Abstract
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.—Fanani, M. L., and B. Maggio. Kinetic steps for the hydrolysis of sphingomyelin by Bacillus cereus sphingomyelinase in lipid monolayers. J. Lipid Res. 2000. 41: 1832–1840.
Highlights
The sphingomyelinase (Sphmase) activity degrading sphingomyelin (Sphm) monolayers shows a slowreaction latency period before exhibiting constant rate catalysis
On the basis of the different cross-sectional molecular areas of sphingomyelin (Sphm) and ceramide in lipid monolayers, we previously showed that the activity of Bacillus cereus Sphmase can be followed in real time, while the catalytic reaction is taking place, with the intermolecular organization of the lipid substrate precisely controlled and continuously known [15]
We showed that the modulation of Sphmase by different lipids and products of the phosphohydrolytic reaction catalyzed by phospholipase A2 (PLA2) takes place at different levels that affect independently the steady state rate of catalysis and the lag-time period [16]
Summary
The sphingomyelinase (Sphmase) activity degrading sphingomyelin (Sphm) monolayers shows a slowreaction 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. 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. On the basis of the different cross-sectional molecular areas of sphingomyelin (Sphm) and ceramide in lipid monolayers, we previously showed that the activity of Bacillus cereus Sphmase can be followed in real time, while the catalytic reaction is taking place, with the intermolecular organization of the lipid substrate precisely controlled and continuously known [15].
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