Hydrolysis of short acyl chain inositol lipids by phospholipase C-delta 1.

Rebecchi, Mario J.; Eberhardt, R.; Delaney, Tristan J.; Ali, S. Y.; Bittman, Robert

doi:10.1016/s0021-9258(18)53914-4

Cited by 46 publications

(30 citation statements)

References 47 publications

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“…The CMC value for diCftPI is suppressed to 9.0 mM under the buffering conditions (50 mM Tris-aceta te) of the NMR assay. Both of these values were significantly higher than the value reported for D-diCePI prepared enzymatically (Rebecchi et al, 1993). In that study the CMC was determined as 2.6 mM in a higher ionic strength medium (100 mM KC1 and 25 mM HEPES); such a drop in CMC would be expected since the PI compound is negatively charged.…”

Section: Resultsmentioning

confidence: 61%

Substrate requirements of bacterial phosphatidylinositol-specific phospholipase C

Lewis

Garigapati²,

Zhou³

et al. 1993

Biochemistry

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A series of symmetric short-chain phosphatidylinositols (PI), including dihexanoyl-PI, diheptanoyl-PI (racemic as well as D and L forms), and 2-methoxy inositol-substituted diheptanoyl-PI, have been synthesized, characterized, and used to investigate key mechanistic questions about phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis. Key results include the following: (i) bacterial PI-PLC exhibits a 5-6-fold "interfacial activation" when its substrate is present in an interface as opposed to existing as a monomer in solution (in fact, the similarity to the activation observed with nonspecific PLC enzymes suggests a similarity in activation mechanisms); (ii) the 2-OH must be free since the enzyme cannot hydrolyze diheptanoyl-2-O-methyl-PI (this is most consistent with the formation of inositol cyclic 1,2-phosphate as a necessary step in catalysis); (iii) the inositol ring must have the D stereochemistry (the L-inositol attached to the lipid moiety is neither a substrate nor an inhibitor); and (iv) the presence of noninhibitory L-PI with the D-PI substrate relieves the diacylglycerol product inhibition detected at approximately 30% hydrolysis.

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Section: Resultsmentioning

confidence: 61%

Substrate requirements of bacterial phosphatidylinositol-specific phospholipase C

Lewis

Garigapati²,

Zhou³

et al. 1993

Biochemistry

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“…This suggests that PI-PLCs make only limited interaction with the DAG moiety. However, kinetic studies of lipid substrates with short acyl chains at concentrations below the critical micellar concentration (Rebecchi et al, 1993) indicate that the efficiency with which substrates are hydrolyzed is profoundly increased with increasing acyl chain length. A convex hydrophobic ridge located at one end of the active site opening that consists of three loops connecting Tβ1 with TR1, Tβ2 with TR2, and Tβ7 with TR6 was shown to bind the hydrophobic portion of a detergent (Essen et al, 1996).…”

Section: Binding Of Substrates and Substrate Analogues To Plc-δ1mentioning

confidence: 99%

“…No measurements of affinities for any of the phospholipid head group or intermediate analogues we have employed are available. However, kinetic measurements with related compounds such as di-C 4 -PI (Rebecchi et al, 1993) and glycerophosphoinositides (Y. Wu, O. Perisic, R. Williams, and M. Roberts, unpublished data) suggest affinities in the millimolar range. Because of the rather low affinities, compounds lacking particular interacting groups may result in partial occupancies or higher than average temperature factors, such as seen with the 4,5-IP 2 and cICH 2 P in comparison with the inositol trisphosphates.…”

Section: Binding Of Substrates and Substrate Analogues To Plc-δ1mentioning

confidence: 99%

Structural Mapping of the Catalytic Mechanism for a Mammalian Phosphoinositide-Specific Phospholipase C^,

et al. 1997

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The crystal structures of various ternary complexes of phosphoinositide-specific phospholipase C-delta 1 from rat with calcium and inositol phosphates have been determined at 2.30-2.95 A resolution. The inositol phosphates used in this study mimic the binding of substrates and the reaction intermediate and include D-myo-inositol-1,4,5-trisphosphate, D-myo-inositol-2,4, 5-trisphosphate. D-myo-inositol-4,5-bisphosphate, and D,1-myo-inositol-2-methylene-1,2-cyclićmonophosphonate. The complexes exhibit an almost invariant mode of binding in the active site, each fitting edge-on into the active site and interacting with both the enzyme and the catalytic calcium at the bottom of the active site. Most of the active site residues do not undergo conformational changes upon binding either calcium or inositol phosphates. The structures are consistent with bidentate liganding of the catalytic calcium to the inositol phosphate intermediate and transition state. The complexes suggest explanations for substrate preference, pH optima, and ratio of cyclic to acyclic reaction products. A reaction mechanism is derived that supports general acid/base catalysis in a sequential mechanism involving a cyclic phosphate intermediate and rules out a parallel mechanism where acyclic and cyclic products are simultaneously generated.

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“…The cleavage of the non-hydrophobic inositol phosphodiesters synthesized in this work is still several-fold slower than the hydrolysis of IcP. The large reduction in the cleavage rate of such non-hydrophobic RO-PI is probably due to an absence of the interfacial activation of PI-PLC with non-hydrophobic substrates (low k cat ) .…”

Section: Discussionmentioning

confidence: 67%

“…The large reduction in the cleavage rate of such non-hydrophobic RO-PI is probably due to an absence of the interfacial activation of PI-PLC with non-hydrophobic substrates (low k cat ) . For example, the cleavage of dihexanoyl-PI with the bacterial and mammalian δ-type PI-PLC is activated only 2- 33b and 5-fold,33c respectively, at the critical micelle concentration.…”

Section: Discussionmentioning

confidence: 99%

Synthesis of Inositol Phosphodiesters by Phospholipase C-Catalyzed Transesterification

et al. 1996

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Transesterification of primary alcohols with inositol 1,2-cyclic phosphate (IcP) in the presence of phosphatidylinositol-specific phospholipase C (PI-PLC) resulted in the formation of O-alkyl inositol 1-phosphates. The starting IcP was obtained in a single step by PI-PLC catalyzed cleavage of phosphatidylinositol from the soybean phospholipid. The transesterification reaction was performed with a series of 20 structurally diverse hydroxyl compounds, ranging in the structural complexity from methanol to the serine-containing Ser-Tyr-Ser-Met tetrapeptide, to give the corresponding phosphodiesters with 20−80% yields, depending mainly on the solubility of alcohols in water. The rates of transesterifications, and of the competing hydrolysis of IcP to inositol 1-phosphate (IP), were relatively insensitive to the alcohol structure. With polyhydroxyl compounds such as glycerol and hexitols, the enzyme displayed complete preference toward formation of the inositol phosphate derivatives of the primary hydroxyl groups. On the other hand, PI-PLC did not discriminate between primary hydroxyl groups in different environments and showed low stereoselectivity with racemic alcohols featuring a chiral center at the β-position. The O-alkyl inositol phosphates formed were readily separable from the hydrolytic product, IP, by the anion-exchange chromatography, and were fully characterized by means of 1H and 31P NMR and electrospray MS. Our results provide a new, simple, and efficient two-step synthetic route to substituted O-alkyl inositol phosphates from inexpensive starting materials. The reported reaction was successfully applied to synthesis of complex inositol phosphate derivatives, as illustrated by inositol phosphoesters of mono- and oligosaccharides, nucleosides and peptides. The synthetic usefulness of this reaction, however, is not limited to the examples shown. Because transesterification activity of phospholipase C has not been reported before, its mechanism is discussed in a broad context of mechanisms of phosphoesterases.

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Hydrolysis of short acyl chain inositol lipids by phospholipase C-delta 1.

Cited by 46 publications

References 47 publications

Substrate requirements of bacterial phosphatidylinositol-specific phospholipase C

Substrate requirements of bacterial phosphatidylinositol-specific phospholipase C

Structural Mapping of the Catalytic Mechanism for a Mammalian Phosphoinositide-Specific Phospholipase C^,

Synthesis of Inositol Phosphodiesters by Phospholipase C-Catalyzed Transesterification

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Hydrolysis of short acyl chain inositol lipids by phospholipase C-delta 1.

Cited by 46 publications

References 47 publications

Substrate requirements of bacterial phosphatidylinositol-specific phospholipase C

Substrate requirements of bacterial phosphatidylinositol-specific phospholipase C

Structural Mapping of the Catalytic Mechanism for a Mammalian Phosphoinositide-Specific Phospholipase C,

Synthesis of Inositol Phosphodiesters by Phospholipase C-Catalyzed Transesterification

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Structural Mapping of the Catalytic Mechanism for a Mammalian Phosphoinositide-Specific Phospholipase C^,