共查询到20条相似文献,搜索用时 15 毫秒
1.
2.
Misteli T 《Nature cell biology》2000,2(6):E98-E100
The MKK3/6-p38 pathway has been found to induce the relocalization of premessenger-RNA splicing factors from the nucleus to the cytoplasm. This represents the first physiological mechanism that alters the nuclear ratios of splicing factors and modulates alternative splice-site choice in vivo. 相似文献
3.
The nucleic acids of Escherichia coli cells were uniformly labelled with 32P by growing the cells in [32P]orthophosphoric acid for about four generations. The cells were harvested in the logarithmic phase, resuspended in a buffer containing 6 mM Mg2+, 150 mM NH4+ and polyamines and incubated for 3 min at 37 degrees C in the presence of 3H-labelled amino acids. This procedure preferentially labels growing peptidyl chains. Polysomes were isolated, the fraction in the post-translocational state was assessed by a puromycin reaction and the tRNA content/70S ribosome was quantified in comparison to the amount of 5S rRNA determined after separation by gel electrophoresis. The data revealed that at least 75% of post-translocational ribosomes in isolated native polysomes carry a tRNA in their E site. The results are consistent with the allosteric three-site model for the elongation cycle but disagree with the two-site model. 相似文献
4.
5.
The active site of myo-inositol dehydrogenase (IDH, EC 1.1.1.18) from Bacillus subtilis recognizes a variety of mono- and disaccharides, as well as 1l-4-O-substituted inositol derivatives. It catalyzes the NAD+-dependent oxidation of the axial alcohol of these substrates with comparable kinetic constants. We have found that 4-O-p-toluenesulfonyl-myo-inositol does not act as a substrate for IDH, in contrast to structurally similar compounds such as those bearing substituted benzyl substituents in the same position. X-ray crystallographic analysis of 4-O-p-toluenesulfonyl-myo-inositol and 4-O-(2-naphthyl)methyl-myo-inositol, which is a substrate for IDH, shows a distinct difference in the preferred conformation of the aryl substituent. Conformational analysis of known substrates of IDH suggests that this conformational difference may account for the difference in reactivity of 4-O-p-toluenesulfonyl-myo-inositol in the presence of IDH. A sequence alignment of IDH with the homologous glucose-fructose oxidoreductase allowed the construction of an homology model of inositol dehydrogenase, to which NADH and 4-O-benzyl-scyllo-inosose were docked and the active site energy minimized. The active site model is consistent with all experimental results and suggests that a conserved tyrosine-glycine-tyrosine motif forms the hydrophobic pocket adjoining the site of inositol recognition. Y233F and Y235F retain activity, while Y233R and Y235R do not. A histidine-aspartate pair, H176 and D172, are proposed to act as a dyad in which H176 is the active site acid/base. The enzyme is inactivated by diethyl pyrocarbonate, and the mutants H176A and D172N show a marked loss of activity. Kinetic isotope effect experiments with D172N indicate that chemistry is rate-determining for this mutant. 相似文献
6.
Antithrombin-III Denver, a reactive site variant 总被引:6,自引:0,他引:6
Antithrombin-III Denver is a mutant protein which differs from the normal in being defective in serine protease binding (Sambrano, J. E., Jacobson, L. J., Reeve, E. B., Manco-Johnson, M. J., and Hathaway, W. E. (1986) J. Clin. Invest. 77, 887-893). It was isolated from the blood of an individual heterozygous for the abnormal gene by: affinity separation on heparin-Sepharose to obtain an antithrombin fraction, and gel filtration of the species present following complexing of the antithrombin fraction with a small excess of thrombin. The reduced, S-carboxymethylated protein formed a mixture of soluble tryptic peptides which was fractionated on Vydac C18. A single, unique peptide not present in a parallel experiment with normal antithrombin-III was isolated. This peptide was identified by sequence analysis and synthesis to correspond to residues 394-399 in the known sequence of the inhibitor, with leucine replacing reactive site P'1 residue Ser394. Although chromatograms of the tryptic peptides from the normal and mutant proteins were otherwise indistinguishable, the existence of additional residue replacements is not excluded. Measurements of the rate of thrombin binding by the mutant protein with p-aminobenzamidine as a fluorescent indicator showed that the second-order rate constant is reduced drastically. Meaningful measurements with the mutant protein could only be made in the presence of heparin and revealed a reduction of about 4000-fold in the rate constant. 相似文献
7.
Discovery of the ammonium substrate site on glutamine synthetase, a third cation binding site. 总被引:2,自引:0,他引:2
下载免费PDF全文
![点击此处可从《Protein science : a publication of the Protein Society》网站下载免费的PDF全文](/ch/ext_images/free.gif)
S. H. Liaw I. Kuo D. Eisenberg 《Protein science : a publication of the Protein Society》1995,4(11):2358-2365
Glutamine synthetase (GS) catalyzes the ATP-dependent condensation of ammonia and glutamate to yield glutamine, ADP, and inorganic phosphate in the presence of divalent cations. Bacterial GS is an enzyme of 12 identical subunits, arranged in two rings of 6, with the active site between each pair of subunits in a ring. In earlier work, we have reported the locations within the funnel-shaped active site of the substrates glutamate and ATP and of the two divalent cations, but the site for ammonia (or ammonium) has remained elusive. Here we report the discovery by X-ray crystallography of a binding site on GS for monovalent cations, Tl+ and Cs+, which is probably the binding site for the substrate ammonium ion. Fourier difference maps show the following. (1) Tl+ and Cs+ bind at essentially the same site, with ligands being Glu 212, Tyr 179, Asp 50', Ser 53' of the adjacent subunit, and the substrate glutamate. From its position adjacent to the substrate glutamate and the cofactor ADP, we propose that this monovalent cation site is the substrate ammonium ion binding site. This proposal is supported by enzyme kinetics. Our kinetic measurements show that Tl+, Cs+, and NH4+ are competitive inhibitors to NH2OH in the gamma-glutamyl transfer reaction. (2) GS is a trimetallic enzyme containing two divalent cation sites (n1, n2) and one monovalent cation site per subunit. These three closely spaced ions are all at the active site: the distance between n1 and n2 is 6 A, between n1 and Tl+ is 4 A, and between n2 and Tl+ is 7 A. Glu 212 and the substrate glutamate are bridging ligands for the n1 ion and Tl+. (3) The presence of a monovalent cation in this site may enhance the structural stability of GS, because of its effect of balancing the negative charges of the substrate glutamate and its ligands and because of strengthening the "side-to-side" intersubunit interaction through the cation-protein bonding. (4) The presence of the cofactor ADP increases the Tl+ binding to GS because ADP binding induces movement of Asp 50' toward this monovalent cation site, essentially forming the site. This observation supports a two-step mechanism with ordered substrate binding: ATP first binds to GS, then Glu binds and attacks ATP to form gamma-glutamyl phosphate and ADP, which complete the ammonium binding site. The third substrate, an ammonium ion, then binds to GS, and then loses a proton to form the more active species ammonia, which attacks the gamma-glutamyl phosphate to yield Gln. (5) Because the products (Glu or Gln) of the reactions catalyzed by GS are determined by the molecule (water or ammonium) attacking the intermediate gamma-glutamyl phosphate, this negatively charged ammonium binding pocket has been designed naturally for high affinity of ammonium to GS, permitting glutamine synthesis to proceed in aqueous solution. 相似文献
8.
The E site (exit site for deacyl-tRNA) has been shown to be allosterically linked to the A site (aminoacyl-tRNA binding site), in that occupation of the E site reduces the affinity of the A site, and vice versa, whereas the intervening peptidyl-tRNA binding site (P site) keeps its high affinity. Here the question is analysed of whether or not the low affinity state of the A site caused by an occupied E site is of importance for the ribosomal accuracy of the aminoacyl-tRNA selection. In a poly(U) dependent system with high accuracy in poly(Phe) synthesis, the acceptance of the cognate ternary complex Phe-tRNA--EF-Tu--GTP (which has the correct anticodon with respect to the codon at the A site) was compared with the competing acceptance of ternary complexes with near-cognate Leu-tRNA(Leu) (which has a similar anticodon) or non-cognate Asp-tRNA(Asp) (which has a dissimilar anticodon), by monitoring the formation of AcPhePhe, AcPheLeu or AcPheAsp, respectively. Cognate (but not near-cognate) occupation of the E site reduced synthesis of the 'wrong' dipeptide AcPheLeu only marginally relative to that of the cognate AcPhe2, whereas the formation of AcPheAsp was decreased as much as 14-fold, thereby reducing it to the background level. It follows that the allosteric interplay between E and A sites, i.e. the low affinity of the A site induced by the occupation of the E site, excludes the interference of non-cognate complexes in the decoding process and thus reduces the number of aminoacyl-tRNA species competing for A site binding by an order of magnitude.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
9.
10.
WWOX is a gene that spans an extremely large chromosomal region. It is derived from within chromosomal band 16q23.2 which is a region with frequent deletions and other alterations in a variety of different cancers. This chromosomal band also contains the FRA16D common fragile site (CFS). CFSs are chromosomal regions found in all individuals which are highly unstable. WWOX has also been demonstrated to function as a tumor suppressor that is involved in the development of many cancers. Two other highly unstable CFSs, FRA3B (3p14.2) and FRA6E (6q26), also span extremely large genes, FHIT and PARK2, respectively, and these two genes are also found to be important tumor suppressors. There are a number of interesting similarities between these three large CFS genes. In spite of the fact that they are derived from some of the most unstable chromosomal regions in the genome, they are found to be highly evolutionarily conserved and the chromosomal region spanning the mouse homologs of both WWOX and FHIT are also CFSs in mice. Many of the other CFSs also span extremely large genes and many of these are very attractive tumor suppressor candidates. WWOX is therefore a member of a very interesting family of very large CFS genes. 相似文献
11.
Lumisomes, the cellular site of bioluminescence in coelenterates 总被引:3,自引:0,他引:3
12.
Alignment of the heparin-activated serpins indicates the presence of two binding sites for heparin: a small high-affinity site on the D-helix corresponding in size to the minimal pentasaccharide heparin, and a longer contiguous low-affinity site extending to the reactive center pole of the molecule. Studies of the complexing of antithrombin and its variants with heparin fractions and with reactive center loop peptides including intermolecular loop-sheet polymers all support a 3-fold mechanism for the heparin activation of antithrombin. Binding to the pentasaccharide site induces a conformational change as measured by circular dichroism. Accompanying this, the reactive center becomes more accessible to proteolytic cleavage and there is a 100-fold increase in the kass for factor Xa but only a 10-fold increase for thrombin, to 6.4 x 10(4) M-1 s-1. To obtain a 100-fold increase in the kass for thrombin requires in addition a 4:1 molar ratio of disaccharide to neutralize the charge on the extended low-affinity site. Full activation requires longer heparin chains in order to stabilize the ternary complex between antithrombin and thrombin. Thus, addition of low-affinity but high molecular weight heparin in conjunction with pentasaccharide gives an overall kass of 2.7 x 10(6) M-1 s-1, close to that of maximal heparin activation. 相似文献
13.
Structural analysis of recombinant fibrinogen fragment D revealed that the calcium-binding site (beta2-site) composed of residues BbetaAsp261, BbetaAsp398, BbetaGly263, and gammaGlu132 is modulated by the "B:b" interaction. To determine the beta2-site's role in polymerization, we engineered variant fibrinogen gammaE132A in which calcium binding to the beta2-site was disrupted by replacing glutamic acid at gamma132 with alanine. We compared polymerization of gammaE132A to normal fibrinogen as a function of calcium concentration. Polymerization of gammaE132A at concentrations of calcium 相似文献
14.
Synthesis, purification, and active site mutagenesis of recombinant porcine pepsinogen 总被引:7,自引:0,他引:7
In order to carry out studies on structure and function relationships of porcine pepsinogen using site-directed mutagenesis approaches, the cDNA of this zymogen was cloned, sequenced, expressed in Escherichia coli, and the protein refolded, and purified to homogeneity. Porcine pepsinogen cDNA, obtained from a lambda gt10 cDNA library of porcine stomach contains 1364 base pairs. It contains leader, pro, and pepsin regions of 14, 44, and 326 residues, respectively. In addition, it also contains 5'- and 3'-untranslated regions. Four differences are present between the sequence deduced from the cDNA and the pepsinogen sequence determined previously by protein chemistry methods. Residues P19 (in the pro region) and 263 are asparagines in the cDNA sequence instead of aspartic acids. Isoleucine 230 is not present in the cDNA sequence and residue 242 is a tyrosine in the cDNA instead of an aspartic acid. Porcine pepsinogen cDNA was placed under the control of a tac promoter in a plasmid and expressed in E. coli. The synthesis of pepsinogen was optimized to about 50 mg/liter of culture. The recombinant (r-) pepsinogen, which was insoluble, was recovered by centrifugation, washed, dissolved in 6 M urea in Tris-HCl, pH 8, and refolded by rapid dilution. r-pepsinogen was purified to homogeneity after chromatography on Sephacryl S-300 and fast protein liquid chromatography on a monoQ column. r-pepsinogen contains an additional methionine residue at the NH2 terminus as compared to native (n-) pepsinogen. However, r- and n-pepsinogens are indistinguishable in their intramolecular activation constants. After activation, r- and n-pepsins have the same NH2-terminal sequences as well as Km values. Based on these data, r-pepsinogen was judged suitable for mutagenesis studies. A mutant pepsinogen (D32A) with the active site aspartic acid changed to an alanine was produced and purified. D32A-pepsinogen did not convert to pepsin in acid solution but it bound to pepstatin with an apparent KD of about 5 x 10(-10) M. D32A-pepsinogen possesses no detectable proteolytic activity. These results indicate that (i) intramolecular pepsinogen activation is accomplished by the pepsin active site, and (ii) unlike subtilisin (Carter, P., and Wells, J. A. (1988) Nature 332, 564-568), the active site mutant of pepsin is not enzymically active. 相似文献
15.
cosN is the site at which terminase, the DNA packaging enzyme of phage lambda, introduces staggered nicks into viral concatemeric DNA to initiate genome packaging. Although the nick positions and many of the base pairs of cosN show 2-fold rotational symmetry, cosN is functionally asymmetric. That is, the cosN G2C mutation in the left half-site (cosNL) causes a strong virus growth defect whereas the symmetrically disposed cosN C11G mutation in the right half-site (cosNR) does not affect virus growth. The experiments reported here test the proposal that the genetic asymmetry of cosN results from terminase interactions with cosB, a binding site to the right of cosN. In the presence of cosB, the left half-site mutation, cosN G2C, strongly affected the cos cleavage reaction, while the symmetric right half-site mutation, cosN C11G, had little effect. In the absence of cosB, the two mutations moderately reduced the rate of cos cleavage by the same amount. The results indicated that the functional asymmetry of cosNdepends on the presence of cosB. A model is discussed in which terminase-cosN interactions in the nicking complex are assisted by anchoring of terminase to cosB. 相似文献
16.
17.
18.
The OmpR protein of Escherichia coli is a positive regulator involved in activation of the ompF and ompC genes which encode the major outer membrane proteins OmpF and OmpC, respectively. By employing recombinant DNA techniques, we isolated the N- and C-terminal halves of the OmpR molecule. From the results of biochemical analyses of these fragments, it was concluded that the N-terminal portion contains a site involved in phosphorylation by an OmpR-specific protein kinase EnvZ, whereas the C-terminal part possesses a DNA-binding site for the ompC and ompF promoters. 相似文献
19.
20.
先商文化一直是学术界探索的重点,商代文明的诸多特征在先商文化时期已萌芽或得到加强。然而,对这一时期的经济与生业模式,相关研究尚有欠缺。本文对河南安阳鄣邓遗址先商文化时期的大植物遗存进行了分析,结果表明,粟是该时期先民最重要的作物,黍其次;小麦、大豆已被利用,但只是处于辅助地位。这些是先商文化时期农业的最直接证据,与文献记载及考古研究得出的先商农业发展状况基本一致。发轫于河北中部地区的商族最初的生计方式为渔猎畜牧,在南下的过程中逐渐学习并采纳了中原地区的生计方式,这对商族的发展壮大起到了积极的推动作用。 相似文献