Linked Life Data

2888482

Source:http://linkedlifedata.com/resource/pubmed/id/2888482

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
13
pubmed:dateCreated
1987-11-16
pubmed:abstractText
We have shown by kinetic and magnetic resonance measurements that a spin-labeled substrate analogue, spiro[doxyl-2,3'-5' alpha-androstan]-17'beta-ol, binds at the substrate site of crystalline delta 5-3-ketosteroid isomerase (steroid delta-isomerase; EC 5.3.3.1) of Pseudomonas testosteroni. The spin-labeled steroid is a linear competitive inhibitor with a Ki value (25 +/- 5 microM) that is consistent with dissociation constants obtained by direct binding measurements based on changes in the electron paramagnetic resonance spectrum of the nitroxide, longitudinal relaxation rates of water protons, and longitudinal and transverse relaxation rates of carbon-bound protons of the isomerase. These binding studies yield a stoichiometry for the nitroxide of 1 per subunit of the enzyme. Measurements of the longitudinal relaxation rates of water protons indicate that the 3-doxyl portion of the spin-label is highly immobilized yet is exposed to solvent. Paramagnetic effects of the nitroxide on T1 defined distances to several previously assigned [Benisek, W. F., & Ogez, J. R. (1982) Biochemistry 21, 5816-5825] and newly assigned protons of the enzyme. These distances were then used to locate (with an accuracy of +/- 2 A) the nitroxide moiety at a unique position in a partially refined 2.5-A resolution X-ray structure of native isomerase. Three of five additional proton resonance peaks, attributed to ring-shielded methyl groups, could be assigned to specific residues on the basis of distances from the spin-label in the X-ray structure. The remaining portion of the spin-labeled steroid was then docked into the X-ray structure in a hydrophobic cavity of the enzyme. This position of the steroid is consistent with the steroid binding site previously proposed [Westbrook, E. M., Piro, O. E., & Sigler, P. B. (1984) J. Biol. Chem. 259, 9096-9103]. However, the rotational orientation of this steroid about its long axis could not be unambiguously established. If we assume that steroid substrates and the spin-labeled inhibitor bind to the same site, but with reversal of the 3- and 17-positions, then the phenolic hydroxyl of Tyr-55 is optimally positioned to function as the general acid that protonates the 3-keto group of the substrate, facilitated by the negative end of the dipole of a 10-residue alpha-helix, the only helix in the molecule.(ABSTRACT TRUNCATED AT 400 WORDS)
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jun
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
30
pubmed:volume
26
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
3927-37
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
1987
pubmed:articleTitle
Positioning of a spin-labeled substrate analogue into the structure of delta 5-3-ketosteroid isomerase by combined kinetic, magnetic resonance, and X-ray diffraction methods.
pubmed:affiliation
Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S., Research Support, U.S. Gov't, Non-P.H.S., Research Support, Non-U.S. Gov't