Surface dilution kinetics using substrate analog enantiomers as diluents: enzymatic lipolysis by bee venom phospholipase A2.
A novel assay employing D-enantiomers of phospholipids as diluents for characterizing surface kinetics of lipid hydrolysis by phospholipases is introduced. The rationales of the method are (i) D-enantiomers resist hydrolysis because of the stereoselectivity of the enzymes toward L-enantiomers and (ii) mixtures of L+D-lipids at various L/D ratios but constant L+D-lipid concentrations yield a surface dilution series of variable L-lipid concentration with constant medium properties. Kinetic characterization of bee venom phospholipase A(2) activity at bile salt+phospholipid aggregate-water interfaces was performed using the mixed L+D-lipid surface dilution assay, and interface kinetic parameters were obtained. The assay applies to biomembrane models as well. Activity was measured by pH-stat methods. Aggregation numbers and interface hydration/microviscosity measured by time-resolved fluorescence quenching and electron spin resonance, respectively, confirmed that interface properties were indeed invariant in a surface dilution series, supporting rationale (ii), and were used to calculate substrate concentrations. Activity data show excellent agreement with a kinetic model derived with D-enantiomers as diluents and also that D-phospholipids bind to the enzyme but resist hydrolysis; underscoring rationale (i). The assay is significant for enabling determination of interface-specific kinetic parameters for the first time and thereby characterization of interface specificity of lipolytic enzymes.
Singh
Jasmeet
J
Department of Physics and Center for Supramolecular Studies, California State University, Northridge, CA 91330, USA.
Ranganathan
Radha
R
Hajdu
Joseph
J
D000818:Animals; D001514:Bee Venoms / Q000201:enzymology*; D001516:Bees; D004578:Electron Spin Resonance Spectroscopy; D057075:Enzyme Assays / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008066:Lipolysis; D008823:Micelles; D008956:Models, Chemical; D054467:Phospholipases A2 / Q000378:metabolism*; D013237:Stereoisomerism; D013379:Substrate Specificity
D001514:Bee Venoms; D008823:Micelles; D054467:Phospholipases A2