title: Mechanism of papain-catalyzed synthesis of oligo-tyrosine peptides.
abstract: 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. 5mM, 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 5mM, 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.
authors.affiliation: Graduate School of Veterinary Medicine and Life Science, Nippon Veterinary and Life Science University, 1-7-1 Kyonan-cho, Musashino-shi, Tokyo 180-8602, Japan.
authors.forename: Jun
authors.initials: J
authors.lastname: Mitsuhashi
authors.affiliation: Graduate School of Veterinary Medicine and Life Science, Nippon Veterinary and Life Science University, 1-7-1 Kyonan-cho, Musashino-shi, Tokyo 180-8602, Japan.
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Nakayama
authors.affiliation: Graduate School of Veterinary Medicine and Life Science, Nippon Veterinary and Life Science University, 1-7-1 Kyonan-cho, Musashino-shi, Tokyo 180-8602, Japan. Electronic address: a-narai@nvlu.ac.jp.
authors.forename: Asako
authors.initials: A
authors.lastname: Narai-Kanayama
mesh_terms: D000215:Acylation; D000806:Angiotensin-Converting Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism; D055162:Biocatalysis; D004151:Dipeptides / Q000096:biosynthesis; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009842:Oligopeptides / Q000096:biosynthesis* / Q000737:chemistry; D010206:Papain / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000806:Angiotensin-Converting Enzyme Inhibitors; D004151:Dipeptides; D009842:Oligopeptides; C042696:tyrosyltyrosine; D014443:Tyrosine; C051772:ethyl tyrosine ester; D010206:Papain
keywords: Oligo-tyrosine; Papain; Peptide synthesis