By applying a newly developed method, cDNAs for the human muscle isoform of fructose-1,6-bisphosphatase were isolated from phage- and plasmid-derived libraries. From these cDNAs and an EST clone, a composite sequence (1302 bp) was deduced that contains an open reading frame encoding a polypeptide of 339 amino acids with an estimated molecular weight of 36 755. After overexpression in E. coli, recombinant human muscle fructose 2,6-bisphosphatase was found to be active in cel-free extracts and could be strongly inhibited by AMP and fructose 2,6-bisphosphate. Sequence comparisons revealed that (1) all amino acids thought to be in contact with substrate molecules, regulatory molecules or metal ions in mammalian liver fructose-1,6-bisphosphatases are, with one exception, conserved in the human muscle enzyme and (2) the human muscle isoform is more homologous to the mouse intestine fructose-1,6-bisphosphatase than to the mammalian liver isoform. This is the first report of the cloning and expression of a muscle fructose-1,6-bisphosphatase isoenzyme.
The chemical reactions and pathways involving fructose, the ketohexose arabino-2-hexulose. Fructose exists in a open chain form or as a ring compound. D-fructose is the sweetest of the sugars and is found free in a large number of fruits and honey.
By applying a newly developed method, cDNAs for the human muscle isoform of fructose-1,6-bisphosphatase were isolated from phage- and plasmid-derived libraries. From these cDNAs and an EST clone, a composite sequence (1302 bp) was deduced that contains an open reading frame encoding a polypeptide of 339 amino acids with an estimated molecular weight of 36 755. After overexpression in E. coli, recombinant human muscle fructose 2,6-bisphosphatase was found to be active in cel-free extracts and could be strongly inhibited by AMP and fructose 2,6-bisphosphate. Sequence comparisons revealed that (1) all amino acids thought to be in contact with substrate molecules, regulatory molecules or metal ions in mammalian liver fructose-1,6-bisphosphatases are, with one exception, conserved in the human muscle enzyme and (2) the human muscle isoform is more homologous to the mouse intestine fructose-1,6-bisphosphatase than to the mammalian liver isoform. This is the first report of the cloning and expression of a muscle fructose-1,6-bisphosphatase isoenzyme.
Adenosine 5'-monophosphate (AMP) inhibits muscle fructose 1,6-bisphosphatase (FBPase) about 44 times stronger than the liver isozyme. The key role in strong AMP binding to muscle isozyme play K20, T177 and Q179. Muscle FBPase which has been mutated towards the liver enzyme (K20E/T177M/Q179C) is inhibited by AMP about 26 times weaker than the wild-type muscle enzyme, but it binds the fluorescent AMP analogue, 2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-monophosphate (TNP-AMP), similarly to the wild-type liver enzyme. The reverse mutation of liver FBPase towards the muscle isozyme significantly increases the affinity of the mutant to TNP-AMP. High affinity to the inhibitor but low sensitivity to AMP of the liver triple mutant suggest differences between the isozymes in the mechanism of allosteric signal transmission.
Subject to complex allosteric regulation. The enzyme can assume an active R-state, or an inactive T-state. Intermediate conformations may exist. AMP acts as allosteric inhibitor. Fructose 2,6-bisphosphate acts as competitive inhibitor.
Adenosine 5'-monophosphate (AMP) inhibits muscle fructose 1,6-bisphosphatase (FBPase) about 44 times stronger than the liver isozyme. The key role in strong AMP binding to muscle isozyme play K20, T177 and Q179. Muscle FBPase which has been mutated towards the liver enzyme (K20E/T177M/Q179C) is inhibited by AMP about 26 times weaker than the wild-type muscle enzyme, but it binds the fluorescent AMP analogue, 2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-monophosphate (TNP-AMP), similarly to the wild-type liver enzyme. The reverse mutation of liver FBPase towards the muscle isozyme significantly increases the affinity of the mutant to TNP-AMP. High affinity to the inhibitor but low sensitivity to AMP of the liver triple mutant suggest differences between the isozymes in the mechanism of allosteric signal transmission.
Protein participating in biochemical reactions in which carbohydrates are involved. Carbohydrate is a general term for sugars and related compounds with the general formula Cn(H2O)n. The smallest are monosaccharides (e.g. glucose); polysaccharides (e.g. starch, cellulose, glycogen) can be large and vary in length.
Protein involved in the biosynthesis of "new" glucose from such noncarbohydrate precursors as pyruvate, lactate, certain amino acids and intermediates of the tricarboxylic acid cycle.
Enzyme which catalyzes hydrolysis reaction, i.e. the addition of the hydrogen and hydroxyl ions of water to a molecule with its consequent splitting into two or more simpler molecules.
Enzyme whose activity is modified by the noncovalent binding of an allosteric effector at a site other than the active site. This binding mediates conformational changes, altering its catalytic or binding properties.
A reference proteome is a set of protein sequences derived from a complete proteome which constitutes a defined standard for a particular user community. Reference proteomes are manually defined according to a number of criteria. They cover the proteomes of well- studied model organisms and other proteomes of interest for biomedical and biotechnological research. Reference proteomes have been selected to provide broad coverage of the tree of life, and constitute a representative cross-section of the taxonomic diversity to be found within UniProtKB.
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