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Site-directed mutagenesis of porcine pepsin: Possible role of Asp32, Thr33, Asp215 and Gly217 in maintaining the nuclease activity of pepsin

Paper ID Volume ID Publish Year Pages File Format Full-Text
16786 42612 2016 7 PDF Available
Title
Site-directed mutagenesis of porcine pepsin: Possible role of Asp32, Thr33, Asp215 and Gly217 in maintaining the nuclease activity of pepsin
Abstract

•The active sites of pepsin towards NA were the same as those for protein digestion.•Asp32, Thr33, Asp215 and Gly217 were the pepsin active sites for NA digestion.•Residues Tyr189 and Val214 were essential for pepsin binding and catalysis of dsDNA digestion.•The Gly34 residue was essential for pepsin binding and catalysis of ssDNA digestion.

Site-directed mutagenesis of porcine pepsin was performed to identify its active sites that regulate nucleic acid (NA) digestion activity and to analyze the mechanism pepsin-mediated NA digestion. The mutation sites were distributed at the catalytic center of the enzyme (T33A, G34A, Y75H, T77A, Y189H, V214A, G217A and S219A) and at its active site (D32A and D215A) for protein digestion. Mutation of the active site residues Asp32 and Asp215 led to the inactivation of pepsin (both the NA and protein digestion activity), which demonstrated that the active sites of the pepsin protease activity were also important for its nuclease activity. Analysis of the variants revealed that T33A and G217A mutants showed a complete loss of NA digestion activity. In conclusion, residues Asp32, Thr33, Asp215 and Gly217 were related to the pepsin active sites for NA digestion. Moreover, the Y189H and V214A variants showed a loss of digestion activity on double-strand DNA (dsDNA) but only a decrease in digestion activity on single-strand DNA (ssDNA). On the contrary, the G34A variant showed a loss of digestion activity on ssDNA but only a decrease in digestion activity on dsDNA. Our findings are the first to identify the active sites of pepsin nuclease activity and lay the framework for further study of the mechanism of pepsin nuclease activity.

Keywords
NA, nucleic acid; dsDNA, double-strand DNA; ssDNA, single-strand DNA; Hb, hemoglobinPepsin; Nucleic acids; Active sites; Site-directed mutagenesis
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Site-directed mutagenesis of porcine pepsin: Possible role of Asp32, Thr33, Asp215 and Gly217 in maintaining the nuclease activity of pepsin
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 89, July 2016, Pages 69–75
Authors
, , , , , ,
Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us