Salt effect on substrate specificity of a subtilisin-like halophilic protease.
Enzyme-substrate interaction under the presence of high concentration of salts is of great interest for biotechnology applications and basic enzymology. In our previous work, the salt effect on halophilic subtilase SR5-3 was evaluated with Suc-AAPF-MCA and with the FRET peptide Abz-AAPFSSKQ-EDDnp. It was demonstrated that the magnitude of catalytic activity enhancement was affected by the presence of the prime site residues (Okamoto et al., 2009). In this work, a detailed analysis of the salt effect on SR5-3 protease substrate specificity was performed using chromogenic and coumarin substrates as well as FRET peptides derived from Abz-KLRSSKQ-EDDnp. The followings were demonstrated: 1) Preference of amino acid of SR5-3 protease at the P(3), P(2), P(1), P(1)' or P(2)' position of FRET substrates was almost similar with that of subtilisin. 2) Under the presence of the salts (3M NaCl or 1M Na(2)SO(4)), SR5-3 protease showed higher kcat values, lower Km values and totally 2-6 times higher kcat/Km values compared with those of control for FRET substrates, and salts did not significantly affect the preference of amino acid residues at the primary positions (P1 and P1'), but it affected the preference at the P(2) and P(2)' position. In contrast, for smaller substrates with only non-prime sites, SR5-3 protease showed 20-75 times higher kcat/Km values compared with those of control. These findings are in agreement with the notion that increases in enzyme-substrate interactions in subtilases alter the rate-determining step in peptide hydrolysis.
Okamoto
Debora N
DN
Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo. Rua Três de Maio 100, 04044-20 São Paulo, Brazil. gouvea@biofis.epm.br.
Kondo
Marcia Y
MY
Hiraga
Kazumi
K
Juliano
Maria A
MA
Juliano
Luiz
L
Gouvea
Iuri E
IE
Oda
Kohei
K
D000595:Amino Acid Sequence; D001406:Bacillaceae / Q000201:enzymology*; D001426:Bacterial Proteins / Q000378:metabolism*; D001665:Binding Sites; D055162:Biocatalysis / Q000187:drug effects; D006868:Hydrolysis / Q000187:drug effects; D010455:Peptides / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000378:metabolism*; D012965:Sodium Chloride / Q000494:pharmacology; D013379:Substrate Specificity
D001426:Bacterial Proteins; D010455:Peptides; D012965:Sodium Chloride; D012697:Serine Endopeptidases