Linked Life Data

3015923

Source:http://linkedlifedata.com/resource/pubmed/id/3015923

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
22
pubmed:dateCreated
1986-9-17
pubmed:abstractText
Ubiquitin (Ub) carboxyl-terminal hydrolase (E) catalyzes the hydrolysis, at the Ub-carboxyl terminus, of a wide variety of C-terminal Ub derivatives. We show that the enzyme is inactivated by millimolar concentrations of either sodium borohydride or hydroxylamine, but only if Ub is present. We have interpreted these results on the assumption that the hydrolase mechanism is one of nucleophilic catalysis with an acyl-Ub-E intermediate. The borohydride-inactivated enzyme has the following properties. It is a stoichiometric complex of E and Ub containing tritium from sodium boro[3H]hydride. This complex is stable at neutral pH in 5 M urea and can be isolated on the basis of size on a sieving column, but a labeled product the size of Ub is released under more strongly denaturing conditions. The "Ub" released in acid is Ub-carboxyl-terminal aldehyde, based on the observations that: it contains the tritium present in the reduced complex and it is able to form the inactive enzyme from a stoichiometric amount of fresh enzyme, and inactivation is accompanied by E-Ub adduct formation; it has chemical properties expected of an aldehyde: after a second reduction of the Ub released with boro[3H]hydride and complete acid hydrolysis, tritium counts are found in ethanolamine (the carboxyl-terminal residue of Ub is glycine). These results suggest that enzyme and Ub combine in an equilibrium reaction to form an ester or thiol ester adduct (at the Ub-carboxyl terminus), and that this adduct is trapped by borohydride to give a very stable inactive E-Ub (thio) hemiacetal which is unable to undergo a second reduction step and which can release Ub-aldehyde in mild acid. Inactivation in the presence of hydroxylamine of hydrolase occurs once during hydrolysis of 1200 molecules of Ub-hydroxamate by the enzyme. The hydrolysis/inactivation ratio is constant over the range of 10-50 mM hydroxylamine showing that forms of E-Ub with which hydroxylamine and water react are different and not in rapid equilibrium. The inactive enzyme may be an acylhydroxamate formed from an E-Ub mixed anhydride generated from the E-Ub (thiol) ester inferred from the borohydride study. A direct radioactive assay for the hydrolase has been developed using the Ub-C-terminal amide of [3H]butanol-4-amine as substrate.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Aug
pubmed:issn
0021-9258
pubmed:author
pubmed:issnType
Print
pubmed:day
5
pubmed:volume
261
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
10210-7
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
1986
pubmed:articleTitle
Mechanism of ubiquitin carboxyl-terminal hydrolase. Borohydride and hydroxylamine inactivate in the presence of ubiquitin.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S., Research Support, Non-U.S. Gov't