Characterization of the kinetics of phospholipase C activity toward mixed micelles of sodium deoxycholate and dimyristoylphosphatidylcholine

Abstract

Phospholipase C catalyzed hydrolysis of dimyristoyl phosphatidylcholine (DMPC) in phospholipid–bile salt mixed micelles was studied with particular attention on the relationship between interfacial enzyme activity and the physicochemical properties of substrate aggregates. Steady state kinetics is observed and it is argued that conditions for steady state exist because the enzyme encounters a steady supply of substrate by hopping between micelles at a rate faster than the chemical reaction rate. An existing kinetic model is reformulated to a more usable form. This presents a new approach to treating the kinetic data and allows extraction of the kinetic parameters of the model from the activity dependence on micellar lipid substrate surface concentration. The kinetic parameters were found to depend on the physicochemical properties of substrate aggregates, but remain constant over a range of lipid and bile salt concentrations. The substrate aggregates were characterized by time-resolved fluorescence quenching (TRFQ). The activity values and the micelle sizes group into two sets: (i) larger micelles for bile salt/lipid ≤ 5 showing higher activity and shorter steady state duration (≤ 4 min) and (ii) smaller micelles for bile salt/lipid > 5 with lower activity and longer steady state (≈ 10 min). At least two sets of parameters, for bile salt/lipid ≤ 5 and > 5, characterize the kinetics. Higher enzyme–micelle dissociation constant and lower catalytic rate are found for the group of smaller micelles. An explanation supporting our finding is that as micelles become smaller the overlap area for enzyme–micelle binding decreases, leading to weaker binding. Consequently the enzyme dissociation constant increases. Extension of the present approach to other phospholipases and substrates to establish its generality and correlation between micelle size and the catalytic rate are areas for future investigations.