title: Kinetic basis for the substrate specificity during hydrolysis of phospholipids by secreted phospholipase A2.
abstract: 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.
authors: Department of Chemistry and Biochemistry, University of Delaware, Newark 19716, USA.
authors: J
authors: J
authors: Rogers
authors: B Z
authors: BZ
authors: Yu
authors: S V
authors: SV
authors: Serves
authors: G M
authors: GM
authors: Tsivgoulis
authors: D N
authors: DN
authors: Sotiropoulos
authors: P V
authors: PV
authors: Ioannou
authors: M K
authors: MK
authors: Jain
mesh_terms: D000818:Animals; D001152:Arsenicals / Q000378:metabolism; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D006868:Hydrolysis; D007700:Kinetics; D050356:Lipid Metabolism; D008055:Lipids; D008823:Micelles; D008956:Models, Chemical; D015394:Molecular Structure; D010179:Pancreas / Q000201:enzymology*; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D010743:Phospholipids / Q000378:metabolism*; D011485:Protein Binding; D013053:Spectrophotometry; D013379:Substrate Specificity; D013552:Swine
chemical_list: D001152:Arsenicals; D008055:Lipids; D008823:Micelles; D010743:Phospholipids; C028299:phosphonolipids; C015518:sulfolipids; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium