DISSECTING THE CATALYTIC MECHANISM OF STAPHYLOCOCCAL LIPASES USING CARBAMATE SUBSTRATES - CHAIN-LENGTH SELECTIVITY, INTERFACIAL ACTIVATION,AND COFACTOR DEPENDENCE

p-Nitrophenyl N-alkylcarbamates with different alkyl chains were used as substrates to determine separately the carbamylation and decarbamyl ation rates of the lipases from Staphylococcus hyicus and S. aureus. B oth enzymes are reversibly inhibited by these compounds due to a rapid carbamylation of their active site serines followed by a slow decarba mylation. The carbamylation reaction is strongly pH-dependent and the pH profile suggests that an unprotonated histidine is required for thi s reaction. In contrast, the decarbamylation is pH-independent suggest ing the presence of a hydrogen bond between the active site histidine and the carbamyl moiety. S. hyicus lipase preferably reacts with mediu m to long chain carbamates with an optimum for eight carbon atoms. In contrast, S. aureus lipase is highly specific for short chain carbamat es. These results are in agreement with the respective substrate prefe rences of both lipases toward natural lipids. The decarbamylation rate s of both enzymes hardly depend on the alkyl chain length, and from th is it is concluded that chain length selectivity is expressed in the f irst step of catalysis. Both the carbamylation and decarbamylation rea ction rates of S. hyicus lipase are enhanced in the presence of micell es, the activation effect being most pronounced in the first step. For the S. aureus lipase only a small influence of interfaces on both rea ction steps was observed. These results are discussed in view of a pos sible role of a lid covering the active site. Kinetic experiments in t he presence and absence of calcium strongly suggest that calcium ions are important for the structural stabilization of the unmodified as we ll as of the carbamylated enzymes. This structural function of calcium was supported by urea unfolding experiments, from which it appeared t hat for both enzymes the free energy for unfolding is significantly lo wer in the absence of calcium, In conclusion our results show that the kinetic differences between both lipases reside in the acylation step , and that calcium is important for the structural stabilization of th e unmodified, and moreover, the acylated enzymes.