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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)

Limited chymotrypsin proteolysis of CTP:phosphocholine cytidylyltransferase (CT; EC 2.7.7.15) produced several distinct fragments which were mapped to the N terminus of CT using antibodies directed against the N and C terminus and the conserved central domain. A time course of chymotrypsin proteolysis showed a progression in digestion as follows: 42-->39-->35-->30-->28-->26 kDa. The binding of CT and of the chymotrypsin fragments to lipid vesicles was assessed by floatation analysis. The ability of the fragments to bind to activating lipid vesicles correlated with the presence of a putative amphipathic alpha-helix, helix-1, between residues 236 and 293. Fragments lacking this helix could, however, bind to phosphatidylcholine/sphingosine vesicles, which inhibit CT activity, and were capable of dimer formation. The degree of resistance to chymotrypsin degradation increased when CT was bound to the strongly activating lipid vesicles phosphatidylcholine/oleic acid (1:1) and phosphatidylcholine/phosphatidylglycerol (1:1). Conversion of the 39- and 35-kDa fragments, which contain the intact helix-1, to the 30-, 28-, and 26-kDa bands, which lack helix-1, required longer proteolysis times, suggesting that this helical domain is more shielded from solvent upon membrane binding. These results support the theory that CT has a bipartite tertiary structure composed of a globular N-terminal domain and an extended C-terminal domain and that CT interacts with membranes via its putative amphipathic helix which intercalates into the membrane bilayer of activating phospholipids.
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PMID:Identification of the membrane-binding domain of rat liver CTP:phosphocholine cytidylyltransferase using chymotrypsin proteolysis. 810 70

The effect of mutations in the proposed catalytic domain of CTP:phosphocholine cytidylyltransferase was investigated by constructing the single mutants CT-S91 and CT-C114 from the double mutant CT-S91C114, previously shown to have 4-fold lower than wild-type activity [Walkey, C.R., Kalmar, G. B., & Cornell, R. B. (1994) J. Biol. Chem. 269, 5742-5749]. The constructs were overexpressed in COS cells. The mutation Gly-91 to Ser-91 was found to be responsible for the decreased activity, whereas Ser-114 to Cys-114 had no effect. An alanine substitution at position 91, CT-A91, had a lesser effect on cytidylyltransferase activity. CT-S91 and CT-WT were purified from COS cells, and their kinetic constants were determined. CT-S91 had a 4-fold lower Vmax, and a K(m) for CTP 25-fold higher than the wild-type enzyme, suggesting that substitution of Gly-91 with serine interferes with CTP binding. The K(m) for phosphocholine was not affected in the CT-S91 mutant. There was no difference in the chymotrypsin sensitivities of CT-S91 and CT-WT, indicating that the mutation did not cause a global change in protein structure. However, the CT-S91 activity was more susceptible to inhibition by the denaturant urea than that of CT-WT, indicative of a perturbation of the active site folding. Gly-91 resides in the local sequence HSGH, which has been proposed to be a CTP-binding motif in the novel cytidylyltransferase superfamily [Bork, P., Holm, L., Koonin, E.V., & Sander, C. (1995) Proteins: Struct., Funct., Genet. 22, 259-266]. Our results represent the first experimental validation of this hypothesis.
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PMID:Substitution of serine for glycine-91 in the HXGH motif of CTP:phosphocholine cytidylyltransferase implicates this motif in CTP binding. 871 64

CTP:phosphocholine cytidylyltransferase (CT), the rate controlling enzyme in phosphatidylcholine biosynthesis, is activated by reversible membrane binding. To investigate the membrane binding mechanism of CT, we have used the photoreactive hydrophobic probe 3-(trifluoromethyl)-3-(m-[l25I]iodophenyl)diazirine ([125I]TID). Association of CT with phosphatidylcholine/oleic acid (1:1) vesicles was first demonstrated by gel filtration analysis. Upon irradiation, CT was covalently labeled by [125I]TID presented in phosphatidylcholine/oleic acid vesicles. This demonstrates an intercalation of part of the protein into the hydrophobic core of the membrane. To identify the membrane-embedded domain, the chymotrypsin digestion products of [125I]TID labeled CT were analysed. Chymotrypsin digestion produced a set of previously defined N-terminal fragments (Craig, L., Johnson, J.E. and Cornell, R.B. (1994) J. Biol. Chem. 269, 3311), as well as several small C-terminal fragments which react with an anti-peptide antibody raised against the proposed amphipathic alpha-helix. All fragments containing the amphipathic helical region of the enzyme had [125I]TID label associated, while the chymotryptic fragment which lacked this region was not highly labeled. Similar fragment labeling patterns were produced when [125I]TID was presented in phosphatidylcholine/oleic acid or phosphatidylcholine/diacylglycerol vesicles, suggesting that the same domain of CT mediates binding to membranes containing either of the two lipid activators. A 62-residue synthetic peptide corresponding in sequence to the amphipathic helical region of CT was labeled with [125I]TID, demonstrating its ability to intercalate independently of the rest of the protein. These results indicate a membrane-binding mechanism for cytidylyltransferase involving the intercalation of the amphipathic alpha-helix region into the hydrophobic acyl chain core of the activating membrane.
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PMID:An amphipathic alpha-helix is the principle membrane-embedded region of CTP:phosphocholine cytidylyltransferase. Identification of the 3-(trifluoromethyl)-3-(m-[125I]iodophenyl) diazirine photolabeled domain. 909 14

CTP:phosphocholine cytidylyltransferase (CCT) is a multi-domain enzyme that regulates phosphatidylcholine synthesis. It converts to an active form upon binding cell membranes, and interdomain dissociations have been hypothesized to accompany this process. To identify these interdomain and membrane interactions, the tertiary structures of three forms of CCTalpha were probed by monitoring accessibility to proteases. Time-limited digestion with chymotrypsin or arginine C of soluble CCTalpha (CCT(sol)), phospholipid vesicle-bound CCT (CCT(mem)), and a soluble constitutively active CCT truncated at amino acid 236 generated complex mixtures of peptides that were resolved and identified by gel electrophoresis/immunoblotting and by matrix-assisted laser desorption/ionization-mass spectrometry, with or without coupling to capillary liquid chromatography. Identification of cleavage sites enabled assembly of peptide bond accessibility maps for each CCT form. Our results reveal a approximately 80-residue core within the catalytic domain (domain C) as the most inaccessible region in all three forms and the C-terminal phosphorylation domain as the most accessible. Membrane binding has little effect on the protease accessibility of these domains. To map the protease sites onto the catalytic domain, its three-dimensional structure was modeled from the atomic coordinates of glycerol-phosphate cytidylyltransferase (Protein Data Bank code 1COZ). The protease inaccessibility of most sites in domain C could be explained by burial or location within secondary structural elements. The accessibility of the N-terminal domain (domain N) was enhanced upon membrane binding. Residues Phe(234)-Leu(303) were inaccessible in CCT(mem), suggesting burial in the membrane. Surprisingly, residues Leu(274)-Leu(303) of this domain were also inaccessible in CCT(sol). We propose that this region is buried by interdomain contacts with domain N in CCT(sol). Membrane binding and burial of domain M in the lipid bilayer may disrupt this interaction, leading to increased exposure of sites in domain N.
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PMID:Interdomain and membrane interactions of CTP:phosphocholine cytidylyltransferase revealed via limited proteolysis and mass spectrometry. 1571 72