Switch to
| Predicate | Object |
|---|---|
| rdf:type | |
| lifeskim:mentions | |
| pubmed:issue |
23
|
| pubmed:dateCreated |
1992-9-10
|
| pubmed:abstractText |
Lysine 356 has been implicated by protein modification studies as a fructose-2,6-bisphosphate binding site residue in the 6-phosphofructo-2-kinase domain of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (Kitajima, S., Thomas, H., and Uyeda, K. (1985) J. Biol. Chem. 260, 13995-14002). However, Lys-356 is found in the fructose-2,6-bisphosphatase domain (Bazan, F., Fletterick, R., and Pilkis, S. J. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9642-9646). In order to ascertain whether Lys-356 is involved in fructose-2,6-bisphosphatase catalysis and/or domain/domain interactions of the bifunctional enzyme, Lys-356 was mutated to Ala, expressed in Escherichia coli, and then purified to homogeneity. Circular dichroism experiments indicated that the secondary structure of the Lys-356-Ala mutant was not significantly different from that of the wild-type enzyme. The Km for fructose 2,6-bisphosphate and the Ki for the noncompetitive inhibitor, fructose 6-phosphate, for the fructose-2,6-bisphosphatase of the Lys-356-Ala mutant were 2700- and 2200-fold higher, respectively, than those of the wild-type enzyme. However, the maximal velocity and the Ki for the competitive product inhibitor, inorganic phosphate, were unchanged compared to the corresponding values of the wild-type enzyme. Furthermore, in contrast to the wild-type enzyme, which exhibits substrate inhibition, there was no inhibition by substrate of the Lys-356-Ala mutant. In the presence of saturating substrate, inorganic phosphate, which acts by relieving fructose-6-phosphate and substrate inhibition, is an activator of the bisphosphatase. The Ka for inorganic phosphate of the Lys-356-Ala mutant was 1300-fold higher than that of the wild-type enzyme. The kinetic properties of the 6-phosphofructo-2-kinase of the Lys-356-Ala mutant were essentially identical with that of the wild-type enzyme. The results demonstrate that: 1) Lys-356 is a critical residue in fructose-2,6-bisphosphatase for binding the 6-phospho group of fructose 6-phosphate/fructose 2,6-bisphosphate; 2) the fructose 6-phosphate binding site is responsible for substrate inhibition; 3) Inorganic phosphate activates fructose-2,6-bisphosphatase by competing with fructose 6-phosphate for the same site; and 4) Lys-356 is not involved in 6-phosphofructo-2-kinase substrate/product binding or catalysis.
|
| pubmed:grant | |
| pubmed:language |
eng
|
| pubmed:journal | |
| pubmed:citationSubset |
IM
|
| pubmed:chemical |
http://linkedlifedata.com/resource/pubmed/chemical/Fructosediphosphates,
http://linkedlifedata.com/resource/pubmed/chemical/Lysine,
http://linkedlifedata.com/resource/pubmed/chemical/Phosphofructokinase-2,
http://linkedlifedata.com/resource/pubmed/chemical/Phosphoric Monoester Hydrolases,
http://linkedlifedata.com/resource/pubmed/chemical/Phosphotransferases,
http://linkedlifedata.com/resource/pubmed/chemical/Recombinant Proteins,
http://linkedlifedata.com/resource/pubmed/chemical/fructose 2,6-diphosphate
|
| pubmed:status |
MEDLINE
|
| pubmed:month |
Aug
|
| pubmed:issn |
0021-9258
|
| pubmed:author | |
| pubmed:issnType |
Print
|
| pubmed:day |
15
|
| pubmed:volume |
267
|
| pubmed:owner |
NLM
|
| pubmed:authorsComplete |
Y
|
| pubmed:pagination |
16669-75
|
| pubmed:dateRevised |
2007-11-14
|
| pubmed:meshHeading |
pubmed-meshheading:1322913-Amino Acid Sequence,
pubmed-meshheading:1322913-Animals,
pubmed-meshheading:1322913-Binding Sites,
pubmed-meshheading:1322913-Circular Dichroism,
pubmed-meshheading:1322913-Cloning, Molecular,
pubmed-meshheading:1322913-Escherichia coli,
pubmed-meshheading:1322913-Fructosediphosphates,
pubmed-meshheading:1322913-Kinetics,
pubmed-meshheading:1322913-Liver,
pubmed-meshheading:1322913-Lysine,
pubmed-meshheading:1322913-Models, Molecular,
pubmed-meshheading:1322913-Mutagenesis, Site-Directed,
pubmed-meshheading:1322913-Phosphofructokinase-2,
pubmed-meshheading:1322913-Phosphoric Monoester Hydrolases,
pubmed-meshheading:1322913-Phosphotransferases,
pubmed-meshheading:1322913-Protein Conformation,
pubmed-meshheading:1322913-Rats,
pubmed-meshheading:1322913-Recombinant Proteins
|
| pubmed:year |
1992
|
| pubmed:articleTitle |
Lysine 356 is a critical residue for binding the C-6 phospho group of fructose 2,6-bisphosphate to the fructose-2,6-bisphosphatase domain of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.
|
| pubmed:affiliation |
Department of Physiology & Biophysics, State University of New York, Stony Brook 11794.
|
| pubmed:publicationType |
Journal Article,
Research Support, U.S. Gov't, P.H.S.,
Research Support, U.S. Gov't, Non-P.H.S.
|