Linked Life Data

11682630

Source:http://linkedlifedata.com/resource/pubmed/id/11682630

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
11
pubmed:dateCreated
2001-10-29
pubmed:abstractText
Human estrogenic 17beta-hydroxysteroid dehydrogenase is an NADP(H)-preferring enzyme. It possesses 11- and 4-fold higher specificity toward NADP(H) over NAD(H) for oxidation and reduction, respectively, as demonstrated by kinetic studies. To elucidate the roles of the amino acids involved in cofactor specificity, we generated variants by site-directed mutagenesis. The results showed that introducing a positively charged residue, lysine, at the Ser12 position increased the enzyme's preference for NADP(H) more than 20-fold. Substitution of the negatively charged residue, aspartic acid, into the Leu36 position switched the enzyme's cofactor preference from NADPH to NAD with a 220-fold change in the ratio of the specificity toward the two cofactors in the case of oxidation. This variant dramatically abolished the enzyme's reductase function and stimulated its dehydrogenase activity, as shown by enzyme activity in intact cells. The substrate-binding pocket was also studied with four variants: Ser142Gly, Ser142Cys, His221Ala, and Glu282Ala. The Ser142Gly variant abolished most of the enzyme's oxidation and reduction activities. The residual reductase activity in vitro is less than 2% that of the wild-type enzyme. However, the Ser142Cys variant was fully inactive, both as a partially purified protein and in intact cells. This suggests that the bulky sulfhydryl group of cysteine entirely disrupted the catalytic triad and that the Ser142 side chain is important for maintaining the integrity of this triad. His221 variation weakened the apparent affinity for estrone, as demonstrated by a 30-fold increase in Michaelis-Menten constant, supporting its important role in substrate binding. This residue may play an important role in substrate inhibition via the formation of a dead-end complex. The formerly suggested importance of Glu282 could not be confirmed.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Nov
pubmed:issn
0888-8809
pubmed:author
pubmed:issnType
Print
pubmed:volume
15
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
2010-20
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed:year
2001
pubmed:articleTitle
Critical residues for the specificity of cofactors and substrates in human estrogenic 17beta-hydroxysteroid dehydrogenase 1: variants designed from the three-dimensional structure of the enzyme.
pubmed:affiliation
Medical Research Council Group in Molecular Endocrinology, Oncology, and Molecular Endocrinology Research Center, Laval University Medical Center, Québec, Québec G1V 4G2, Canada.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't