Dissecting the Catalytic Mechanism of Staphylococcal Lipases Using Carbamate Substrates:  Chain Length Selectivity, Interfacial Activation, and Cofactor Dependence

Abstract

p-Nitrophenyl N-alkylcarbamates with different alkyl chains were used as substrates to determine separately the carbamylation and decarbamylation rates of the lipases from Staphylococcus hyicus and S. aureus. Both enzymes are reversibly inhibited by these compounds due to a rapid carbamylation of their active site serines followed by a slow decarbamylation. The carbamylation reaction is strongly pH-dependent and the pH profile suggests that an unprotonated histidine is required for this reaction. In contrast, the decarbamylation is pH-independent suggesting the presence of a hydrogen bond between the active site histidine and the carbamyl moiety. S. hyicus lipase preferably reacts with medium to long chain carbamates with an optimum for eight carbon atoms. In contrast, S. aureus lipase is highly specific for short chain carbamates. These results are in agreement with the respective substrate preferences of both lipases toward natural lipids. The decarbamylation rates of both enzymes hardly depend on the alkyl chain length, and from this it is concluded that chain length selectivity is expressed in the first step of catalysis. Both the carbamylation and decarbamylation reaction rates of S. hyicus lipase are enhanced in the presence of micelles, the activation effect being most pronounced in the first step. For the S. aureus lipase only a small influence of interfaces on both reaction steps was observed. These results are discussed in view of a possible role of a lid covering the active site. Kinetic experiments in the presence and absence of calcium strongly suggest that calcium ions are important for the structural stabilization of the unmodified as well as of the carbamylated enzymes. This structural function of calcium was supported by urea unfolding experiments, from which it appeared that for both enzymes the free energy for unfolding is significantly lower in the absence of calcium. In conclusion our results show that kinetic differences between both lipases reside in the acylation step, and that calcium is important for the structural stabilization of the unmodified, and moreover, the acylated enzymes.

Biochemical characterization of a new thermostable lipase from Bacillus pumilus strain

“…The N-terminal sequence of purified BPL was determined by automated Edman_s degradation, using an Applied Biosystems 470 A protein sequencer equipped with PTH 120A analyser [18]. The sequence was kindly determined by Dr. Reinbolt (IBMC, UPR 9002, CNRS-Strasbourg, France).…”

“…Lipase activities were measured as a function of various substrate (TC 4 , TC 8 or TC 18 ) concentrations (0-40 mM). The Michaelis-Menten constant (KMapp.)…”

“…The Michaelis-Menten constant (KMapp.) and the maximum velocity (Vmax) for the reaction with TC 4 , TC 8 or TC 18 as substrate were calculated by Lineweaver-Burk plot.…”

The synthesis and biological evaluation of para-substituted phenolic N-alkyl carbamates as endocannabinoid hydrolyzing enzyme inhibitors

“…The majority of the carbamates were obtained by treating various phenols with suitable alkyl isocyanates in the presence of triethylamine; the O-phenyl carbamates 9-29, 30 [51], 31-33, 35 [53,54], 37-39 [55], 40 [52,55], 41, 42 [53,56], 43 [57], 44, 46, and 48-51 were prepared from the corresponding phenols, and in the case of N-phenyl carbamate 52, from cyclohexanol by a route described in Schemes 1 and 2. On the contrary, the O-phenyl carbamates 34 [52,55,[58][59][60] and 36 [61] were prepared from 4-nitrophenyl chloroformate and the appropriate amine, yielding the desired products in 32-99% yield, with shorter reaction time than in the isocyanate method. The O-phenyl N-n-hexylcarbamates 45 and 47 were prepared from the corresponding methyl benzoates (Scheme 2).…”

Characterization and Heterologous Gene Expression of a Novel Esterase fromLactobacillus caseiCL96

“…A common characteristic of these enzymes is that they contain a catalytic triad, composed of Ser, His, and Asp or Glu (15,48). In addition, most of these enzymes have a structural motif, G-X-S-X-G, which contains the active-site serine residue (6).…”