Kinetic steps for the hydrolysis of sphingomyelin by Bacillus cereus sphingomyelinase in lipid monolayers.

The sphingomyelinase (Sphmase) activity degrading sphingomyelin (Sphm) monolayers shows a slow-reaction latency period before exhibiting constant rate catalysis. These two kinetic regions are regulated independently by the lateral surface pressure and by lipids that are biomodulators of cell function such as ceramide, glycosphingolipids, fatty acids, and lysophospholipids. Knowledge of the interfacial adsorption of Sphmase, precatalytic activation, initiation of effective catalysis, and the corresponding kinetic parameters is necessary for studying the level at which different lipids modulate the activity. We dissected some kinetic steps and determined the rate constants for degradation of Sphm, under controlled intermolecular organization, by Sphmase. Six models, adapted to two dimensions, were used to elucidate possible mechanisms for the interfacial activation of Sphmase during the lag time. The models consider enzyme binding to the substrate monolayer and a subsequent, essentially irreversible interfacial activation; this is supported experimentally by monolayer transfer experiments. Some mechanisms involve enzyme-substrate binding and associated states of the enzyme in the bulk subphase or at the interface, prior to complete activation. The activity of Sphmase is consistent with kinetics involving enzyme partitioning into the interface followed by substrate association, and by a process that proceeds with bimolecular kinetic dependence on the interfacial Sphmase concentration, and a subsequent slow step of activation. A possible equilibrium between the apparent monomolecular and bimolecular activated states of the interfacial enzyme, coupled to a slow activation, constitute rate-limiting steps that can explain the existence of lag time and the achievement of a maximum constant rate of degradation of Sphm monolayers by Sphmase.