Linked Life Data

7588765

Source:http://linkedlifedata.com/resource/pubmed/id/7588765

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
1
pubmed:dateCreated
1995-12-14
pubmed:databankReference
pubmed:abstractText
Properties of purified recombinant adenosine 3'-phosphate 5'-phosphosulfate (PAdoPS) reductase from Escherichia coli were investigated. The Michaelis constants for reduced thioredoxin and PAdoPS are 23 microM and 10 microM, respectively; the enzyme has a Vmax of 94-99 mumol min-1 mg-1 and a molecular activity/catalytically active dimer of 95 s-1. Adenosine 3',5'-bisphosphate (PAdoP) inhibits competitively (Ki 4 microM) with respect to PAdoPS; adenosine 2',5'-bisphosphate and sulfite are not inhibitory. Alkylation by SH-group inhibitors irreversibly inactivates the enzyme. The structural gene (cysH) encodes for a small polypeptide with a single Cys residue located in a conserved cluster (KXECGI/LH) of amino acids. Involvement of the only Cys and of Tyr209 in the reduction of PAdoPS to sulfite was investigated by site-specific mutagenesis: cysH was mutated by single-strand-overlay extension PCR; the mutated genes were cloned in pBTac1 and expressed in E. coli RL 22 (delta cysHIJ). Homogenous Cys239Ser and Tyr209Phe mutant PAdoPS reductases were investigated for altered catalytic properties. Mutation of the single Cys reduced Vmax by a factor of 4.5 x 10(3) (Vmax = 0.02-0.013 mumol min-1 mg-1) with marginal effects on Km for PAdoPS (19 microM) and reduced thioredoxin (14 microM). Mutation of Tyr209 drastically affected saturation with thioredoxin (Km 1.5 microM) and decreased Vmax (0.22-0.25 mumol min-1 mg-1) in addition to a small increase in Km for PAdoPS (31 microM). Chromophores as prosthetic groups were absent from recombinant PAdoPS reductase. Difference absorption spectra between reduced and oxidized forms of wild-type and mutated proteins indicated that, in addition to Cys239 and Tyr209, an unidentified Trp (delta lambda max 292 nm) appears to be involved in the reduction. The data suggest a special ping-pong mechanism with PAdoPS reacting with the reduced enzyme isomer in a Theorell-Chance type mechanism.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Oct
pubmed:issn
0014-2956
pubmed:author
pubmed:issnType
Print
pubmed:day
1
pubmed:volume
233
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
347-56
pubmed:dateRevised
2007-11-15
pubmed:meshHeading
pubmed:year
1995
pubmed:articleTitle
Reaction mechanism of thioredoxin: 3'-phospho-adenylylsulfate reductase investigated by site-directed mutagenesis.
pubmed:affiliation
Biochemistry of Plants, Ruhr University Bochum, Germany.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't