Interfacial catalysis by phospholipase A2: determination of the interfacial kinetic rate constants

Berg, Otto G.; Bao, Yu; Rogers, Joseph M.; Jain, Mahendra Kumar

doi:10.1021/bi00243a034

Cited by 193 publications

(437 citation statements)

References 34 publications

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“…Such a process would be more efficient than direct binding of remnant particles in solution to the LRP, because remnant particles concentrated on the cell surface could diffuse in two dimensions by rapidly exchanging between HSPG binding sites until they collide with the LRP to form the LRP-remnant particle complex. This lateral diffusion on the cell surface is reminiscent of the "scooting mode" proposed for the interfacial reaction of phospholipases (56). It is also possible that some initial complexes of HSPG-remnant particle would undergo the second binding step, forming a more stable complex for internalization without LRP involvement.…”

Section: Implications For the Sequestration Role Of Hspg In Remnant Lmentioning

confidence: 88%

Two-step Mechanism of Binding of Apolipoprotein E to Heparin

Futamura

Dhanasekaran

Handa

et al. 2005

Journal of Biological Chemistry

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The interaction of apolipoprotein E (apoE) with cellsurface heparan sulfate proteoglycans is an important step in the uptake of lipoprotein remnants by the liver. ApoE interacts predominantly with heparin through the N-terminal binding site spanning the residues around 136 -150. In this work, surface plasmon resonance analysis was employed to investigate how amphipathic ␣-helix properties and basic residue organization in this region modulate binding of apoE to heparin. The apoE/heparin interaction involves a twostep process; apoE initially binds to heparin with fast association and dissociation rates, followed by a step exhibiting much slower kinetics. Circular dichroism and surface plasmon resonance experiments using a disulfide-linked mutant, in which opening of the Nterminal helix bundle was prevented, demonstrated that there is no major secondary or tertiary structural change in apoE upon heparin binding. Mutations of Lys-146, a key residue for the heparin interaction, greatly reduced the favorable free energy of binding of the first step without affecting the second step, suggesting that electrostatic interaction is involved in the first binding step. Although lipid-free apoE2 tended to bind less than apoE3 and apoE4, there were no significant differences in rate and equilibrium constants of binding among the apoE isoforms in the lipidated state. Discoidal apoE3-phospholipid complexes using a substitution mutant (K143R/K146R) showed similar binding affinity to wild type apoE3, indicating that basic residue specificity is not required for the effective binding of apoE to heparin, unlike its binding to the low density lipoprotein receptor. In addition, disruption of the ␣-helix structure in the apoE heparin binding region led to an increased favorable free energy of binding in the second step, suggesting that hydrophobic interactions contribute to the second binding step. Based on these results, it seems that cellsurface heparan sulfate proteoglycan localizes apoEenriched remnant lipoproteins to the vicinity of receptors by fast association and dissociation.

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Section: Implications For the Sequestration Role Of Hspg In Remnant Lmentioning

confidence: 88%

Two-step Mechanism of Binding of Apolipoprotein E to Heparin

Futamura

Dhanasekaran

Handa

et al. 2005

Journal of Biological Chemistry

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“…All kinetic measurements were carried out on a pH-stat titration system to monitor reaction progress as established previously (Jain et al, 1986;Berg et al, 1991). Activities toward micellar DC 7 PC substrates were carried out in 100 mM NaCl and 10 mM CaCl 2 , at 24°C and pH 8.0.…”

Section: Methodsmentioning

confidence: 99%

“…The Michaelis constant for catalysis in the scooting mode at the interface (K M *) 2 was determined (a) by deconvoluting the reaction progress in terms of the integrated Michaelis-Menten equation to describe catalytic turnover at the interface (Berg et al, 1991), (b) from the kinetic dissociation constant for calcium [K Ca *(S)] obtained from the dependence of V 0 on the concentration of calcium , or (c) from the kinetics of inhibition by active site-directed inhibitors because the mole fraction of inhibitor required for 50% inhibition, X I (50), is related to K I * and K M * (Jain et al, 1991a,d). This requires knowledge of dissociation constants for the products of hydrolysis (K p *), calcium (K ca *), the ether analog of the substrate (K S *), or inhibitors (K I *) bound to the enzyme at the interface of deoxy-LPC as a neutral diluent as determined by the protection described below.…”

Section: Methodsmentioning

confidence: 99%

Phospholipase A2 Engineering. Structural and Functional Roles of the Highly Conserved Active Site Residue Aspartate-49

Yu²,

Zhu³

et al. 1994

Biochemistry

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The aspartate-99 of secreted phospholipase A 2 (PLA2) has been proposed to be critical for the catalytic mechanism and interfacial activation of PLA2. Aspartate-99 connects the catalytic machinery (including the catalytic diad, the putative catalytic waters W5 and W6, and the calcium cofactor) to the hydrogen-bonding network. The latter involves Y52, Y73, the structural water, and the N-terminal region putatively required for the interfacial activation. A triple mutant of bovine pancreatic PLA2 with substitutions aspartate plus adjacent tyrosine residues (Y52,73F/D99N) was constructed, its X-ray structure was determined, and kinetic characteristics were analyzed. The kinetic properties of the D99N mutant constructed previously were also further analyzed. The X-ray structure of the Y52,73F/D99N mutant indicated a substantial disruption of the hydrogen-bonding network including the loss of the structural water similar to that seen in the structure of the D99N mutant published previously [Kumar, A., Sekharudu, Y. C., Ramakrishnan, B., Dupureur, C. M., Zhu, H., Tsai, M.-D., & Sundaralingam, M. (1994) Protein Sci. 3, 2082-2088. Kinetic analysis demonstrated that these mutants possessed considerable catalytic activity with a k cat value of about 5% compared to WT. The values of the interfacial Michaelis constant were also little perturbed (ca. 4-fold lower for D99N and marginally higher for Y52,73F/D99N). The results taken together suggest that the hydrogen-bonding network is not critically important for interfacial activation. Instead, it is the chemical step that is perturbed, though only modestly, in the mutants.Secreted phospholipases A 2 (PLA2) 1 are small calciumdependent lipolytic enzymes that catalyze the stereospecific hydrolysis of the sn-2 ester bond of phospholipids at the interfaces . The activity of PLA2 on aggregated substrates is considerably higher than on monomolecularly dispersed zwitterionic substrates. This phenomenon is known as interfacial activation. The molecular basis of interfacial activation is not known, although the hydrogenbonding network (Figure 1) has been proposed to serve as a link between the active site and the interfacial recognition site (Verheij et al., 1981;Dijkstra et al., 1983).The roles of Tyr-52, Tyr-73, and the N-terminal residues have been investigated by mutagenises (Dupureur et al., 1992a;Maliwal et al., 1994;Liu et al., 1995). The results showed that the hydrogen-bonding network involving the N-terminal region and the conserved water molecule (W11) does not play a significant role in the chemical step of the catalytic cycle at the anionic interface. The crystal structure of the double mutant Y52,73F (Sekharudu et al., 1992) showed that although the loss of hydrogen bonds of the tyrosines was compensated by the increase in the hydrophobic contacts of the phenyl groups, the structural water was retained. However, in the single mutant D99N (Kumar et al., 1994), the structural water was absent.To gain a better understanding of the role of the conserved structura...

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“…However, the analysis of phospholipase catalysis at the interface has received considerable investigation and has led to kinetic schemes describing the action of phospholipases (22,23). The kinetics can be described using classical Michaelis-Menten kinetic theory applied to interfacial catalysis (24), which facilitates determination of numerous kinetic parameters and binding constants (11,25).…”

mentioning

confidence: 99%

Competitive, Reversible Inhibition of Cytosolic Phospholipase A2 at the Lipid-Water Interface by Choline Derivatives That Partially Partition into the Phospholipid Bilayer

Burke¹,

Witmer²,

Zusi

et al. 1999

Journal of Biological Chemistry

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Cytosolic phospholipase A 2 (cPLA 2 ) catalyzes the selective release of arachidonic acid from the sn-2 position of phospholipids and is believed to play a key cellular role in the generation of arachidonic acid. When assaying the human recombinant cPLA 2 using membranes isolated from [ 3 H]arachidonate-labeled U937 cells as substrate, 2-(2-benzyl-4-chlorophenoxy)ethyl-dimethyln-octadecyl-ammonium chloride (compound 1) was found to inhibit the enzyme in a dose-dependent manner (IC 50 ‫؍‬ 5 M). It was over 70 times more selective for the cPLA 2 as compared with the human nonpancreatic secreted phospholipase A 2 , and it did not inhibit other phospholipases. Additionally, it inhibited arachidonate production in N-formyl-methionyl-leucyl-phenylalanine-stimulated U937 cells. To further characterize the mechanism of inhibition, an assay in which the enzyme is bound to vesicles of 1,2-dimyristoyl-sn -glycero-3-phosphomethanol containing 6 -10 mol % of 1-palmitoyl-2-[1-14 C]arachidonoyl-sn-glycero-3-phosphocholine was employed. With this substrate system, the dose-dependent inhibition could be defined by kinetic equations describing competitive inhibition at the lipid-water interface. The apparent equilibrium dissociation constant for the inhibitor bound to the enzyme at the interface (K I * app ) was determined to be 0.097 ؎ 0.032 mol % versus an apparent dissociation constant for the arachidonatecontaining phospholipid of 0.3 ؎ 0.1 mol %. Thus, compound 1 represents a novel structural class of inhibitor of cPLA 2 that partitions into the phospholipid bilayer and competes with the phospholipid substrate for the active site. Shorter n-alkyl-chained (C-4, C-6, C-8) derivatives of compound 1 were shown to have even smaller K I * app values. However, these short-chained analogs were less potent in terms of bulk inhibitor concentration needed for inhibition when using the [ 3 H]arachidonate-labeled U937 membranes as substrate. This discrepancy was reconciled by showing that these shorter-chained analogs did not partition into the [ 3 H]arachidonate-labeled U937 membranes as effectively as compound 1. The implications for in vivo efficacy that result from these findings are discussed.

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Interfacial catalysis by phospholipase A2: determination of the interfacial kinetic rate constants

Cited by 193 publications

References 34 publications

Two-step Mechanism of Binding of Apolipoprotein E to Heparin

Two-step Mechanism of Binding of Apolipoprotein E to Heparin

Phospholipase A2 Engineering. Structural and Functional Roles of the Highly Conserved Active Site Residue Aspartate-49

Competitive, Reversible Inhibition of Cytosolic Phospholipase A2 at the Lipid-Water Interface by Choline Derivatives That Partially Partition into the Phospholipid Bilayer

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