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Mechanism of papain-catalyzed synthesis of oligo-tyrosine peptides

Paper ID Volume ID Publish Year Pages File Format Full-Text
16974 42628 2015 8 PDF Available
Title
Mechanism of papain-catalyzed synthesis of oligo-tyrosine peptides
Abstract

•Oligotyrosines (oligo-Tyr) are potential bioactive peptides.•Oligo-Tyr can be synthesized by papain-catalyzed reaction with tyrosine ethyl ester.•The initial substrate concentration has a great influence on the yield of oligo-Tyr.•We kinetically analyzed the reaction and proposed its mechanism for the first time.•Understanding the mechanism would allow effective synthesis of oligo-Tyr.

Di-, tri-, and tetra-tyrosine peptides with angiotensin I-converting enzyme inhibitory activity were synthesized by papain-catalyzed polymerization of l-tyrosine ethyl ester in aqueous media at 30 °C. Varying the reaction pH from 6.0 to 7.5 and the initial concentration of the ester substrate from 25 to 100 mM, the highest yield of oligo-tyrosine peptides (79% on a substrate basis) was produced at pH 6.5 and 75 mM, respectively. In the reaction initiated with 100 mM of the substrate, approx. 50% yield of insoluble, highly polymerized peptides accumulated. At less than 15 mM, the reaction proceeded poorly; however, from 30 mM to 120 mM a dose-dependent increase in the consumption rate of the substrate was observed with a sigmoidal curve. Meanwhile, each of the tri- and tetra-tyrosine peptides, even at approx. 5 mM, was consumed effectively by papain but was not elongated to insoluble polymers. For deacylation of the acyl-papain intermediate through which a new peptide bond is made, l-tyrosine ethyl ester, even at 5 mM, showed higher nucleophilic activity than di- and tri-tyrosine. These results indicate that the mechanism through which papain polymerizes l-tyrosine ethyl ester is as follows: the first interaction between papain and the ester substrate is a rate-limiting step; oligo-tyrosine peptides produced early in the reaction period are preferentially used as acyl donors, while the initial ester substrate strongly contributes as a nucleophile to the elongation of the peptide product; and the balance between hydrolytic fragmentation and further elongation of oligo-tyrosine peptides is dependent on the surrounding concentration of the ester substrate.

Keywords
Peptide synthesis; Oligo-tyrosine; PapainACE, angiotensin I-converting enzyme; BA, Nα-benzoyl-l-arginine; BAEE, Nα-benzoyl-l-arginine ethyl ester; BA-Tyr, Nα-benzoyl-l-arginyl-l-tyrosine; DP, degree of polymerization; RP-HPLC, reverse-phase high-performan
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Mechanism of papain-catalyzed synthesis of oligo-tyrosine peptides
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volumes 75–76, July–August 2015, Pages 10–17
Authors
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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