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 A2 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.