Synthesis and characterization of ubiquitin ethyl ester, a new substrate for ubiquitin carboxyl-terminal hydrolase.
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)
D004912:Erythrocytes / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D012156:Reticulocytes / Q000378:metabolism; D013379:Substrate Specificity; D013869:Thiolester Hydrolases / Q000378:metabolism*; D014357:Trypsin; D043222:Ubiquitin Thiolesterase; D014452:Ubiquitins / Q000031:analogs & derivatives* / Q000097:blood / Q000302:isolation & purification / Q000378:metabolism
D014452:Ubiquitins; C051078:ubiquitin ethyl ester; D013869:Thiolester Hydrolases; D043222:Ubiquitin Thiolesterase; D014357:Trypsin