| Pancreatic lipase (PPL), pancreatic phospholipase A2 (PLA2) and bacterial sphingomyelinase (SMase) were investigated for their hydrolytic activity in monolayers and under controlled relative humidity (RH or a w) conditions. Dynamic specific activities as a function of the monolayer surface pressure ∏, SA(t, ∏), of these enzymes were estimated by developing new experiments that allowed a simultaneous measurement of enzyme hydrolysis and enzyme adsorption. Steady state specific activities (SA ss(aw)) as a function of vapor phase water activity, a w, were estimated by developing protocols for measuring the amount of product formed by known amount of enzyme in a mixture under controlled relative humidity (RH). SA(t, ∏) and SAss(aw) for all the enzymes were strongly dependent on ∏ of the monolayer, and RH of the vapor phase respectively. To analyze SA(t, ∏) in monolayers in terms of interfacial water behavior, a relation between ∏ and the interfacial water, proposed previously, was substantiated by developing new theory that related ∏ of adsorbing protein and activity of the interfacial water. Analysis of SA(t, ∏) in monolayer systems revealed critical role of water in the dynamic behavior of enzymes. New kinetic models for dynamic behaviors that entailed sequential interfacial activation, and interfacial catalysis were proposed and mathematically solved. The successive interfacial processing of enzymes that embodied the rate constants for interfacial activation, and catalysis were defined in terms of interfacial processing parameter P cat. Pcat for the monolayer studies, and SAss for the controlled relative humidity experiments were found to behave in a similar manner for SMase and PLA2. Given the fact that surface pressure was controlled in the monolayer experiments and interfacial water activity was calculated from it and the actual relative humidity was controlled in the other experiments, their similar behavior lent support to the hypothesis that surface pressure indeed is a measure of interfacial water activity. The conclusions of this study strongly suggest that water activity in interfacial regions may modulate enzyme and protein functionalities via hydration-dehydration processes. Particularly for biological membranes, control in water activity in the interfacial regions may provide a mechanism to regulate protein functionality. |
