophan Residues in Interfacial Binding of Phosphatidylinositol-specific Phospholipase C

Abstract

The phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis exhibits several types of interfacial activation. In the crystal structure of the closely related Bacillus cereus PI-PLC, the rim of the active site is flanked by a short helix B and a loop that show an unusual clustering of hydrophobic amino acids. Two of the seven tryptophans in PI-PLC are among the exposed residues. To test the importance of these residues in substrate and activator binding, we prepared several mutants of Trp-47 (in helix B) and Trp-242 (in the loop). Two other tryptophans, Trp-178 and Trp-280, which are not near the rim, were mutated as controls. Kinetic (both phosphotransferase and cyclic phosphodiesterase activities), fluorescence, and vesicle binding analyses showed that both Trp-47 and Trp-242 residues are important for the enzyme to bind to interfaces, both activating zwitterionic and substrate anionic surfaces. Partitioning of the enzyme to vesicles is decreased more than 10-fold for either W47A or W242A, and removal of both tryptophans (W47A/W242A) yields enzyme with virtually no affinity for phospholipid surfaces. Replacement of either tryptophan with phenylalanine or isoleucine has moderate effects on enzyme affinity for surfaces but yields a fully active enzyme. These results are used to describe how the enzyme is activated by interfaces. The phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis exhibits several types of interfacial activation. In the crystal structure of the closely related Bacillus cereus PI-PLC, the rim of the active site is flanked by a short helix B and a loop that show an unusual clustering of hydrophobic amino acids. Two of the seven tryptophans in PI-PLC are among the exposed residues. To test the importance of these residues in substrate and activator binding, we prepared several mutants of Trp-47 (in helix B) and Trp-242 (in the loop). Two other tryptophans, Trp-178 and Trp-280, which are not near the rim, were mutated as controls. Kinetic (both phosphotransferase and cyclic phosphodiesterase activities), fluorescence, and vesicle binding analyses showed that both Trp-47 and Trp-242 residues are important for the enzyme to bind to interfaces, both activating zwitterionic and substrate anionic surfaces. Partitioning of the enzyme to vesicles is decreased more than 10-fold for either W47A or W242A, and removal of both tryptophans (W47A/W242A) yields enzyme with virtually no affinity for phospholipid surfaces. Replacement of either tryptophan with phenylalanine or isoleucine has moderate effects on enzyme affinity for surfaces but yields a fully active enzyme. These results are used to describe how the enzyme is activated by interfaces. Phosphatidylinositol-specific phospholipase C (PI-PLC) 1The abbreviations used are: PI-PLCphosphatidylinositol-specific phospholipase CcIPd-myo-inositol 1,2-cyclic-phosphateI1Pd-myo-inositol 1-phosphateCMCcritical micelle concentrationPCphosphatidylcholinediCnPC1,2-diacylphosphatidylcholine with n carbons in each acyl chainPOPC1-palmitoyl-2-oleoyl-glycero-3-phosphocholineSUVsmall unilamellar vesicleLUVlarge unilamellar vesicleCDcircular dichroismTX-100Triton X-100WTwild type PI-PLCPAphosphatidic acidPMephosphatidylmethanolPGphosphatidyl glyceroliPrOHisopropanol. catalyzes the hydrolysis of PI in two steps: (i) an intramolecular phosphotransferase reaction to form inositol 1,2-cyclic-phosphate (cIP), followed by (ii) a cyclic phosphodiesterase activity that converts cIP to inositol 1-phosphate (I1P). Although the enzymes in eukaryotes play key roles in generating membrane-associated second messengers and in some case water-soluble second messengers (1Berridge M.J. Biochem. J. 1984; 220: 345-360Crossref PubMed Scopus (2493) Google Scholar, 2Nishizuka K. Science. 1986; 233: 305-311Crossref PubMed Scopus (4038) Google Scholar), PI-PLC enzymes in bacteria are secreted and play critical roles in cell infectivity (3Marques M.B. Weller P.F. Parsonnet J. Ransil B.J. Nicholson-Weller A. J. Clin. Microbiol. 1989; 27: 2451-2454Crossref PubMed Google Scholar, 4Mengaud J. Braun-Breton C. Cossart P. Mol. Microbiol. 1991; 5: 367-372Crossref PubMed Scopus (144) Google Scholar). The PI-PLC from Bacillus thuringiensis exhibits several types of kinetic interfacial activation by interfaces. Micellar PI is a better substrate than monomeric PI (5Lewis K.A. Garigapati V.R. Zhou C. Roberts M.F. Biochemistry. 1993; 32: 8836-8841Crossref PubMed Scopus (74) Google Scholar, 6Hendrickson H.S. Hendrickson E.K. Johnson J.L. Khan T.H. Chial H.J. Biochemistry. 1992; 31: 12169-12172Crossref PubMed Scopus (49) Google Scholar), and interfaces of phosphatidylcholine, a nonsubstrate that does not bind at the active site, activate the enzyme for both PI cleavage (7Qian X. Zhou C. Roberts M.F. Biochemistry. 1998; 37: 6513-6522Crossref PubMed Scopus (28) Google Scholar) and cIP hydrolysis (8Zhou C., Wu, Y. Roberts M.F. Biochemistry. 1997; 36: 347-355Crossref PubMed Scopus (61) Google Scholar). In the available crystal structure (9Heinz D.W. Ryan M. Bullock T.L. Griffith O.H. EMBO J. 1995; 14: 3855-3863Crossref PubMed Scopus (147) Google Scholar) of the closely related Bacillus cereus PI-PLC, bound myo-inositol localized the active site inside the βα barrel. However, the orientation of PI substrate side chains and any other sites for interfacial PC were not defined. phosphatidylinositol-specific phospholipase C d-myo-inositol 1,2-cyclic-phosphate d-myo-inositol 1-phosphate critical micelle concentration phosphatidylcholine 1,2-diacylphosphatidylcholine with n carbons in each acyl chain 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine small unilamellar vesicle large unilamellar vesicle circular dichroism Triton X-100 wild type PI-PLC phosphatidic acid phosphatidylmethanol phosphatidyl glycerol isopropanol. The rim of the active site of bacterial PI-PLC (9Heinz D.W. Ryan M. Bullock T.L. Griffith O.H. EMBO J. 1995; 14: 3855-3863Crossref PubMed Scopus (147) Google Scholar) has a short helix B and one particular loop (residues 237–243) that show an unusual clustering of hydrophobic amino acids that are fully exposed to solvent (Fig. 1). This structural characteristic could contribute to the binding of substrate fatty acyl chains (for PI) but also to the binding of the PC activator. Tryptophan residues are often elements inserted into bilayers when a peripheral protein binds (e.g. annexin V (10Pigault C. Follenius-Wund A. Chabbert M. Biochem. Biophys. Res. Commun. 1999; 254: 484-489Crossref PubMed Scopus (10) Google Scholar) or phospholipase A2 (11Sumandea M. Das S. Sumandea C. Cho W. Biochemistry. 1999; 38: 16290-16297Crossref PubMed Scopus (54) Google Scholar)). Two of the seven tryptophans (Trp-47 and Trp-242) in PI-PLC are among the exposed hydrophobic amino acids. Their structural orientation is consistent with previous spectroscopic data (12Volwerk J.J. Filthuth E. Griffith O.H. Jain M.K. Biochemistry. 1994; 33: 3464-3474Crossref PubMed Scopus (63) Google Scholar, 13Zhou C. Roberts M.F. Biochemistry. 1998; 37: 16430-16439Crossref PubMed Scopus (22) Google Scholar), which showed enzyme intrinsic fluorescence increases significantly upon binding to micelles or vesicles. However, the details of any in the by activating surfaces not for activator substrate acyl chains bind is to how the kinetic activation helix B and loop at the of active site are in activation of the of the tryptophans in each PI-PLC intrinsic fluorescence and the kinetic activation by PC surfaces. To test we prepared the W242A, the and These were for kinetic activation of PI and cIP hydrolysis by and to bind to PC interfaces both intrinsic fluorescence and a and any in structure by two other tryptophans were and Trp-178 a in PI-PLC by the of the active site from the of the protein and by a with the side chain of a to critical for substrate The of Trp-178 by the Trp-178 and and has to the of PI hydrolysis Ryan M. Griffith O.H. D.W. Biochemistry. 1997; 36: PubMed Scopus Google Scholar). prepared to how enzyme fluorescence as as kinetic activation by interfaces. is in the loop that with the and helix of the is from the activator binding and the active of play in substrate and activator of these show that the two tryptophan residues Trp-47 and Trp-242 are critical for the enzyme to bind to zwitterionic activating interfaces. Trp-242 is also the to micelle and PI were from PI for cIP from prepared from the phospholipase C. Roberts M.F. Biochemistry. 1998; 37: 16430-16439Crossref PubMed Scopus (22) Google Scholar). cIP prepared from PI as (8Zhou C., Wu, Y. Roberts M.F. Biochemistry. 1997; 36: 347-355Crossref PubMed Scopus (61) Google Scholar). from the from into from of the protein by of to the E. at to an in and a of PI-PLC protein in the the cell by and the The to two a followed by a to the were used to the of the of the PI-PLC were by J. C. A. Mol. Google Scholar, W. 1999; PubMed Scopus Google Scholar) a from Two from the in were to the on of the The and were used for and for W242A, and the were and of the a second into the W47A the The and were used for the of and were by and structure and as by the were by an were in protein in a and the at as the from to in with an of or were at with protein in a of structure with in the to Johnson Biochemistry. 1994; 33: PubMed Scopus Google Scholar, 1992; 5: PubMed Scopus Google Scholar, P. J.J. 1993; PubMed Scopus Google Scholar). PI-PLC intrinsic fluorescence were with a fluorescence were at with protein in with The with both and at The for the in PI-PLC fluorescence the for and the tryptophan in the fluorescence were as is the of protein and is the in the of an PI-PLC activity by two (i) PI as were from the reaction at and with of also the The of cIP and in the water-soluble by as (8Zhou C., Wu, Y. Roberts M.F. Biochemistry. 1997; 36: 347-355Crossref PubMed Scopus (61) Google Scholar, 13Zhou C. Roberts M.F. Biochemistry. 1998; 37: 16430-16439Crossref PubMed Scopus (22) Google Scholar) a (ii) hydrolysis by by the of substrate and in as a of with the enzyme. The of protein that no more than substrate hydrolysis to for activation by PC used cIP in the or of to for PC activation. used to PI-PLC from PI-PLC bound to vesicles. used to S. phospholipase to phospholipid vesicles K. Roberts M.F. J. 1999; PubMed Scopus Google Scholar). of small unilamellar vesicles with an prepared by in unilamellar vesicles were prepared by of an for binding with were prepared with protein in the concentration and for PI-PLC, and and for W47A and In the binding with binding of PI-PLC to the concentration to for the were to and at in a for the the The protein bound to vesicles on the of the protein the The and a enzyme in the of vesicles the and than of the protein on the for were by of and of to the were with the were from and the PI-PLC were of to a vesicles used to the of enzyme is the of The of enzyme as The with is the for the protein with surfaces. The crystal structure of bacterial PI-PLC enzyme a with the active site at the side by of the (Fig. 1). Replacement of tryptophan residues at the of the is to a on structure as as the To of and PI-PLC were and used to for structural and were also by the of structure with in of structure from the by Johnson Biochemistry. 1994; 33: PubMed Scopus Google Scholar, 1992; 5: PubMed Scopus Google Scholar, P. J.J. 1993; PubMed Scopus Google Scholar) with the structure elements in the crystal structure (9Heinz D.W. Ryan M. Bullock T.L. Griffith O.H. EMBO J. 1995; 14: 3855-3863Crossref PubMed Scopus (147) Google Scholar). the tryptophan mutants the of structure also any in PC binding and are to to protein that has a significantly structure from of structure of and tryptophan PI-PLC the and data for the from the of at with is the in the of the of The in each is than the and data for the The from the of at with is the in the of the of The in each is than in a the decreased from for the to and also showed a in and in The side chain of at the of active site, is critical for and hydrophobic that the and active of these to PI-PLC, and the enzyme The other with a significantly is at a near the C of the of the side chain not to structure elements as in However, the of is decreased with that of tryptophan does contribute to of the Replacement of either tryptophan at the rim effects on the protein The of W47A the of the for large not Trp-47 is at the of helix However, the of to Replacement of the side chain of the tryptophan in the loop at the of the the This with a small in the of loop also a of the loop that structure and is that to a small of the PI-PLC has seven tryptophan residues that contribute to fluorescence that the binding of PC activator micelles (12Volwerk J.J. Filthuth E. Griffith O.H. Jain M.K. Biochemistry. 1994; 33: 3464-3474Crossref PubMed Scopus (63) Google Scholar, C. X. Roberts M.F. Biochemistry. 1997; 36: PubMed Scopus Google Scholar) or vesicles H.S. Hendrickson E.K. Johnson J.L. Khan T.H. Chial H.J. Biochemistry. 1992; 31: 12169-12172Crossref PubMed Scopus (49) Google Scholar, J.J. Filthuth E. Griffith O.H. Jain M.K. Biochemistry. 1994; 33: 3464-3474Crossref PubMed Scopus (63) Google Scholar) an in PI-PLC intrinsic fluorescence that bind to the active site a in fluorescence (12Volwerk J.J. Filthuth E. Griffith O.H. Jain M.K. Biochemistry. 1994; 33: 3464-3474Crossref PubMed Scopus (63) Google C. Roberts M.F. Biochemistry. 1998; 37: 16430-16439Crossref PubMed Scopus (22) Google Scholar). a tryptophan has by some or of that particular tryptophan were for of the upon interfacial W242A, and were as a of and and with The the for and by the of PC micelles to or in the in fluorescence of at showed a small the micelles were and bound to the of the tryptophan and (Fig. However, showed significantly to micelle The fluorescence with micelle but the of fluorescence that of The the tryptophan in helix B and the showed no in intrinsic fluorescence upon micelle binding (Fig. The of micelles on PI-PLC intrinsic fluorescence to that for in fluorescence the of with the that W47A as as also showed a significantly in fluorescence (Fig. However, the in fluorescence upon micelle binding the for the no to PC micelle Micellar PI-PLC hydrolysis of cIP monomeric activate the enzyme C. Roberts M.F. Biochemistry. 1998; 37: 16430-16439Crossref PubMed Scopus (22) Google Scholar). of monomeric a in PI-PLC fluorescence that with binding of to the active as micelles the intrinsic fluorescence in of decreased PI-PLC fluorescence for the PI-PLC mutants active site binding of is not by the tryptophan The in fluorescence the for and the micelle of on the and of the and is to these V.R. J. Roberts M.F. J. 1995; Scopus Google and the showed large increases in fluorescence consistent with micelle binding as as active site binding of the This could that Trp-242 not micelle binding but to the in fluorescence as bind to the active is a of PI-PLC J.J. Griffith O.H. Biophys. PubMed Scopus Google Scholar). is not has no to form interfaces. other that bind to the PI-PLC active a in the fluorescence of PI-PLC (Fig. Tryptophan mutants that more myo-inositol to a decreased fluorescence also a to PC the in fluorescence for W242A, and the than that with PI-PLC, that the interfacial site is to substrate the showed a more in fluorescence with myo-inositol than both W47A and the decreased fluorescence is with binding, that removal of both tryptophan residues the binding of small water-soluble inositol at the active However, the in fluorescence with myo-inositol could also in the of the or removal of that are the of small binding to the active To the of the tryptophans in the interfacial activation of PI-PLC, cIP hydrolysis and PI cleavage were for and with and PC activator in either Trp-47 or which at the rim of the the PI-PLC activity cIP to and of mutants kinetic activation by but not to the as the in the of PC activation is by activation of which the as is near the of the protein and from the active site and interfacial activation site is near the of the In the of an the activity of the both Trp-47 and Trp-242 cIP that of the However, of the to with showed a The of kinetic activation of by PC micelles that the two tryptophan residues are key in the interfacial binding site of the enzyme. of these side chains with the phospholipid key to the protein to a the of PC activation for the of in the intrinsic fluorescence when into the no fluorescence the micelle binding site activity of PI-PLC and mutants cIP and the of in a the tryptophan the one with the activity cIP Trp-178 is at the of the active site side chain a with the side chain of a to critical for substrate Replacement of Trp-178 with significantly the of and in could the of the to bind The activity of cIP is of with PI in Triton the activity is that of previous of to showed a activity Although is a activated by The of activity of with and activator is the as PI-PLC for both cIP hydrolysis and PI of the tryptophans Trp-47 and Trp-242 to roles in activator Although myo-inositol is a of PI-PLC, the of as an used to how mutants bind of the as substrate and no activating the PI-PLC activity in myo-inositol the to the The that the of myo-inositol on W47A and activity cIP that the removal of Trp-47 or Trp-242 the interfacial binding not the active site inositol binding of activating PI-PLC is to moderate of Y. Roberts M.F. Biochemistry. 1997; 36: PubMed Scopus Google Scholar). has that a (e.g. PI-PLC by the of the active activated and the mutants W242A, and for hydrolysis of The of activation the activity with to cIP hydrolysis by that in for the mutants for However, removal of both Trp-47 and Trp-242 PI-PLC that could activated by that solvent were cleavage of in or PI in micelles is a better substrate than in micelles (7Qian X. Zhou C. Roberts M.F. Biochemistry. 1998; 37: 6513-6522Crossref PubMed Scopus (28) Google Scholar). of the is more both the activating phospholipid and the substrate acyl However, one a of tryptophan removal PI cleavage as as PC activation of the of PI-PLC in removal of either rim tryptophan to a in PI-PLC activity PI in both these mutants could activated for cIP cleavage of PI to cIP to the that activated for cIP hydrolysis and PI cleavage were of both tryptophans to enzyme that activity PI and not activated by In the activity for PI in than PI in that for the the two rim tryptophan residues are critical for PI cleavage as as cIP This is consistent with the The inositol of PI binds at the active site, the acyl chains are with hydrophobic side chains of the rim to the PI binding with Replacement of the tryptophan side chain with a is a is that the roles of these residues in interfacial activation and substrate binding are not for tryptophan but a hydrophobic side and phenylalanine residues are hydrophobic and than with tryptophan the PubMed Scopus Google Scholar), phenylalanine is to tryptophan in isoleucine is significantly of and and fluorescence and kinetic analyses were to The of and PI in were to enzyme and the two Trp-242 mutants were the PI activity decreased when the rim tryptophans were PI cleavage is consistent with structural for interfacial activation. both isoleucine and phenylalanine mutants is consistent with hydrophobic to interfacial activity of isoleucine and phenylalanine mutants at Trp-47 and PI in The in the are PI in The in the are in a these mutants PI activity to used as a better of the of each tryptophan in to the intrinsic fluorescence to micelle in and intrinsic fluorescence show a on concentration in to but with the fluorescence in upon micelle The are when the enzymes were with the were no increases in the intrinsic fluorescence of and The of activation by the that removal of both tryptophan residues the affinity of for activating surfaces. This by the of and PI-PLC enzymes to PC a were used PI-PLC showed activity small large vesicles (7Qian X. Zhou C. Roberts M.F. Biochemistry. 1998; 37: 6513-6522Crossref PubMed Scopus (28) Google Scholar, C. X. Roberts M.F. Biochemistry. 1997; 36: PubMed Scopus Google Scholar). the of PI-PLC as the concentration of PC is in The in the of PI-PLC to PC with a to an This is not a but an a of of the for PC for PI-PLC binding to is the concentration of PC for of binding is to the for PI-PLC on PC in from the to the were of PI-PLC mutants binding to PC to for protein the in for the side chains in the of and This is the concentration of PC for of binding is to the for PI-PLC on PC in from the to the were The of PI-PLC mutants binding to PC to for protein the in for the side chains in the of and PubMed Scopus Google Scholar). in a The of PI-PLC for PC binding of PI-PLC to PC the of tryptophan residues on binding to PC vesicles with for W47A and were than for The of and for PC were than but both were than the data in an to the protein binding of to could with when the concentration of PC to This a on the of the of for PC surfaces. Trp-242 and Trp-47 a in interfacial binding of of these two residues to the interfacial binding of Replacement of tryptophan with the phenylalanine side chain the affinity of the enzyme for PC surfaces. The binding are consistent with the kinetic of residues Trp-47 and Trp-242 are critical for interfacial activation. Tryptophan residues in in in for a of the amino acids 1999; PubMed Google Scholar). This large hydrophobic side chains the for into PC as by the of and PubMed Scopus Google Scholar). of crystal for a of show that these side chains to the and the hydrophobic acyl chain of bilayers 1999; PubMed Google Scholar). has that play a in into the as as in 1999; PubMed Google Scholar). peripheral tryptophan residues are also critical for of the protein with the In the case of a tryptophan in a loop to binding of the to interfaces A. J.J. J. 1997; PubMed Scopus Google Scholar). phospholipase A2 has two tryptophans and in the that are important for phospholipase binding to phospholipid interfaces (11Sumandea M. Das S. Sumandea C. Cho W. Biochemistry. 1999; 38: 16290-16297Crossref PubMed Scopus (54) Google Scholar). These residues to into the the interfacial of However, for as as tryptophan are critical for binding and interfacial activation Cho W. 1999; PubMed Scopus Google Scholar). is the interfacial activation of PI-PLC thuringiensis PI-PLC has seven tryptophan two of at the hydrophobic rim of the active site in helix B and a loop (residues at the of the active site two near the of the protein and and two to the of the protein and The two near the of the are of particular near the for to the active site (Fig. 1). Replacement of either or both of these tryptophans with and or both (W47A/W242A) effects on activity a in of PI-PLC monomeric substrate However, a more in PC activation for two of these W47A and W242A, that with the The of PC activation by the not with structure of the is that both side chains are in binding to a phospholipid of the mutants to PC vesicles showed that of either tryptophan with decreased the affinity of the enzyme for an activating PC and and W242A, The PubMed Scopus Google Scholar) used to in of a of related a a tryptophan is by the of the to that of of both tryptophans The for Trp-47 and Trp-242 mutants with show the as these at Trp-47 effects than at could several for Trp-47 is in a short helix and has a Trp-242 is in a more The is on short that no structure either in or to a PC in mutants at the two could for a in a helix a loop into the Trp-47 not into the PC Trp-242 is The that Trp-242 the to the in fluorescence upon PI-PLC binding to PC surfaces is consistent with a is that the rim loop with Trp-242 a or upon into the PC on the of the hydrophobic side of the both helix B and loop tryptophans play critical roles in thuringiensis PI-PLC binding to PC interfaces. in binding affinity of the and PI-PLC are in activity of PI-PLC is used as the and the of the enzyme for by However, PC micelle binding to with PC PC binding for a of the kinetic activation but not as as which is is The showed no to PC vesicles and a small in activity cIP when an activating the binding of PI-PLC to an activating PC is by both Trp-47 and Trp-242 residues. However, the for tryptophan at is not hydrophobic or for tryptophan at these with the side chain generating protein activity is to Trp-47 and Trp-242 roles in PC activation of PI-PLC hydrolysis of water-soluble also PI cleavage to are important for the enzyme at any either activating interfaces as PC or a substrate of both rim tryptophans an enzyme that is to both activating and substrate interfaces. the cleavage of PI by the mutants showed activity PI in the by This is from for hydrolysis by with the for the mutants The could in the of the for the the as that of to PC activation when the the of the active site and rim, is a in the orientation of one or both tryptophans that is critical for substrate binding and The of tryptophan residues into interfacial of often by in the E. to bilayers into the to form a not the is by an in protein intrinsic fluorescence Biophys. PubMed Scopus Google Scholar). of any of the tryptophans activity of the Biophys. PubMed Scopus Google Scholar). of other show tryptophan into as key in (e.g. E. Biochemistry. 1998; 37: PubMed Scopus Google Scholar) and M. K. Y. J. Biochem. 1998; PubMed Scopus Google Scholar)). PI-PLC has seven tryptophan residues that fluorescence at of Trp-242 PI-PLC that the to the of PC both and that in is for of the in fluorescence upon micelle of Trp-47 also enzyme that a to binding to interfaces for W47A and more for and of these tryptophan mutants fluorescence upon binding to with micelles are small and Roberts M.F. J. 1986; Scopus Google Scholar), micelles Roberts M.F. J. Scopus Google Scholar). The of Trp-242 and Trp-47 to PC interfaces from orientation of the residues on the is that the side chain of Trp-47 is in a more of the Trp-242 into the of the of loop a in orientation could with a small micelle in a at the The micelles a for Trp-47 that with Trp-242 to the in fluorescence upon micelle In either binding of to a PC than W47A consistent with a of Trp-242 with PC surfaces. Trp-242 also to the protein intrinsic fluorescence when myo-inositol or monomeric bind to the removal a protein that is to active site binding as as activator micelle However, and W47A also a in fluorescence upon inositol binding at the active the fluorescence to a binding at the active site has from at of the The crystal structure of PI-PLC from a to PI-PLC from a of J. J. J. D.W. J. Mol. 1997; PubMed Scopus Google Scholar). The site and hydrophobic rim are in both crystal is one tryptophan near the of helix B in PI-PLC (Trp-47 is at the C of helix B in thuringiensis and a phenylalanine in a rim loop that to to Trp-242 in the thuringiensis is one the of PC interfacial activation for bacterial enzyme. The crystal of M. J. 1998; PubMed Scopus Google Scholar, Biophys. 1999; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar) show the active site to in a to thuringiensis The rim of the bacterial has a of hydrophobic residues and This hydrophobic one of the active site and is to the the to binding and is with substrate of the hydrophobic in the enzyme of to decreased the activity from to the bacterial PI-PLC a to how in binding helix B and the rim loop are to both phosphotransferase and cyclic phosphodiesterase for the the