Kinetic analysis of salting activation of a subtilisin-like halophilic protease.
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.
Okamoto
Débora N
DN
Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo. Rua Três de Maio 100, 04044-20 São Paulo, Brazil.
Kondo
Marcia Y
MY
Santos
Jorge A N
JA
Nakajima
Sawa
S
Hiraga
Kazumi
K
Oda
Kohei
K
Juliano
Maria A
MA
Juliano
Luiz
L
Gouvea
Iuri E
IE
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
D001426:Bacterial Proteins; D012492:Salts; D012697:Serine Endopeptidases; D020860:Subtilisin