Synthesis and characterization of ubiquitin ethyl ester, a new substrate for ubiquitin carboxyl-terminal hydrolase.

Full Abstract

A new substrate for ubiquitin carboxyl-terminal hydrolase, the carboxyl-terminal ethyl ester of ubiquitin, has been synthesized by a trypsin-catalyzed transpeptidation. In the presence of 1.6 M glycylglycine ethyl ester, trypsin removes the carboxyl-terminal glycylglycine of ubiquitin and replaces it with the dipeptide ester. The equilibrium mixture under these conditions contains 30% ubiquitin ethyl ester and 70% hydrolysis product, the 74-residue fragment of ubiquitin. Ubiquitin ethyl ester can be purified by gel filtration and ion-exchange chromatography. The structure of this product has been verified by identification of the products of base hydrolysis, tryptic cleavage in aqueous solution, and peptide mapping. When ubiquitin ethyl ester is incubated with purified ubiquitin carboxyl-terminal hydrolase, specific cleavage of the ester linkage is observed. A rapid, sensitive assay is described utilizing high-performance liquid chromatography. By use of this assay, it has been shown that ubiquitin carboxyl-terminal hydrolase is inactivated in the absence of thiols. Optimal protective effects are seen with 10 mM dithiothreitol. The rate of catalysis is maximal at pH 8.5, with evidence for catalytically important groups with pK values of 5.2, 7.6, and 9.5. These findings are consistent with the participation of a thiol group in the active site. Native ubiquitin is a competitive inhibitor of ubiquitin ethyl ester hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Author information

Author/s: Wilkinson, K D (KD); Cox, M J (MJ); Mayer, A N (AN); Frey, T (T);

Grants: GM30308 (Agency:NIGMS NIH HHS)

Journal and publication information

Publication Type: Journal Article; Research Support, U.S. Gov't, P.H.S.

Journal: Biochemistry (Biochemistry), published in UNITED STATES. (Language: eng)

Reference: 1986-Oct; vol 25 (issue 21) : pp 6644-9

Dates: Created 1987/02/11; Completed 1987/02/11; Revised 2007/11/14;

PMID: 3024715, status: MEDLINE (last retrieval date: 2/18/2009, IMS Date: )

Sourced from the National Library of Medicine. Abstract text and other information may be subject to copyright.

External Links for this article
(including full text providers, if available):

Click Electronic Full-text Provider Links to see options for finding the electronic full text links to this article. Note there may be a subscription or fee required for access to the full text. See our FAQ for information on finding FREE full text articles.

This article may also be located in paper journal collections available in many libraries. Use the Journal and Publication Information above to find the full article.

MeSH headings (categories)

This article was linked to the MESH Headings shown below.

Substrate Specificity
Thiolester Hydrolases - metabolism
Trypsin
Ubiquitin Thiolesterase
Ubiquitins - analogs & derivatives; blood; isolation & purification; metabolism

Associated Chemicals: Ubiquitins (0) ; ubiquitin ethyl ester (0) ; Thiolester Hydrolases (EC 3.1.2.-) ; Ubiquitin Thiolesterase (EC 3.1.2.15) ; Trypsin (EC 3.4.21.4)

These are the highest related articles currently in the database: