Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.27.5 (RNase)
17,967 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Two conserved Trp-Cys-Gly-His-Cys (WCGHC) sequences are assigned to act as catalytic sites for protein disulfide isomerase. Peptides containing the active site sequence, Ala-Pro-Trp-Cys-Gly-His-Cys-Lys(APWCGHCK), were synthesized both in a mono-molecular form and on multiple antigen peptide (MAP) resin or Wang resin by the 9-fluoroenylmethoxycarbonyl (Fmoc)-based solid-phase method. With scrambled RNase as a substrate, the (APWCGHCK)8-MAP was first shown to mimic the PDI activity, which was one thousandth of that of bovine PDI and comparable to that of thioredoxin. APWCGPCK and APWCGHCK, however, did not display a disulfide isomerase activity even at a concentration 8 times higher than that of (APWCGHCK)8-MAP. It was assumed that a sterically proper proximity of at least two active site peptides with CXXC motif was required for the expression of PDI activity.
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PMID:Active site peptides with CXXC motif on map-resin can mimic protein disulfide isomerase activity. 765 33

Glutaredoxin (Grx) contains a redox-active disulfide and catalyzes thiol-disulfide interchange reactions with specificity for GSH. The dithiol form of Grx reduces mixed disulfides involving GSH or protein disulfides. During oxidative refolding of 8 microM reduced and denatured ribonuclease RNase-(SH)8 in a redox buffer of 1 mM GSH and 0.2 mM GSSG to yield native RNase-(S2)4, a large number of GSH-mixed disulfide species are formed. A lag phase that precedes formation of folded active RNase at a steady-state rate was shortened or eliminated by the presence of a catalytic concentration (0.5 microM) of Escherichia coli Grx together with protein disulfide-isomerase (PDI), its procaryotic equivalent E. coli DsbA, or the PDI analogue the E. coli thioredoxin mutant protein P34H. A mutant Grx in which one of the active site cysteine residues (Cys-11 and Cys-14) had been replaced by serine, C14S Grx, had similar effect compared with its wild-type counterpart. This demonstrated that Grx acted by a monothiol mechanism involving only Cys-11 and that RNase-S-SG-mixed disulfides were the substrates. Grx displayed synergistic activity together with PDI only in GSH/GSSG redox buffers with sufficiently low redox potential (E'0 of -208 or -181 mV) to allow reduction of the active site of Grx. In refolding systems that do not depend on glutathione, like cystamine/cysteamine or in the presence of selenite (SeO3(2-)), no synergistic activity of Grx was observed with PDI. We conclude that Grx acts by reducing mixed disulfides between GSH and RNase that are rate-limiting in enzyme-catalyzed refolding.
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PMID:Glutaredoxin accelerates glutathione-dependent folding of reduced ribonuclease A together with protein disulfide-isomerase. 771 72

Protein disulfide isomerase (PDI), a foldase of the endoplasmic recticulum, is a multifunctional protein that catalyzes the formation and isomerization of disulfide bonds during protein folding. The wild-type protein contains two redox active thiol/disulfide sites near the N and C terminus that are homologous to the redox center of thioredoxin. Using site-directed mutagenesis, both cysteines of each of the thioredoxin-like centers, (C35S,C38S) and (C379S,C382S) were replaced by serines. In addition, a mutant PDI was constructed with all four of the active cysteines mutated to serine (C35S,C38S,C379S,C382S). The activity of the wild-type and mutant proteins in the oxidative renaturation of reduced, denatured RNase was analyzed over a wide range of RNase concentrations, PDI concentrations, and glutathione redox buffers compositions. All mutants, including the construct with no functional thioredoxin centers, have measurable disulfide isomerase activity. Both of the thioredoxin-like sites contribute some to apparent steady-state binding (Km) and catalysis at saturating substrate concentrations (kcat); however, their contributions are not equivalent. At saturating concentrations of RNase, the mutant with an inactivated C-terminal active site (kcat = 0.72 +/- 0.06 min-1) retains near wild-type activity (kcat = 0.76 +/- 0.02 min-1), while the N-terminal mutant exhibits a significantly lower kcat (0.24 +/- 0.01 min-1). The Km for RNase is elevated for the C-terminal mutant (Km = 29 +/- 4 microM) while the N-terminal mutant (Km = 7.1 +/- 1.1 microM) exhibits a wild-type Km (6.9 +/- 0.8 microM). The larger Km for the C-terminal mutant (4.2 times wild-type) and the lower kcat of N-terminal mutant (32% of wild-type) suggest that the C-terminal region contributes more to apparent steady-state substrate binding, and the N-terminal region contributes more to catalysis at saturating concentrations of substrate. Despite their complementary roles in catalysis, the thioredoxin-like centers exhibit the same dependence on the glutathione redox buffer composition as evidenced by the equivalent K(ox) values for the wild-type (47 +/- 1 microM), N-terminal mutant (43 +/- 3 microM), and C-terminal mutant (44 +/- 1 microM). The mutant with both thioredoxin sites mutated displays a low but detectable level of disulfide-isomerase activity (0.5% of wild-type) that can be observed at high PDI concentrations. At high RNase concentrations (> or = 26 microM), wild-type PDI and all of the mutants catalyze intermolecular RNase aggregation in a nucleation growth reaction that is first order in PDI but fourth order with respect to RNase.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Mutations in the thioredoxin sites of protein disulfide isomerase reveal functional nonequivalence of the N- and C-terminal domains. 798 29

Protein disulfide isomerase (PDI), a very abundant protein in the endoplasmic reticulum, facilitates the formation and rearrangement of disulfide bonds using two nonequivalent redox active-sites, located in two different thioredoxin homology domains [Lyles, M. M., & Gilbert, H. F. (1994) J. Biol. Chem. 269, 30946-30952]. Each dithiol/disulfide active-site contains the thioredoxin consensus sequence CXXC. Four mutants of protein disulfide isomerase were constructed that have only a single active-site cysteine. Kinetic analysis of these mutants show that the first (more N-terminal) cysteine in either active site is essential for catalysis of oxidation and rearrangement during the refolding of reduced bovine pancreatic ribonuclease A (RNase). Mutant active sites with the sequence SGHC show no detectable activity for disulfide formation or rearrangement, even at concentrations of 25 microM. The second (more C-terminal) cysteine is not essential for catalysis of RNase disulfide rearrangements, but it is essential for catalysis of RNase oxidation, even in the presence of a glutathione redox buffer. Mutant active sites with the sequence CGHS show 12%-50% of the kcat activity of wild-type active sites during the rearrangement phase of RNase refolding but < 5% activity during the oxidation phase. In addition, mutants with the sequence CGHS accumulate significant levels of a covalent PDI-RNase complex during steady-state turnover while the wild-type enzyme and mutants with the sequence SGHC do not. Since both active-site cysteines are essential for catalysis of disulfide formation, the dominant mechanism for RNase oxidation may involve direct oxidation by the active-site PDI disulfide. Although it is not essential for catalysis of RNase rearrangements, the more C-terminal cysteine does contribute 2-8-fold to the rearrangement activity. A mechanism for substrate rearrangement is suggested in which the second active-site cysteine provides PDI with a way to "escape" from covalent intermediates that do not rearrange in a timely fashion. The second active-site cysteine may normally serve the wild-type enzyme as an internal clock that limits the time allowed for intramolecular substrate rearrangements.
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PMID:Catalysis of oxidative protein folding by mutants of protein disulfide isomerase with a single active-site cysteine. 863 81

Protein disulfide isomerase has broad specificity in the catalysis of the formation and rearrangement of native disulfide bonds in proteins. This enzyme has two independent thioredoxin-like active sites (-CGHC-) and a peptide binding site. However, the mechanisms involving the catalytic processes are not clearly understood. It was reported that the enzyme associates with scrambled pancreatic ribonuclease A in vitro, and with misfolded human lysozyme in vivo. In the present study, recombinant human interleukin 2 has been chosen to probe the reaction intermediate in the reaction with the enzyme. We have identified and characterized a covalent associate formed in vitro by SDS-PAGE and Western blot analysis. This associate has a molecular weight of 71-72 kDa, the approximate sum of the molecular weights of the enzyme and the substrate. Western blot analysis confirmed that it formed via an intermolecular disulfide bond. Upon treatment with 2-mercaptoethanol, this bond was cleaved.
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PMID:Covalent association of protein disulfide isomerase with recombinant human interleukin 2 in vitro. 866 Mar 62

A catalyst of disulfide formation and isomerization during protein folding, protein-disulfide isomerase (PDI) has two catalytic sites housed in two domains homologous to thioredoxin, one near the N terminus and the other near the C terminus. The thioredoxin domains, by themselves, can catalyze disulfide formation, but they are unable to catalyze disulfide isomerizations (Darby, N. J. and Creighton, T. E. (1995) Biochemistry 34, 11725-11735). A 21-kDa, C-terminal fragment of PDI (amino acids 308-491), termed weePDI, comprises the C-terminal third of the molecule. The kcat for ribonuclease oxidative folding by weePDI is 0.26 +/- 0.02 min-1, 3-fold lower than the wild-type enzyme but indistinguishable from the activity of a full-length mutant of PDI in which both active site cysteines of the N-terminal thioredoxin domain have been mutated to serine. Eliminating the ability of weePDI to escape easily from covalent complexes with substrate by mutating the active site cysteine nearer the C terminus to serine has a large effect on the isomerase activity of weePDI compared with its effect on the full-length enzyme. weePDI also displays chaperone and anti-chaperone activity characteristic of the full-length molecule. As isolated, weePDI is a disulfide-linked dimer in which the single cysteine (Cys-326) outside active site cross-links two weePDI monomers. The presence of the intermolecular disulfide decreases the activity by more than 2-fold. The results imply that the functions of the core thioredoxin domains of PDI and other members of the thioredoxin superfamily might be modified quite easily by the addition of relatively small accessory domains.
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PMID:A 21-kDa C-terminal fragment of protein-disulfide isomerase has isomerase, chaperone, and anti-chaperone activities. 940 79

The practical exploitation of the vast numbers of sequences in the genome sequence databases is crucially dependent on the ability to identify the function of each sequence. Unfortunately, current methods, including global sequence alignment and local sequence motif identification, are limited by the extent of sequence similarity between sequences of unknown and known function; these methods increasingly fail as the sequence identity diverges into and beyond the twilight zone of sequence identity. To address this problem, a novel method for identification of protein function based directly on the sequence-to-structure-to-function paradigm is described. Descriptors of protein active sites, termed "fuzzy functional forms" or FFFs, are created based on the geometry and conformation of the active site. By way of illustration, the active sites responsible for the disulfide oxidoreductase activity of the glutaredoxin/thioredoxin family and the RNA hydrolytic activity of the T1 ribonuclease family are presented. First, the FFFs are shown to correctly identify their corresponding active sites in a library of exact protein models produced by crystallography or NMR spectroscopy, most of which lack the specified activity. Next, these FFFs are used to screen for active sites in low-to-moderate resolution models produced by ab initio folding or threading prediction algorithms. Again, the FFFs can specifically identify the functional sites of these proteins from their predicted structures. The results demonstrate that low-to-moderate resolution models as produced by state-of-the-art tertiary structure prediction algorithms are sufficient to identify protein active sites. Prediction of a novel function for the gamma subunit of a yeast glycosyl transferase and prediction of the function of two hypothetical yeast proteins whose models were produced via threading are presented. This work suggests a means for the large-scale functional screening of genomic sequence databases based on the prediction of structure from sequence, then on the identification of functional active sites in the predicted structure.
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PMID:Method for prediction of protein function from sequence using the sequence-to-structure-to-function paradigm with application to glutaredoxins/thioredoxins and T1 ribonucleases. 971 46

EF-Tu is involved in the binding and transport of the appropriate codon-specified aminoacyl-tRNA to the aminoacyl site of the ribosome. We and others have recently shown that the Escherichia coli EF-Tu, in additon to its acknowledged role in translation elongation, displays chaperone-like properties. We report here that EF-Tu, like thioredoxin, protein disulfide isomerase, and DsbA, catalyzes protein disulfide formation (oxidative renaturation of reduced RNase), reduction (reduction of insulin disulfides), and isomerization (refolding of randomly oxidized RNase). In contrast with most protein disulfide isomerases which possess vicinal cysteines and form an intramolecular disulfide upon oxidation, EF-Tu, which does not possess vicinal cysteines, forms intermolecular disulfides upon oxidation, resulting in the appearance of multimeric forms.
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PMID:Protein-disulfide isomerase activity of elongation factor EF-Tu. 981 62

Assembly and degradation of fibronectin-containing extracellular matrices are dynamic processes that are up-regulated during wound healing, embryogenesis, and metastasis. Although several of the early steps leading to fibronectin deposition have been identified, the mechanisms leading to the accumulation of fibronectin in disulfide-stabilized multimers are largely unknown. Disulfide-stabilized fibronectin multimers are thought to arise through intra- or intermolecular disulfide exchange. Several proteins involved in disulfide exchange reactions contain the sequence Cys-X-X-Cys in their active sites, including thioredoxin and protein-disulfide isomerase. The twelfth type I module of fibronectin (I12) contains a Cys-X-X-Cys motif, suggesting that fibronectin may have the intrinsic ability to catalyze disulfide bond rearrangement. Using an established protein refolding assay, we demonstrate here that fibronectin has protein-disulfide isomerase activity and that this activity is localized to the carboxyl-terminal type I module I12. I12 was as active on an equal molar basis as intact fibronectin, indicating that most of the protein-disulfide isomerase activity of fibronectin is localized to I12. Moreover, the protein-disulfide isomerase activity of fibronectin appears to be partially cryptic since limited proteolysis of I10-I12 increased its isomerase activity and dramatically enhanced the rate of RNase refolding. This is the first demonstration that fibronectin contains protein-disulfide isomerase activity and suggests that cross-linking of fibronectin in the extracellular matrix may be catalyzed by a disulfide isomerase activity contained within the fibronectin molecule.
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PMID:Identification of protein-disulfide isomerase activity in fibronectin. 1006 58

Hydroxymethylacylfulvene (HMAF, MGI 114) is a novel antitumor drug and a potent pro-apoptotic agent that has the potential to alkylate cellular nucleophiles. The objective of these studies was to characterize drug uptake and cellular targets for drug binding in human leukemia CEM cells. The uptake of [14C]HMAF had two components: a rapid phase (0-10 min) and a slow phase. At 10 microM drug (37 degrees), the rapid and slower phase amounted to 0.86 and 0.13 pmol/min/10(6)cells, respectively. HMAF uptake was inhibited 82% by low temperature (4 degrees) at 4 hr. Cell-associated HMAF localized to nuclear (50%), cytoplasmic (37%), and membrane fractions (10%). Continued drug uptake appeared to be driven by covalent binding to cellular macromolecules. Approximately 1/4 and 2/3 of cell-associated HMAF formed covalent adducts after 10 min and 4 hr, respectively, as found by perchloric acid precipitation. Drug adducts were not readily reversible; 77% of the covalently bound radiolabel was retained by the cells 20 hr after drug treatment. Combinations of DNase, RNase, and proteinase K with perchloric acid precipitation showed that approximately 60, 30, and 10% of the covalently bound drug was associated with the protein, DNA, and RNA fractions, respectively. Incubation of 100 microM [14C]HMAF (24 hr) with purified DNA, serum albumin, thioredoxin, and thioredoxin reductase resulted in 6, 22, 14, and 11 pmol [14C]HMAF/microg DNA or protein, respectively. Results indicate that multiple targets for HMAF binding may contribute to the pro-apoptotic and antiproliferative action of the drug.
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PMID:Drug uptake and cellular targets of hydroxymethylacylfulvene (HMAF). 1042 61


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