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| Predicate | Object |
|---|---|
| rdf:type | |
| lifeskim:mentions | |
| pubmed:issue |
35
|
| pubmed:dateCreated |
1992-10-16
|
| pubmed:abstractText |
A molecular graphics analysis of the features which prevent cytosolic malate dehydrogenase dimers from forming tetramers was evaluated by its success in predicting the synthesis of a version of the LDH framework which is a stable dimer. Surface residues responsible for malate dehydrogenases being dimers were revealed by superimposing the structures of two dimers of pig cytosolic malate dehydrogenase on one homologous tetramer of L-lactate dehydrogenase from Bacillus stearothermophilus. Four regions were identified as composing the P-axis dimer-dimer interface. Two regions of the dimer were surface loops that collided when built as a tetramer: a large loop (residues 203-207, KNOBI) and a small loop (residues 264-269, KNOBII), and these were candidates to explain the dimeric character of malate dehydrogenase. The analysis was tested by constructing a synthetic B. stearothermophilus lactate dehydrogenase (KNOBI) containing the large malate dehydrogenase loop (residues 203-207 being AYIKLQAKE, and extra four amino acids). The new construct was thermotolerant (90 degrees C) and enzymically active with kcat and KM (pyruvate) values similar to those of the wild-type enzyme. However, whereas the allosteric activator fructose 1,6-bisphosphate decreased KM 100 times for wild type, it had no influence on KNOBI. The molecular volumes of 1-120 microM concentrations of the construct were measured by time-resolved decay of tryptophan fluorescence anisotropy and by gel filtration. Both methods showed the molecular weight of wild type increased from dimer to tetramer with Kd about 20 microM dimer. KNOBI remained a dimer under these conditions.(ABSTRACT TRUNCATED AT 250 WORDS)
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| pubmed:language |
eng
|
| pubmed:journal | |
| pubmed:citationSubset |
IM
|
| pubmed:chemical |
http://linkedlifedata.com/resource/pubmed/chemical/L-Lactate Dehydrogenase,
http://linkedlifedata.com/resource/pubmed/chemical/Macromolecular Substances,
http://linkedlifedata.com/resource/pubmed/chemical/Oligodeoxyribonucleotides,
http://linkedlifedata.com/resource/pubmed/chemical/Recombinant Proteins
|
| pubmed:status |
MEDLINE
|
| pubmed:month |
Sep
|
| pubmed:issn |
0006-2960
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| pubmed:author | |
| pubmed:issnType |
Print
|
| pubmed:day |
8
|
| pubmed:volume |
31
|
| pubmed:owner |
NLM
|
| pubmed:authorsComplete |
N
|
| pubmed:pagination |
8307-14
|
| pubmed:dateRevised |
2009-11-19
|
| pubmed:meshHeading |
pubmed-meshheading:1525168-Amino Acid Sequence,
pubmed-meshheading:1525168-Animals,
pubmed-meshheading:1525168-Base Sequence,
pubmed-meshheading:1525168-Chromatography, Gel,
pubmed-meshheading:1525168-Enzyme Stability,
pubmed-meshheading:1525168-Escherichia coli,
pubmed-meshheading:1525168-Geobacillus stearothermophilus,
pubmed-meshheading:1525168-L-Lactate Dehydrogenase,
pubmed-meshheading:1525168-Macromolecular Substances,
pubmed-meshheading:1525168-Models, Molecular,
pubmed-meshheading:1525168-Molecular Sequence Data,
pubmed-meshheading:1525168-Mutagenesis, Site-Directed,
pubmed-meshheading:1525168-Oligodeoxyribonucleotides,
pubmed-meshheading:1525168-Protein Conformation,
pubmed-meshheading:1525168-Recombinant Proteins,
pubmed-meshheading:1525168-Sequence Homology, Nucleic Acid,
pubmed-meshheading:1525168-Spectrometry, Fluorescence,
pubmed-meshheading:1525168-Swine
|
| pubmed:year |
1992
|
| pubmed:articleTitle |
Construction of a stable dimer of Bacillus stearothermophilus lactate dehydrogenase.
|
| pubmed:affiliation |
Molecular Recognition Centre, University of Bristol School of Medical Sciences, U.K.
|
| pubmed:publicationType |
Journal Article,
Comparative Study,
Research Support, Non-U.S. Gov't
|