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941.
We studied whether the peptides of nine amino acids (9-mers) that are typically used in MHC class I presentation are sufficiently unique for self:nonself discrimination. The human proteome contains 28,783 proteins, comprising 107 distinct 9-mers. Enumerating distinct 9-mers for a variety of microorganisms we found that the average overlap, i.e., the probability that a foreign peptide also occurs in the human self, is about 0.2%. This self:nonself overlap increased when shorter peptides were used, e.g., was 30% for 6-mers and 3% for 7-mers. Predicting all 9-mers that are expected to be cleaved by the immunoproteasome and to be translocated by TAP, we find that about 25% of the self and the nonself 9-mers are processed successfully. For the HLA-A*0201 and HLA-A*0204 alleles, we predicted which of the processed 9-mers from each proteome are expected to be presented on the MHC. Both alleles prefer to present processed 9-mers to nonprocessed 9-mers, and both have small preference to present foreign peptides. Because a number of amino acids from each 9-mer bind the MHC, and are therefore not exposed to the TCR, antigen presentation seems to involve a significant loss of information. Our results show that this is not the case because the HLA molecules are fairly specific. Removing the two anchor residues from each presented peptide, we find that the self:nonself overlap of these exposed 7-mers resembles that of 9-mers. Summarizing, the 9-mers used in MHC class I presentation tend to carry sufficient information to detect nonself peptides amongst self peptides. 相似文献
942.
943.
Physicochemical and saccharide-binding studies have been performed on Trichosanthes cucumerina seed lectin (TCSL). The agglutination activity of TCSL is highest in the pH range 8.0-11.0, whereas below pH 7.0 it decreases quite rapidly, which is consistent with the involvement of imidazole side chains of His residues, which titrate in this pH range, in the sugar-binding activity of the lectin. The lectin activity is unaffected between 0 and 60 degrees C, but a sharp decline occurs at higher temperatures. Isoelectric focusing studies show that TCSL has three isoforms with pI values of 5.3, 6.2, and 7.1, with the isoform of pI 6.2 being the most abundant. Circular dichroism spectroscopic studies reveal that TCSL contains about 28.4% beta-sheet, 10.6% beta-turns, 7% polyproline type 2 structure, with the remainder comprising unordered structure; the alpha-helix content is negligible. Binding of 4-methylumbelliferyl-beta-D-galactopyranoside (MeUmbbetaGal) to TCSL results in a significant increase in the fluorescence intensity of the ligand, and this change has been used to obtain the association constant for the interaction. At 25 degrees C, the association constant, K(a), for the TCSL-MeUmbbetaGal interaction was determined as 6.9 x 10(4)M(-1). Binding of nonfluorescent, inhibitory sugars was studied by monitoring their ability to reverse the fluorescence changes observed when MeUmbbetaGal was titrated with TCSL. 相似文献
944.
Sio CF Otten LG Cool RH Quax WJ 《Biochemical and biophysical research communications》2003,312(3):755-760
Residue Phe375 of cephalosporin acylase has been identified as one of the residues that is involved in substrate specificity. A complete mutational analysis was performed by substituting Phe375 with the 19 other amino acids and characterising all purified mutant enzymes. Several mutations cause a substrate specificity shift from the preferred substrate of the enzyme, glutaryl-7-ACA, towards the desired substrate, adipyl-7-ADCA. The catalytic efficiency ( [Formula: see text] (cat)/ [Formula: see text] (m)) of mutant SY-77(F375C) towards adipyl-7-ADCA was increased 6-fold with respect to the wild-type enzyme, due to a strong decrease of [Formula: see text] (m). The [Formula: see text] (cat) of mutant SY-77(F375H) towards adipyl-7-ADCA was increased 2.4-fold. The mutational effects point at two possible mechanisms by which residue 375 accommodates the long side chain of adipyl-7-ADCA, either by a widening of a hydrophobic ring-like structure that positions the aliphatic part of the side chain of the substrate, or by hydrogen bonding to the carboxylate head of the side chain. 相似文献
945.
Noguchi A Nakayama T Hemmi H Nishino T 《Biochemical and biophysical research communications》2003,304(4):684-690
Dextran glucosidases show high sequence identity (50%) to Bacillus sp. SAM1606 alpha-glucosidase, which is more specific for short-chain substrates. Sequence comparison of these enzymes as well as molecular modeling studies predicted that the extension of loop 4 of the (beta/alpha)(8)-barrel fold may be responsible for the narrower specificity of SAM1606 alpha-glucosidase with respect to substrate chain length. Indeed, deletion mutants of SAM1606 alpha-glucosidase that lack this extension showed higher relative activities toward dextran and long-chain isomaltooligosaccharides. Kinetic and thermodynamic analyses of oligosaccharide hydrolysis catalyzed by SAM1606 alpha-glucosidase and its deletion mutants suggested that the loss of such extension(s) in loop 4 should energetically destabilize the Michaelis complexes with long-chain substrates to result in smaller differences between the activation free energies for the enzymatic hydrolyses of isomaltoheptaose and isomaltose than those observed for the wild-type enzyme. This is the reason that dextran glucosidase, whose loop 4 is shorter in length, shows broader substrate chain-length specificity than does SAM1606 alpha-glucosidase. 相似文献
946.
El Tamer MK Lücker J Bosch D Verhoeven HA Verstappen FW Schwab W van Tunen AJ Voragen AG de Maagd RA Bouwmeester HJ 《Archives of biochemistry and biophysics》2003,411(2):196-203
Monoterpene cyclases are the key enzymes in the monoterpene biosynthetic pathway, as they catalyze the cyclization of the ubiquitous geranyl diphosphate (GDP) to the specific monoterpene skeletons. From Citrus limon, four monoterpene synthase-encoding cDNAs for a beta-pinene synthase named Cl(-)betaPINS, a gamma-terpinene synthase named ClgammaTS, and two limonene synthases named Cl(+)LIMS1 and Cl(+)LIMS2 were recently isolated [J. Lücker et al., Eur. J. Biochem. 269 (2002) 3160]. The aim of our work in this study was to identify domains within these monoterpene synthase enzymes determining the product specificity. Domain swapping experiments between Cl(-)betaPINS and ClgammaTS and between Cl(+)LIMS2 and ClgammaTS were conducted. We found that within the C-terminal domain of these monoterpene synthases, a region comprising 200 amino acids, of which 41 are different between Cl(-)betaPINS and ClgammaTS, determines the specificity for the formation of beta-pinene or gamma-terpinene, respectively, while another region localized further downstream is required for a chimeric enzyme to yield products in the same ratio as in the wild-type ClgammaTS. For Cl(+)LIMS2, the two domains together appear to be sufficient for its enzyme specificity, but many chimeras were inactive probably due to the low homology with ClgammaTS. Molecular modeling was used to further pinpoint the amino acids responsible for the differences in product specificity of ClgammaTS and Cl(-)betaPINS. 相似文献
947.
Seo GJ Kim SE Lee YM Lee JW Lee JR Hahn MJ Kim ST 《Biochemical and biophysical research communications》2003,304(2):339-343
Chk2/hCds1, the human homolog of Saccharomyces cerevisiae Rad53p and Schizosaccharomyces pombe Cds1p, plays a critical role in the DNA damage checkpoint pathway. While several in vivo targets of Chk2 have been identified, the other target proteins of Chk2 responsible for multiple functions, such as cell cycle arrest, DNA repair, and apoptosis, remain to be elucidated. We utilized the GST-peptide approach to identify physiological substrates for Chk2. Mutational analyses using GST-linked Cdc25A containing serine 123 revealed that residues at positions -5 and -3 are critical determinants for the recognition of the Chk2 substrate. We determined the general phosphorylation consensus sequence and identified in vitro targets of Chk2 using GST peptides as substrates. The newly identified in vitro target proteins include Abl1, Bub1R, Bub1, Bub3, Psk-H1, Smc3, Plk1, Cdc25B, Dcamkl1, Mre11, Pms1, and Xrcc9. 相似文献
948.
Specificity of Staphylococcus aureus 209P cell wall hydrolysis by the L1 and L2-bacteriolytic enzymes from lysoamidase lytic complex was studied. L1-peptidase was shown to display both glycyl-glycine endopeptidase and N-acetylmuramyl-L-alanine amidase enzymatic activities on the S. aureus peptidoglycan molecule, whereas L2-peptidase acts as N-acetylmuramyl-L-alanine amidase. 相似文献
949.
Frankenberg RJ Andersson M Clark DS 《Extremophiles : life under extreme conditions》2003,7(5):353-360
The hydrolytic specificity of the recombinant 20S proteasome from the deep-sea thermophile Methanococcus jannaschii was evaluated toward oxidized insulin B-chain across a range of temperatures (35°, 55°, 75°, and 90°C) and hydrostatic pressures (1, 250, 500, and 1,000 atm). Of the four temperatures considered, the same maximum overall hydrolysis rate was observed at both 55° and 75°C, which are much lower than the Topt of 116°C previously observed for a small amide substrate (Michels and Clark 1997). At 35°C the rates of cleavage were highest at the carboxyl side of glutamine and leucine, whereas at the three higher temperatures, the most rapid cleavages occurred after leucine and glutamic acid residues. The distribution of proteolytic fragments and the cleavage sequence also varied between the lowest and higher temperatures. Application of hydrostatic pressure did not increase proteasome activity, as observed previously for the amide substrate (Michels and Clark 1997), but instead significantly reduced the overall conversion of the polypeptide substrate. Overall cleavage patterns observed for the recombinant M. jannaschii proteasome were similar to those reported previously for Thermoplasma acidophilum (Akopian et al. 1997) and human proteasomes (Dick et al. 1991), indicating that proteasome specificity has been conserved despite significant environmental diversity.Communicated by G. Antranikian 相似文献
950.
Comparative Characteristics of Carbohydrate Binding by Lectins from Broad Bean,Pea, Common Vetch,and Lentil Seeds 总被引:1,自引:0,他引:1
Lectins from the seeds of broad bean (Vicia faba L.), pea (Pisum sativum L.), common vetch (V. sativa L.), and lentil (Lens culinaris Medik.) were isolated and purified by affinity chromatography. The hemagglutinating activity of lectins was most effectively inhibited by methyl--D-mannopyranoside, trehalose, and D-mannose. Other carbohydrate haptens, such as methyl--D-glucopyranoside, maltose, and alginic and D-glucuronic acids were less effective. Two lectins obtained from different lentil cultivars, unlike other lectins, had a relatively high affinity for melecitose, N-acetyl-D-glucosamine, L-sorbose, and sucrose. Furthermore, these lectins interacted with soluble starch. All the lectins examined had similar, but not identical, carbohydrate-binding properties. Because of their similar D-mannose/D-glucose specificity, these lectins interacted with lipopolysaccharides and exopolysaccharides of Rhizobium leguminosarum bv. viciae, root nodule bacteria that infect broad-bean, pea, common-vetch, and lentil plants with the formation of nitrogen-fixing symbiosis. However, owing to individual distinctions of carbohydrate-binding properties, these lectins showed a higher affinity for the polysaccharides of those microsymbionts within the R. leguminosarum bv. viciae species that were better specialized towards one or the other host plant from the cross inoculation group of legumes. 相似文献