Substrate specificity for interfacial catalysis by phospholipase A2 in the scooting mode

Jain, Mahendra Kumar; Rogers, Joseph M.

doi:10.1016/0005-2760(89)90104-5

Cited by 35 publications

(45 citation statements)

References 19 publications

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“…As shown, the most remarkable feature of the hydrolysis of DC 8 PM is that the rate of hydrolysis shows no anomalous change in the observed rate at the cmc. Such a behavior is quite unlike that seen for the hydrolysis of zwitterionic short chain phosphatidylcholines (de Haas et al, 1971;Pieterson et al, 1974;Jain & Rogers, 1989). The initial rate of hydrolysis increases with the chain length and the nature of the head group.…”

Section: Kinetic Features Of the Hydrolysis Of Short-chain Anionicmentioning

confidence: 69%

“…As is the case with phospholipids, the long chain arsonolipids form lamellar phases which show the characteristic gel-fluid thermotropic transition (Serves et al, 1992(Serves et al, , 1993. Also, as is the case with the phosphatidylmethanol analogs (Jain & Rogers, 1989), the short chain phosphono-and arsonolipids in aqueous dispersions are in monomeric form below their respective cmc's, and above cmc there will be an equilibrium between the monomeric and micellar form of lipid. Although there appears to be no effect of cmc on the profile of V 0 versus concentration of anionic substrates, cmc appears to have an effect on certain interfacial parameters, such as X I -(50).…”

Section: Methodsmentioning

confidence: 95%

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Kinetic Basis for the Substrate Specificity during Hydrolysis of Phospholipids by Secreted Phospholipase A₂

Rogers

Serves

et al. 1996

Biochemistry

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Kinetics of hydrolysis of aqueous dispersions of arsono-, sulfo-, phosphono- and phospholipids by phospholipase A2 from pig pancreas are characterized in terms of interfacial rate and equilibrium parameters. The enzyme with or without calcium binds with high affinity to the aqueous dispersions of the four classes of anionic lipids and shows the same general kinetic behavior. The rate of hydrolysis of anionic substrates does not show an anomalous change at the critical micelle concentration because the enzyme is present in aggregates even when bulk of the substrate is dispersed as a solitary monomer. Apparent affinities of the enzyme for the interface of different anionic lipids are virtually the same. Also, affinities of these substrates for the active site of the enzyme at the interface are comparable. However, a significant change in the catalytic turnover rate is seen as the sn-3 phosphodiester group is modified; the apparent maximum rate at saturating bulk substrate concentration, V(M)app values, increase in the order: homo- and arsonolipids < sulfo- < phosphono- < phospholipids. Not only the basis for the sn-2 enantiomeric selectivity but also the decrease in the rate of hydrolysis with the increasing chain length is due to a decrease in the value of V(M)app. Results show that even when the bulk concentration of anionic phospholipid is below cmc, hydrolysis occurs in aggregates of enzyme and substrate where the chemical step of the turnover cycle remains rate-limiting, which provides a basis for the assumption that V(M)app is directly related to Kcat. The fact that Kcat depends on the nature of the head group (phosphate, phosphonate, sulfate, arsonate) implies that the head group plays a critical role in the rate-limiting chemical step of the catalytic cycle, possibly during the decomposition of the tetrahedral intermediate. The significance of these results for the microscopic steady-state condition for hydrolysis at the micellar interface, mechanism of esterolysis by phospholipase A2, and inhibitor design are discussed.

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Section: Kinetic Features Of the Hydrolysis Of Short-chain Anionicmentioning

confidence: 69%

Section: Methodsmentioning

confidence: 95%

Kinetic Basis for the Substrate Specificity during Hydrolysis of Phospholipids by Secreted Phospholipase A₂

Rogers

Serves

et al. 1996

Biochemistry

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“…DC n PCs and the corresponding ethers (custom synthesis) were from Avanti Polar Lipids. MJ33 (Jain et al, 1991b), DC n PM (Jain and Rogers, 1989) and DC 14 PC-ether (Jain et al, 1986c) were synthesized previously. Preparation and characterization of 4-hexadecyl-2,6-dimethylbenzenediazonium tetrafluoroborate (16-ArN 2 BF 4 ) and the reaction products, 16-ArOH, 16-ArH, and 16-ArCl, are described elsewhere (Chaudhuri et al, 1993).…”

Section: Methodsmentioning

confidence: 99%

“…For example, studies with site-directed mutants have shown that Tyr-52, Phe-22, and Phe-106 participate in substrate binding (Dupureur et al, 1992a,b); that Asp-99 affects k cat * (Sekar et al, 1997); that the N-terminal residues contribute to the binding of the enzyme to the interface (Maliwal et al, 1994;Liu et al, 1995); and that the C-terminal segment participates in coupling of the chemical step to the anionic charge at the interface (Huang et al, 1996). Scheme 1 also accommodates kinetics with anionic micelles, where PLA2 binds with high affinity, substrate exchange with excess micelles is rapid, and the catalytic turnover is limited by the chemical step (Jain & Rogers, 1989;Rogers et al, 1996). Also, direct vesicle to vesicle exchange of phospholipid mediated by peptides and proteins provides a kinetic basis for rationalizing nonspecific inhibition (Jain & Berg, 1989) and the apparent activation of PLA2 under conditions leading to increased substrate replenishment (Jain et al, 1991c;Cajal et al, 1996aCajal et al, ,b, 1997.…”

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confidence: 99%

Thermodynamic and Kinetic Basis of Interfacial Activation: Resolution of Binding and Allosteric Effects on Pancreatic Phospholipase A₂ at Zwitterionic Interfaces^,

et al. 1997

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A general kinetic model for catalysis by interfacial enzymes is developed. It couples the Michaelis-Menten catalytic turnover cycle at the interface with that in the aqueous phase through the distribution equilibria between the interface and the surrounding aqueous phase. Analysis under two limiting conditions fully describes the steady-state kinetics of hydrolysis and resolves the allosteric effects from apparent modes of interfacial activation in terms of the primary rate and equilibrium parameters for pig pancreatic phospholipase A2 (PLA2). One limit is observed in dispersions of anionic phospholipid vesicles, in which intervesicle exchange of enzyme, substrate, and hydrolysis products is absent and reaction occurs only on vesicles containing enzyme. A complete analysis at this highly processive limit, called kinetics in the scooting mode, has been published [Berg et al. (1991) Biochemistry 30, 7283]. Here is reported the analysis in the other limit, PLA2-catalyzed hydrolysis of zwitterionic micelles of short-chain phosphatidylcholines, at which substrate and products are in rapid exchange. Hydrolysis occurs either in bulk aqueous solution with phospholipid monomers or at the micellar interface. Above the critical micelle concentration (cmc), the hydrolysis rate shows a hyperbolic dependence on the bulk substrate concentration present as micelles. This dependence, characterized by the fitting parameters KMapp and VMapp, is analyzed in terms of the primary rate and equilibrium constants. The kinetic analysis is based on the assumption that the microscopic steady-state condition is satisfied because substrate replenishment in the micro-environment of the enzyme is fast relative to the catalytic turnover time. Added NaCl and anionic interface increase the hydrolysis rate in zwitterionic micelles dramatically. The overall interfacial rate enhancement is attributed to three factors: (a) promotion of PLA2 binding by net anionic charge of the interface, (b) enhancement of substrate affinity of PLA2 at the interface (Ks* allostery), and (c) stimulation of the rate-limiting chemical step (kcat* allostery).

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“…PAF possesses an acetate group at the sn-2 position, which is essential for its biological activities. Removal of this acetate group by specific phospholipases A2 12,13 to produce lyso-PAF, provides a natural mechanism for blocking its toxic effects in the various tissues.…”

Section: Introductionmentioning

confidence: 99%

Evidence for Lipid Packaging in the Crystal Structure of the GM2-Activator Complex with Platelet Activating Factor

Wright

Rastinejad

2004

Journal of Molecular Biology

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Substrate specificity for interfacial catalysis by phospholipase A2 in the scooting mode

Cited by 35 publications

References 19 publications

Kinetic Basis for the Substrate Specificity during Hydrolysis of Phospholipids by Secreted Phospholipase A₂

Kinetic Basis for the Substrate Specificity during Hydrolysis of Phospholipids by Secreted Phospholipase A₂

Thermodynamic and Kinetic Basis of Interfacial Activation: Resolution of Binding and Allosteric Effects on Pancreatic Phospholipase A₂ at Zwitterionic Interfaces^,

Evidence for Lipid Packaging in the Crystal Structure of the GM2-Activator Complex with Platelet Activating Factor

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Substrate specificity for interfacial catalysis by phospholipase A2 in the scooting mode

Cited by 35 publications

References 19 publications

Kinetic Basis for the Substrate Specificity during Hydrolysis of Phospholipids by Secreted Phospholipase A2

Kinetic Basis for the Substrate Specificity during Hydrolysis of Phospholipids by Secreted Phospholipase A2

Thermodynamic and Kinetic Basis of Interfacial Activation: Resolution of Binding and Allosteric Effects on Pancreatic Phospholipase A2 at Zwitterionic Interfaces,

Evidence for Lipid Packaging in the Crystal Structure of the GM2-Activator Complex with Platelet Activating Factor

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Kinetic Basis for the Substrate Specificity during Hydrolysis of Phospholipids by Secreted Phospholipase A₂

Kinetic Basis for the Substrate Specificity during Hydrolysis of Phospholipids by Secreted Phospholipase A₂

Thermodynamic and Kinetic Basis of Interfacial Activation: Resolution of Binding and Allosteric Effects on Pancreatic Phospholipase A₂ at Zwitterionic Interfaces^,