title: 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.
authors.affiliation: Department of Enzymology and Protein Engineering, CBLE, Utrecht University, The Netherlands.
authors.forename: J W
authors.initials: JW
authors.lastname: Simons
authors.forename: J W
authors.initials: JW
authors.lastname: Boots
authors.forename: M P
authors.initials: MP
authors.lastname: Kats
authors.forename: A J
authors.initials: AJ
authors.lastname: Slotboom
authors.forename: M R
authors.initials: MR
authors.lastname: Egmond
authors.forename: H M
authors.initials: HM
authors.lastname: Verheij
mesh_terms: D002219:Carbamates / Q000378:metabolism*; D002384:Catalysis; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008049:Lipase / Q000737:chemistry* / Q000378:metabolism*; D008956:Models, Chemical; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation; D017510:Protein Folding; D012995:Solubility; D013210:Staphylococcus / Q000201:enzymology*; D013211:Staphylococcus aureus / Q000201:enzymology*; D013379:Substrate Specificity; D013816:Thermodynamics; D014508:Urea / Q000494:pharmacology
chemical_list: D002219:Carbamates; D014508:Urea; D008049:Lipase