Linked Life Data

2036366

Source:http://linkedlifedata.com/resource/pubmed/id/2036366

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
20
pubmed:dateCreated
1991-7-2
pubmed:abstractText
The delta 5-3-ketosteroid isomerase (EC 5.3.3.1) of Pseudomonas testosteroni catalyzes the conversion of androst-5-ene-3,17-dione to androst-4-ene-3,17-dione by a stereospecific transfer of the 4 beta-proton to the 6 beta-position. The reaction involves two steps: (a) a rate-limiting concerted enolization, comprising protonation of the 3-carbonyl oxygen by the phenolic hydroxyl group of Tyr-14 and abstraction of the 4 beta-proton by the carboxylate group of Asp-38, and (b) rapid reketonization of the dienol, which may or may not be concerted. The active-site mutant D38N, which lacks the base responsible for proton transfer, is about 10(6.0)-fold less active catalytically than the wild-type enzyme. With the D38N mutant it was demonstrated spectroscopically that the enzymatic reaction involves the conversion of the substrate to both the dienol and its anion as tightly enzyme-bound intermediates, which are then converted much more slowly to the alpha,beta-unsaturated product. In contrast to the mechanism of the wild-type enzyme, the enolization reaction promoted by the D38N mutant is not stereospecific with respect to removal of the 4 beta-proton and shows primary kinetic isotope effects on enolization when either 4 alpha or 4 beta or both of these protons are replaced by deuterium. Kinetic isotope effects obtained with deuterated substrates, solvent, or combinations of the two indicate that, unlike in the wild-type enzyme, protonation of the carbonyl oxygen and removal of the C-4 proton are not concerted in the D38N mutant.(ABSTRACT TRUNCATED AT 250 WORDS)
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
May
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
21
pubmed:volume
30
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
4991-7
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
1991
pubmed:articleTitle
Catalytic mechanism of an active-site mutant (D38N) of delta 5-3-ketosteroid isomerase. Direct spectroscopic evidence for dienol intermediates.
pubmed:affiliation
Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S.