Kinetics of Bacterial Phospholipase C Activity at Micellar Interfaces: Effect of Substrate Aggregate Microstructure and a Model for the Kinetic Parameters

Overview

Journal J Phys Chem B

Publisher American Chemical Society

Specialty Chemistry

Date 2009 Apr 16

PMID 19367944

Citations 5

Authors

Jasmeet Singh

Radha Ranganathan

Joseph Hajdu

Affiliations

Soon will be listed here.

Abstract

Activity at micellar interfaces of bacterial phospholipase C from Bacillus cereus on phospholipids solubilized in micelles was investigated with the goal of elucidating the role of the interface microstructure and developing further an existing kinetic model. Enzyme kinetics and physicochemical characterization of model substrate aggregates were combined, thus enabling the interpretation of kinetics in the context of the interface. Substrates were diacylphosphatidylcholine of different acyl chain lengths in the form of mixed micelles with dodecyldimethylammoniopropanesulfonate. An early kinetic model, reformulated to reflect the interfacial nature of the kinetics, was applied to the kinetic data. A better method of data treatment is proposed, use of which makes the presence of microstructure effects quite transparent. Models for enzyme-micelle binding and enzyme-lipid binding are developed, and expressions incorporating the microstructural properties are derived for the enzyme-micelle dissociation constant K(s) and the interface Michaelis-Menten constant, K(M). Use of these expressions in the interface kinetic model brings excellent agreement between the kinetic data and the model. Numerical values for the thermodynamic and kinetic parameters are determined. Enzyme-lipid binding is found to be an activated process with an acyl chain length dependent free energy of activation that decreases with micelle lipid molar fraction with a coefficient of about -15RT and correlates with the tightness of molecular packing in the substrate aggregate. Thus, the physical insight obtained includes a model for the kinetic parameters that shows that these parameters depend on the substrate concentration and acyl chain length of the lipid. Enzyme-micelle binding is indicated to be hydrophobic and solvent mediated with a dissociation constant of 1.2 mM.

Citing Articles

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Keshavarz A, Zelaya L, Singh J, Ranganathan R, Hajdu J Chem Phys Lipids. 2016; 202:38-48.

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Surface dilution kinetics of phospholipase A(2) catalyzed lipid-bilayer hydrolysis.

Singh J, Ranganathan R J Phys Chem B. 2014; 118(8):2077-83.

PMID: 24491041 PMC: 3972039. DOI: 10.1021/jp411512c.

Synthesis of mixed-chain phosphatidylcholines including coumarin fluorophores for FRET-based kinetic studies of phospholipase A(2) enzymes.

Wang M, Pinnamaraju S, Ranganathan R, Hajdu J Chem Phys Lipids. 2013; 172-173:78-85.

PMID: 23727005 PMC: 3718315. DOI: 10.1016/j.chemphyslip.2013.05.003.

Quantitation of lysolipids, fatty acids, and phospholipase A2 activity and correlation with membrane polarity.

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