Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The complete amino acid sequence of 6-phospho-fructo-2-kinase/fructose-2,6-bisphosphatase from rat liver was determined by direct analysis of the S-carboxamidomethyl protein. A complete set of nonoverlapping peptides was produced by cleavage with a combination of cyanogen bromide and specific proteolytic enzymes. The active enzyme is a dimer of two identical polypeptide chains composed of 470 amino acids each. The NH2-terminal amino acid residue of the polypeptide chain was shown to be N-acetylserine by fast atom bombardment mass spectrometry of the purified N-terminal tetradecapeptide isolated after cleavage of the intact S-carboxamidomethylated protein with lysyl endoproteinase (Achromobacter protease I). Alignment of the set of unique peptides was accomplished by the analysis of selected overlapping peptides generated by proteolytic cleavage of the intact protein and the larger purified cyanogen bromide peptides with trypsin, Staphylococcus aureus V8 protease, and lysyl endoproteinase. Four nonoverlapping peptides were aligned by comparison with the amino acid sequence predicted from a partial cDNA clone encoding amino acid positions 166-470 of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (Colosia, A.D., Lively, M., El-Maghrabi, M. R., and Pilkis, S. J. (1987) Biochem. Biophys. Res. Commun. 143, 1092-1098). The nucleotide sequence of the cDNA corroborated the peptide sequence determined by direct methods. A search of the Protein Identification Resource protein sequence database revealed that the overall amino acid sequence appears to be unique since no obviously homologous sequences were identified. However, a 100-residue segment of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (residues 250-349), including the active site histidine residue of the bisphosphatase domain, was found to be homologous to the active site regions of yeast phosphoglycerate mutase and human bisphosphoglycerate mutase.
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PMID:Complete amino acid sequence of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. 282 64

Some physicochemical properties of a homogeneous preparation of a bifunctional enzyme, fructose-6-phosphate 2-kinase:fructose-2,6-bisphosphatase, were determined. The molecular weight of the enzyme is 101 000 as determined by high-speed sedimentation equilibrium. The molecular weight of dissociated enzyme is 55 000 in 6 M guanidinium chloride by sedimentation equilibrium and in sodium dodecyl sulfate by polyacrylamide gel electrophoresis. A value of 4.7 was observed for the isoelectric point. Tryptic peptide maps and high-performance liquid chromatography of the trypsin-digested enzyme revealed approximately 60 peptides. Amino acid analysis of the enzyme shows that it contains 27 lysine and 36 arginine residues per 55 000 daltons. No free N-terminal amino acid residue was detectable, suggesting that it is blocked. Hydrolysis of the enzyme by carboxypeptidases A and B releases tyrosine followed by histidine and arginine, indicating that the amino acid sequence at the carboxyl terminus is probably -Arg-His-Tyr. Tryptic digestion of [32P]phosphofructose-6-phosphate 2-kinase:fructose-2,6-bisphosphatase yields a 32P-labeled peptide detected by tryptic peptide mapping and high-performance liquid chromatography. Thermolysin digestion of CNBr-cleaved 32P-enzyme also yields a single 32P-peptide. These results indicate that fructose-6-phosphate 2-kinase:fructose-2,6-bisphosphatase is a dimer of 55 000 daltons and the subunits are very similar, if not identical.
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PMID:Studies of the structure of fructose-6-phosphate 2-kinase:fructose-2,6-bisphosphatase. 300 Apr 38

The reaction mechanism of rat hepatic fructose-2,6-bisphosphatase involves the formation of a phosphohistidine intermediate. In order to determine the sequence around the active site histidine, the enzyme was incubated with [2-32P]fructose 2,6-bisphosphate, denatured, and treated with trypsin or endoproteinase Lys-C. The resultant labeled 32P-phosphopeptides were purified by gel filtration, anion exchange chromatography, and reverse phase high pressure liquid chromatography. The sequence of the tryptic peptide was determined to be HGESELNLR, while the partial sequence of the endoproteinase Lys-C peptide was IFDVGTRYMVNRVQDHVQSRTAYYLMNIHVTPRSIYLRHGESEL. The active site sequence was compared with the active site sequence of other enzymes that catalyze phospho group transfer via a phosphohistidine intermediate. Active site sequences of phosphoglycerate mutase and bisphosphoglycerate synthase were highly homologous with the active site of fructose-2,6-bisphosphatase implying a structural similarity and a common evolutionary origin.
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PMID:Active site sequence of hepatic fructose-2,6-bisphosphatase. Homology in primary structure with phosphoglycerate mutase. 304 Jul 62

Limited proteolysis and photoaffinity labeling of fructose-6-P,2-kinase and fructose-2,6-bisphosphatase were studied. Proteolysis by trypsin proceeds in two stages in which the first cleavage yields a product, Mr about 53,000, which has lost 90% of fructose-6-P,2-kinase, but retains nearly 80% of fructose-2,6-bisphosphatase. Further digestion of this product yields a second cleavage product, Mr about 50,000, which is completely devoid of the kinase and most of the phosphatase activities. These results indicate that fructose-6-P,2-kinase resides only in the original ("native") enzyme (Mr = 55,000), but fructose-2,6-bisphosphatase activity is present in both the native enzyme and the cleavage product(s). All three activities of fructose-6-P,2-kinase including the forward, the reverse, and ATP-ADP exchange activities are lost to the same degree by the mild proteolysis. Ki of fructose-6-P for fructose-2,6-bisphosphatase is not altered by the proteolysis. Partial protection against the proteolysis is provided by ATP, fructose-6-P, and fructose-2,6-P2. When the tryptic digestion of fructose-6-P,2-kinase:fructose-2,6-bisphosphatase was performed before and after phosphorylation of the enzyme by cAMP-dependent protein kinase, both the first and the second cleavage products contained the phosphorylation site. 8-Azido-ATP serves as a substrate for fructose-6-P,2-kinase with a Km of about 1 mM. Exposure of the enzyme-8-azido-ATP complex results in covalent incorporation (0.7 mol/mol of subunit) and 90% inactivation of fructose-6-P,2-kinase without loss of fructose 2,6-bisphosphatase. When the native and the first cleavage product of tryptic digestion were photoaffinity labeled with [alpha-32P]8-azido-ATP, the radiolabel occurred only in the native enzyme. These results provide evidence in support of, although not conclusive, the idea that the active sites of this bifunctional enzyme are different and located in two distinct sites.
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PMID:Limited proteolysis and photoaffinity labeling with 8-azido-ATP of fructose-6-phosphate,2-kinase and fructose-2,6-bisphosphatase. 633 Jan 9