Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.1 (chymotrypsin)
 10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The Neurospora crassa glycogen synthase (UDPglucose:glycogen 4-alpha-glucosyltransferase, EC 2.4.1.11) was purified to electrophoretic homogeneity by a procedure involving ultracentrifugation, DEAE-cellulose column chromatography, (NH4)2SO4 fractionation and 3-aminopropyl-Sepharose column chromatography. The final purified enzyme preparation was almost entirely dependent on glucose-6-P and had a specific activity of 6.9 units per mg of protein. The subunit molecular weight of the glycogen synthase was determined by electrophoresis in sodium dodecyl sulfate-polyacrylamide gel to be 88 000--90 000. The native enzyme was shown to have a molecular weight of 270 000 as determined by sucrose density gradient centrifugation. Thus, the glucose-6-P-dependent form of the N. crassa glycogen synthase can exist as trimer of the subunit. Limited proteolysis with trypsin or chymotrypsin converted the glucose-6-P-dependent form of the enzyme into an apparent glucose-6-P-independent form. The enzyme was shown to catalyze transfer of glucose from UDPglucose to glycogen as well as to its phosphorylase limit dextrin, but not to its beta-amylase limit dextrin. Moreover, glucose, maltose and maltotriose were not active as acceptors.
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PMID:Biosynthesis of glycogen in Neurospora crassa. Purification and properties of the UDPglucose:glycogen 4-alpha-glucosyltransferase. 2 41

A glucan-binding domain of 1,6-alpha-glucan synthase (dextransucrase) (GTF-S) was isolated from a trypsin digest of the Streptococcus sobrinus enzyme. The large 60.5-kilodalton peptide had an affinity for dextran comparable to that of the native enzyme, but had no glucan synthesis activity. The domain was produced in high yield compared with other large cleavage products, which allowed easy purification by size exclusion high-pressure liquid chromatography and affinity chromatography. Two other proteases (mouse submaxillaris protease and lysyl endopeptidase) with specificities similar to trypsin generated a distribution of GTF-S peptides that was also greatly enriched in the glucan-binding peptide. Proteases with markedly different specificities (chymotrypsin and Staphylococcus aureus V8 protease) produced a family of peptides with some evidence of the glucan-binding domain but in far lower yield. The tertiary structure of the domain was critical to its resistance to proteolysis; heat denaturation of GTF-S before trypsin digestion resulted in cleavage of the enzyme to small limit peptides leaving no evidence of the glucan-binding domain. The amino acid composition of the peptide was very similar to that of the native enzyme. The common occurrence of proteases in oral streptococcus cultures and reports of glucosyltransferase degradation during purification and storage raises the possibility that some accounts of glucan-binding receptors are peptides derived from glucosyltransferase. Kinetic implications of a glucan-binding domain are discussed.
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PMID:Isolation of a glucan-binding domain of glucosyltransferase (1,6-alpha-glucan synthase) from Streptococcus sobrinus. 296 13

The UDP-Glc:glycoprotein glucosyltransferase (GT), a key player in the endoplasmic reticulum (ER) quality control of glycoprotein folding, only glucosylates glycoproteins displaying non-native conformations. To determine whether GT recognizes folding intermediates or irreparably misfolded species with nearly native structures, we generated and tested as GT substrates neoglycoprotein fragments derived from chymotrypsin inhibitor 2 (GCI2) bearing from 53 to 64 (full-length) amino acids. Fragment conformations mimicked the last stage-folding structures adopted by a glycoprotein entering the ER lumen. GT catalytic efficiency (V(max)/K(m)) remained constant from GCI2-(1-53) to GCI2-(1-58) and then steadily declined to reach a minimal value with GCI2-(1-64). The same parameter showed a direct hyperbolic relationship with solvent accessibility of the single Trp residue but only in fragments exposing hydrophobic amino acid patches. Mutations introduced (GCI2-(1-63)V63S and GCI2-(1-64)V63S) produced slight structural destabilizations but increased GT catalytic efficiency. This parameter presented an inverse exponential relationship with the free energy of unfolding of canonical and mutant fragments. Moreover, the catalytic efficiency showed a linear relationship with the fraction of unfolded species in water. It was concluded that the GT-derived quality control may be operative with nearly native conformers and that no alternative ER-retaining mechanisms are required when glycoproteins approach their proper folding.
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PMID:The endoplasmic reticulum glucosyltransferase recognizes nearly native glycoprotein folding intermediates. 1531 28