Linked Life Data

10406485

Source:http://linkedlifedata.com/resource/pubmed/id/10406485

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
5
pubmed:dateCreated
1999-9-16
pubmed:abstractText
Previous studies have shown that the reduced nicotinamide adenine dinucleotide phosphate (NADPH)- binding domain of rat liver microsomal steroid 5alpha-reductase isozyme-1 (r5alphaR-1) is in a highly conserved region of the polypeptide sequence (residues 160-190). In this study, we investigated, by site-directed mutagenesis, the role of hydroxylated and aromatic amino acids within the NADPH-binding domain. The r5alphaR-1 cDNA was cloned into a pCMV vector, and the double strand site-directed mutagenesis method was used to create mutants Y179F, Y179S, Y189F, Y189S, S164A, S164T, and Y187F, which were subsequently expressed in COS-1 cells. Kinetic studies of the expressed enzymes showed that the mutation Y179F resulted in an approximately 40-fold increase in the Km for NADPH versus wild-type, with only a 2-fold increase in the Km for testosterone. The mutants Y189F and S164A showed smaller increases (4 and 6-fold) in Kms for NADPH and no significant change in the Km for testosterone, whereas Y189S had kinetic properties similar to the wild-type r5alphaR-1. Mutants Y179S and S164T both resulted in inactive enzymes, whereas F187Y showed an approximately 5-fold decrease in Km for NADPH and a significant increase (approximately 18-fold) in the Km for testosterone. The results suggest that the -OH functionality of Y179 is involved in cofactor binding, but is not essential for the activity of the enzyme, whereas the -OH functionalities of Y189 and S164 play lesser roles in cofactor binding to r5alphaR-1 and may not be required for enzyme activity. On the other hand, the residue F187 may be important for the binding of both NADPH and testosterone.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
May
pubmed:issn
0039-128X
pubmed:author
pubmed:issnType
Print
pubmed:volume
64
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
356-62
pubmed:dateRevised
2010-11-18
pubmed:meshHeading
pubmed:year
1999
pubmed:articleTitle
Site-directed mutagenesis studies of the NADPH-binding domain of rat steroid 5alpha-reductase (isozyme-1) I: analysis of aromatic and hydroxylated amino acid residues.
pubmed:affiliation
VAMC and Department of OB/GYN, University of Kentucky, Lexington, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S., Research Support, U.S. Gov't, Non-P.H.S.