Salt Effect on Substrate Specificity of a Subtilisin-Like Halophilic Protease

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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. 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 P3, P2, P1, P1' or P2' position of FRET substrates was almost similar with that of subtilisin. 2) Under the presence of the salts (3M NaCl or 1M Na2SO4), 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 P2 and P2' 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.

Keywords: FRET peptides; Peptidase; kosmotropic salts

Document Type: Research Article


Publication date: June 1, 2010

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  • Protein & Peptide Letters publishes short papers in all important aspects of protein and peptide research, including structural studies, recombinant expression, function, synthesis, enzymology, immunology, molecular modeling, drug design etc. Manuscripts must have a significant element of novelty, timeliness and urgency that merit rapid publication. Reports of crystallisation, and preliminary structure determinations of biologically important proteins are acceptable. Purely theoretical papers are also acceptable provided they provide new insight into the principles of protein/peptide structure and function.
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