title: Kinetic analysis of salting activation of a subtilisin-like halophilic protease.
abstract: The secreted extracellular subtilase SR5-3 from Halobacillus sp. bacterium, isolated from the high-salt environment of Thai fish sauce, was utilized as a model halophilic serine protease. The dependence of salt activation on the size and structure of substrates was evaluated assaying the enzyme with Suc-AAPF-MCA and with the Fluorescence Resonance Energy Transfer (FRET) peptide Abz-AAPFSSKQ-EDDnp. Solvent isotope effects (SIE) and the thermodynamic parameters for activation of the hydrolysis of Suc-AAPF-MCA and Abz-AAPFSSKQ-EDDnp by SR5-3 protease in the presence of salts were also performed. All the obtained results support the notion that the salting out effect is responsible for the halophilic character of SR5-3, and the magnitude of its hydrolytic activity is mainly derived from the improvement of catalytic and/or interaction steps depending on the nature and size of the substrates, principally if they occupy the substrate prime subsites.
authors.affiliation: Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo. Rua Três de Maio 100, 04044-20 São Paulo, Brazil.
authors.forename: Débora N
authors.initials: DN
authors.lastname: Okamoto
authors.forename: Marcia Y
authors.initials: MY
authors.lastname: Kondo
authors.forename: Jorge A N
authors.initials: JA
authors.lastname: Santos
authors.forename: Sawa
authors.initials: S
authors.lastname: Nakajima
authors.forename: Kazumi
authors.initials: K
authors.lastname: Hiraga
authors.forename: Kohei
authors.initials: K
authors.lastname: Oda
authors.forename: Maria A
authors.initials: MA
authors.lastname: Juliano
authors.forename: Luiz
authors.initials: L
authors.lastname: Juliano
authors.forename: Iuri E
authors.initials: IE
authors.lastname: Gouvea
mesh_terms: D001406:Bacillaceae / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D002384:Catalysis; D004789:Enzyme Activation; D031541:Fluorescence Resonance Energy Transfer; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D012492:Salts / Q000737:chemistry / Q000494:pharmacology*; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D020860:Subtilisin / Q000737:chemistry / Q000378:metabolism*; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D012492:Salts; D012697:Serine Endopeptidases; D020860:Subtilisin