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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 structural and functional organization of Escherichia coli polypeptide chain release factors 1 and 2 (RF-1 and RF-2) was investigated by limited proteolysis with trypsin and chymotrypsin. A protease-sensitive site was found in a similar position in both factors at the beginning of a highly conserved region in the C-terminal part of the proteins. Chymotrypsin cleavage of RF-2 yielded a nicked form with the fragments associated. This nicked factor lost in vitro peptidyl-tRNA hydrolysis activity (a peptidyltransferase function) but had enhanced in vitro codon-ribosome binding activity (a decoding site function). It inhibited codon-dependent f[3H]Met-tRNA hydrolysis activity of intact RF-1 and RF-2, presumably as a result of an increased affinity for ribosomes. These data are consistent with a model whereby the release factor acts like a tRNA analog spanning the decoding and peptidyltransferase centers on the ribosome. The proteolytic sensitivity of the RFs most likely reflects an exposed surface loop. We propose that this loop interacts with the ribosomal peptidyltransferase site and that the stabilization of factor:ribosome binding upon cleavage could be explained by conformational coupling between domains on the factor for codon-ribosome binding at the decoding site and interaction with peptidyltransferase.
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PMID:A single proteolytic cleavage in release factor 2 stabilizes ribosome binding and abolishes peptidyl-tRNA hydrolysis activity. 803 46

The measurement of protein kinetics by isotopic techniques has been hindered by the long-standing difficulty of accurately measuring the isotope content of the biosynthetic precursor pool (aminoacyl-tRNA in tissues). Mass isotopomer distribution analysis (MIDA) is a stable isotope-mass spectrometric (MS) technique for measuring biosynthetic precursor enrichments from measurements on a polymeric product, based on combinatorial probabilities of labeled and unlabeled monomeric subunits. Proteins contain complex isotopomer patterns as a result of their relatively high molecular mass, however, so that resolution of the individual mass isotopomers in the polymeric product (an analytic requirement for MIDA) is technically difficult. An approach for measuring protein synthesis by MIDA is described and tested here: First, in vitro, using a synthetic peptide present in human serum albumin; and then, in vivo, for albumin synthetic rate in rats. A peptide contained in human serum albumin (SVVLLLR) and theoretically recoverable from trypsin/chymotrypsin proteolysis was synthesized by solid-phase peptide synthesis using known mixtures of natural abundance and [5,5, 5-2H3]leucine. Additionally, enriched and natural abundance peptides were mixed in vitro to simulate in vivo biosynthesis and to address problems of instrument accuracy, precision, and data management. The mass isotopomer patterns of the synthetic peptides were analyzed using electrospray ionization (ESI) with both magnetic sector and quadrupole mass analyzers. The resolution of the magnetic sector was superior to that of the quadrupole instrument, but accuracy and precision in the measurement of mass isotopomer abundances and kinetic parameters were comparable and both gave values close to those predicted. Next, rats were infused with [5,5,5-2H3]leucine intravenously, and a leucine-rich peptide was isolated and purified from trypsin-digested rat serum albumin (RHPDYSVSLLLR, 1456 Da) and then analyzed by ESI-MS using a magnetic sector instrument. Precursor pool enrichments and fractional synthetic rates (0.45 +/- 0.03 day-1, t1/2 = 1.53 +/- 0.09 days) were calculated. Biosynthetic rates of rat serum albumin were congruent with previously published values. In summary, measurement of protein synthesis and precursor pool enrichments by MIDA is technically feasible and practical in vivo using proteolytically derived peptides and ESI-MS analysis.
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PMID:Measuring protein synthesis by mass isotopomer distribution analysis (MIDA). 991 49

The gene coding for the 3C protease from human rhinovirus strain 1B was efficiently expressed in an Escherichia coli strain which also overexpressed the rare argU tRNA. The protease was isolated from inclusion bodies, refolded, and exhibited a kcat/Km = 3280 M-1 s-1 using an internally quenched peptidyl fluorogenic substrate. This continuous fluorogenic assay was used to measure the kinetics of 3C protease inhibition by several conventional peptidyl chloromethylketones as well as a novel series of compounds, the bromomethylketonehydrazides. Compounds containing the bromomethylketonehydrazide backbone and a glutamine-like side chain at the P1 position were potent, time-dependent inhibitors of rhinovirus 3C protease with kinact/Kinact values as high as 23,400 M-1 s-1. The inhibitory activity of compounds containing modified P1 side chains suggests that the interactions between the P1 carboxamide group and the 3C protease contributes at least 30-fold to the kinact/Kinact rate constants for bromomethylketonehydrazide inhibition of 3C protease. Electrospray ionization mass spectrometry measurements of the molecular weights of native and inhibited 3C protease have established an inhibitory mechanism involving formation of a covalent adduct between the enzyme and the inhibitor with the loss of a bromide ion from the bromomethylketonehydrazide. Tryptic digestion of bromomethylketonehydrazide-inhibited 3C protease established adduct formation to a peptide corresponding to residues 145-154, a region which contains the active site cysteine-148 residue. The bromomethylketonehydrazides were fairly weak inhibitors of chymotrypsin, human elastase, and cathepsin B and several of these compounds also showed evidence for inhibition of human rhinovirus 1B replication in cell culture.
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PMID:Inhibition of 3C protease from human rhinovirus strain 1B by peptidyl bromomethylketonehydrazides. 998 47

The interaction of adenine nucleotides with glycyl-tRNA synthetase was examined by several experimental approaches. ATP and nonsubstrate ATP analogues render glycyl-tRNA synthetase more resistant to digestion by a number of proteases (thrombin, Arg-C, and chymotrypsin) at concentrations that correlate with their Michaelis constants or inhibition constants, consistent with their exerting an effect by binding at the ATP site. Glycine had little effect alone but potentiated the effect of ATP in increasing the resistance to thrombin digestion, consistent with the formation of an enzyme-bound adenylate. No protection from thrombin digestion was afforded by tRNA(gly). Binding constants were determined by isothermal titration calorimetry at 25 degrees C for ATP (2.5 x 10(5) M(-1)), AMPPNP (3.7 x 10(5) M(-1)), and AMPPCP (2.2 x 10(6) M(-1)). The nucleotides had similar values for DeltaH (-71 kJ mol(-1)), with values for TDeltaS that accounted for the differences in the binding constants. Near-ultraviolet CD spectra of the enzyme-nucleotide complexes indicate that the nucleotides are bound in the anti configuration. A glycyl-adenylate analogue, glycine sulfamoyl adenosine (GSAd), bound with a large value for DeltaH (-187 kJ mol(-1)), which was balanced by a large TDeltaS term to give a binding constant (3.7 x 10(6) M(-1)) only slightly larger than that of AMPPCP. Glycine binding to the enzyme could not be detected calorimetrically, and its presence did not change the thermodynamic parameters for binding of AMPPCP. AMPPNP and AMPPCP were not substrates for glycyl-tRNA synthetase. Analysis of the temperature dependence of ATP binding indicated that the heat capacity change is small, whereas the binding of GSAd is accompanied by a large negative heat capacity change (-2.6 kJ K(-1) mol(-1)). Titrations performed in buffers with different ionization enthalpies indicate that the large value for DeltaH for the adenylate analogue does not arise from a coupled protonation event. Differential scanning calorimetry indicated that glycyl-tRNA synthetase is stabilized by nucleotides. Unfolding of the protein is irreversible, and thermodynamic parameters for unfolding could therefore not be determined. The results are consistent with a significant conformational transition in glycyl-tRNA synthetase coupled to the binding of GSAd.
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PMID:Thermodynamic characterization of the binding of nucleotides to glycyl-tRNA synthetase. 1273 74

Mycobacterium tuberculosis glutamyl-tRNA synthetase (Mt-GluRS), encoded by Rv2992c, was overproduced in Escherichia coli cells, and purified to homogeneity. It was found to be similar to the other well-characterized GluRS, especially the E. coli enzyme, with respect to the requirement for bound tRNA(Glu) to produce the glutamyl-AMP intermediate, and the steady-state kinetic parameters k(cat) (130 min(-1)) and K(M) for tRNA (0.7 microm) and ATP (78 microm), but to differ by a one order of magnitude higher K(M) value for L-Glu (2.7 mm). At variance with the E. coli enzyme, among the several compounds tested as inhibitors, only pyrophosphate and the glutamyl-AMP analog glutamol-AMP were effective, with K(i) values in the mum range. The observed inhibition patterns are consistent with a random binding of ATP and L-Glu to the enzyme-tRNA complex. Mt-GluRS, which is predicted by genome analysis to be of the non-discriminating type, was not toxic when overproduced in E. coli cells indicating that it does not catalyse the mischarging of E. coli tRNA(Gln) with L-Glu and that GluRS/tRNA(Gln) recognition is species specific. Mt-GluRS was significantly more sensitive than the E. coli form to tryptic and chymotryptic limited proteolysis. For both enzymes chymotrypsin-sensitive sites were found in the predicted tRNA stem contact domain next to the ATP binding site. Mt-GluRS, but not Ec-GluRS, was fully protected from proteolysis by ATP and glutamol-AMP. Small-angle X-ray scattering showed that, at variance with the E. coli enzyme that is strictly monomeric, the Mt-GluRS monomer is present in solution in equilibrium with the homodimer. The monomer prevails at low protein concentrations and is stabilized by ATP but not by glutamol-AMP. Inspection of small-angle X-ray scattering-based models of Mt-GluRS reveals that both the monomer and the dimer are catalytically active. By using affinity chromatography and His(6)-tagged forms of either GluRS or glutamyl-tRNA reductase as the bait it was shown that the M. tuberculosis proteins can form a complex, which may control the flux of Glu-tRNA(Glu) toward protein or tetrapyrrole biosynthesis.
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PMID:Kinetic and mechanistic characterization of Mycobacterium tuberculosis glutamyl-tRNA synthetase and determination of its oligomeric structure in solution. 1918 40


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