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1.
Substrate specificity of trypsin investigated by using a genetic selection. 总被引:4,自引:5,他引:4
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L B Evnin J R Vsquez C S Craik 《Proceedings of the National Academy of Sciences of the United States of America》1990,87(17):6659-6663
The structural determinants of the primary substrate specificity of rat anionic trypsin were examined by using oligonucleotide-directed mutagenesis coupled to a genetic selection. A library was created that encoded trypsins substituted at amino acid positions 189 and 190 at the base of the substrate binding pocket. A genetic selection, with a dynamic range of 5 orders of proteolytic activity, was used to search 90,000 transformants of the library. Rapid screening for arginyl amidolysis and esterolysis confirmed the activity of the purified isolates. Trypsin and 15 mutant trypsins with partially preserved function were identified and characterized kinetically on arginyl and lysyl peptide substrates. Alternative arrangements of amino acids in the substrate binding pocket sustained efficient catalysis. A negative charge at amino acid position 189 or 190 was shown to be essential for high-level catalysis. With the favored aspartic acid residue at position 189, several amino acids could replace serine at position 190. Modulation of the specificity for arginine and lysine substrates was shown to depend on the amino acid at position 190. The regulatory effect of the amino acid side chain at position 190 on the substrate specificity is also reflected in substrate binding pockets of naturally occurring trypsin homologs. 相似文献
2.
Meyerovitch J Balta Y Ziv E Sack J Shafrir E 《International journal of experimental diabetes research》2002,3(3):199-204
Phosphotyrosine phosphatase (PTPase) activity and its regulation by overnight food deprivation were studied in Psammomys obesus (sand rat), a gerbil model of insulin resistance and nutritionally induced diabetes mellitus. PTPase activity was measured using a phosphopeptide substrate containing a sequence identical to that of the major site of insulin receptor (IR) beta-subunit autophosphorylation. The PTPase activity in membrane fractions was 3.5-, 8.3-, and 5.9-fold lower in liver, fat, and skeletal muscle, respectively, compared with corresponding tissues of albino rat. Western blotting of tissue membrane fractions in Psammomys showed lower PTPase and IR than in albino rats. The density of PTPase transmembrane protein band was 5.5-fold lower in liver and 12-fold lower in adipose tissue. Leukocyte antigen receptor (LAR) and IR were determined by specific immunoblotting and protein bands densitometry and were also found to be 6.3-fold lower in the liver and 22-fold lower in the adipose tissue in the hepatic membrane fractions. Liver cytosolic PTPase activity after an overnight food deprivation in the nondiabetic Psammomys rose 3.7-fold compared with postprandial PTPase activity, but it did not change significantly in diabetic fasted animals. Similar fasting-related changes were detected in the activity of PTPase derived from membrane fraction. In conclusion, the above data demonstrate that despite the insulin resistance, Psammomys is characterized by low level of PTPase activities in membrane and cytosolic fractions in all 3 major insulin responsive tissues, as well as in liver. PTPase activity does not rise in activity as a result of insulin resistance and nutritionally induced diabetes. 相似文献
3.
The complex formation between thrombin and hirudin is unique among other serine proteinase-inhibitor complexes. The serpines occupy the specificity pocket of the active site of the target enzyme with an amino acid residue corresponding to the specificity of the enzyme at the P1 site of the substrate. In contrast, the Thr2 residue of hirudin approaches only the entrance of the pocket. The peptide chain of the inhibitors D-Phe-Pro-ArgCH2Cl and NAPAP is antiparallel to the enzyme backbone, whereas the N-terminal amino acids of hirudin run parallel. These unexpected interactions seem to contribute to a greater extent to the tight binding than the ionic interactions of the hirudin tail with the fibrinogen binding site of thrombin. Obviously, these interactions account for the unique selectivity of hirudin for thrombin. 相似文献
4.
The mechanism of substrate (aglycone) specificity in beta -glucosidases is revealed by crystal structures of mutant maize beta -glucosidase-DIMBOA, -DIMBOAGlc, and -dhurrin complexes
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Czjzek M Cicek M Zamboni V Bevan DR Henrissat B Esen A 《Proceedings of the National Academy of Sciences of the United States of America》2000,97(25):13555-13560
The mechanism and the site of substrate (i.e., aglycone) recognition and specificity were investigated in maize beta-glucosidase (Glu1) by x-ray crystallography by using crystals of a catalytically inactive mutant (Glu1E191D) in complex with the natural substrate 2-O-beta-d-glucopyranosyl-4-hydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOAGlc), the free aglycone DIMBOA, and competitive inhibitor para-hydroxy-S-mandelonitrile beta-glucoside (dhurrin). The structures of these complexes and of the free enzyme were solved at 2.1-, 2.1-, 2.0-, and 2.2-A resolution, respectively. The structural data from the complexes allowed us to visualize an intact substrate, free aglycone, or a competitive inhibitor in the slot-like active site of a beta-glucosidase. These data show that the aglycone moiety of the substrate is sandwiched between W378 on one side and F198, F205, and F466 on the other. Thus, specific conformations of these four hydrophobic amino acids and the shape of the aglycone-binding site they form determine aglycone recognition and substrate specificity in Glu1. In addition to these four residues, A467 interacts with the 7-methoxy group of DIMBOA. All residues but W378 are variable among beta-glucosidases that differ in substrate specificity, supporting the conclusion that these sites are the basis of aglycone recognition and binding (i.e., substrate specificity) in beta-glucosidases. The data also provide a plausible explanation for the competitive binding of dhurrin to maize beta-glucosidases with high affinity without being hydrolyzed. 相似文献
5.
The nitrite reductase from Pseudomonas aeruginosa: essential role of two active-site histidines in the catalytic and structural properties
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Cutruzzola F Brown K Wilson EK Bellelli A Arese M Tegoni M Cambillau C Brunori M 《Proceedings of the National Academy of Sciences of the United States of America》2001,98(5):2232-2237
Cd(1) nitrite reductase catalyzes the conversion of nitrite to NO in denitrifying bacteria. Reduction of the substrate occurs at the d(1)-heme site, which faces on the distal side some residues thought to be essential for substrate binding and catalysis. We report the results obtained by mutating to Ala the two invariant active site histidines, His-327 and His-369, of the enzyme from Pseudomonas aeruginosa. Both mutants have lost nitrite reductase activity but maintain the ability to reduce O(2) to water. Nitrite reductase activity is impaired because of the accumulation of a catalytically inactive form, possibly because the productive displacement of NO from the ferric d(1)-heme iron is impaired. Moreover, the two distal His play different roles in catalysis; His-369 is absolutely essential for the stability of the Michaelis complex. The structures of both mutants show (i) the new side chain in the active site, (ii) a loss of density of Tyr-10, which slipped away with the N-terminal arm, and (iii) a large topological change in the whole c-heme domain, which is displaced 20 A from the position occupied in the wild-type enzyme. We conclude that the two invariant His play a crucial role in the activity and the structural organization of cd(1) nitrite reductase from P. aeruginosa. 相似文献
6.
7.
Kristan K Stojan J Möller G Adamski J Rizner TL 《Molecular and cellular endocrinology》2005,241(1-2):80-87
The 17beta-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus is an NADP(H)-dependent member of the short-chain dehydrogenase/reductase superfamily (SDR) that belongs to the cP1 classical subfamily. Here, we have created several mutants by site-directed mutagenesis, and through these we have studied the amino acid residues that are responsible for coenzyme binding and specificity. The Thr202Val and Thr202Ile mutants were inactive, thus confirming the importance of Thr202 for the appropriate orientation of the coenzyme that enables the hydride transfer. The Ala50Arg and Asn51Arg mutants had increased rates of NADPH dissociation, and thus an enhanced substrate oxidation with NADP+, while the Asn51Arg mutant also showed an increased rate of NADP+ dissociation, and thus an enhanced substrate reduction with NADPH. Addition of a negatively-charged amino acid residue at the first position after the second beta-strand (Tyr49Asp) affected the coenzyme specificity and turned the enzyme into an NAD+-dependent oxidase resembling the cD1d subfamily members. 相似文献
8.
To explore the sequence context-dependent nature of the human immunodeficiency virus type 1 (HIV-1) protease’s specificity and to provide a rationale for viral mutagenesis to study the potential role of the nucleocapsid (NC) processing in HIV-1 replication, synthetic oligopeptide substrates representing the wild-type and modified versions of the proximal cleavage site of HIV-1 NC were assayed as substrates of the HIV-1 protease (PR). The S1′ substrate binding site of HIV-1 PR was studied by an in vitro assay using KIVKCF↓NCGK decapeptides having amino acid substitutions of N17 residue of the cleavage site of the first zinc-finger domain, and in silico calculations were also performed to investigate amino acid preferences of S1′ site. Second site substitutions have also been designed to produce “revertant” substrates and convert a non-hydrolysable sequence (having glycine in place of N17) to a substrate. The specificity constants obtained for peptides containing non-charged P1′ substitutions correlated well with the residue volume, while the correlation with the calculated interaction energies showed the importance of hydrophobicity: interaction energies with polar residues were related to substantially lower specificity constants. Cleavable “revertants” showed one residue shift of cleavage position due to an alternative productive binding mode, and surprisingly, a double cleavage of a substrate was also observed. The results revealed the importance of alternative binding possibilities of substrates into the HIV-1 PR. The introduction of the “revertant” mutations into infectious virus clones may provide further insights into the potential role of NC processing in the early phase of the viral life-cycle. 相似文献
9.
10.
Evidence that autophosphorylation of solubilized receptors for epidermal growth factor is mediated by intermolecular cross-phosphorylation. 总被引:24,自引:4,他引:24
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A M Honegger R M Kris A Ullrich J Schlessinger 《Proceedings of the National Academy of Sciences of the United States of America》1989,86(3):925-929
Structurally distinguishable mutants of human epidermal growth factor receptor (EGFR) were used to investigate the mechanism of EGFR autophosphorylation. Mutant receptors generated by site-directed mutagenesis were expressed in transfected NIH 3T3 cells lacking endogenous receptors. After coincubation of cell lysates in the presence or absence of EGF, receptor immunoprecipitates were incubated with [gamma-32P]ATP. A kinase-negative mutant EGFR (K721A), in which Lys-721 in the ATp binding site was replaced by an alanine residue, was shown to be phosphorylated in an EGF-dependent manner by an enzymatically active EGFR deletion mutant lacking two autophosphorylation sites. A mutant EGFR lacking the EGF-binding domain as well as the phosphorylation sites also phosphorylated the kinase-negative mutant. In both cases the kinase-negative mutant K721A was phosphorylated on sites virtually identical to the sites that are autophosphorylated by wild-type recombinant or native human EGFRs. With four different site-specific anti-EGFR antibodies, it was shown that deletion mutants devoid of epitopes recognized by the antibodies were coimmunoprecipitated together with wild-type or mutant receptors recognized by the antibodies. This indicates that EGFR oligomers were preserved during immunoprecipitation. On the basis of these results, we propose that autophosphorylation of solubilized EGFR is mediated by intermolecular cross-phosphorylation, probably facilitated by receptor oligomerization. 相似文献
11.
Epidermal growth factor-receptor mutant lacking the autophosphorylation sites induces phosphorylation of Shc protein and Shc-Grb2/ASH association and retains mitogenic activity. 总被引:13,自引:3,他引:13
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N Gotoh A Tojo K Muroya Y Hashimoto S Hattori S Nakamura T Takenawa Y Yazaki M Shibuya 《Proceedings of the National Academy of Sciences of the United States of America》1994,91(1):167-171
Epidermal growth factor (EGF) receptor (EGFR) can induce cell growth and transformation in a ligand-dependent manner. To examine whether the autophosphorylation of EGFR correlates with the capacity of the activated EGFR to induce cell growth and transformation, we truncated the human EGFR just after residue 1011, removing all three major autophosphorylation sites (DEL1011). Further, a point mutation was introduced at another autophosphorylation site, Tyr-992-->Phe (DEL1011+F992). The wild-type and mutant receptors were stably expressed in a NIH 3T3 variant cell line that expresses an extremely low level of endogenous EGFR and does not grow with EGF. As expected, DEL1011 and DEL1011+F992 were found to be severely impaired in EGF-induced autophosphorylation, due to the deletion of the appropriate target tyrosines. However, mutant receptors still could induce EGF-dependent DNA synthesis, morphological transformation, and anchorage-independent growth, although the extent of these was significantly reduced when compared with wild-type EGFR. EGF-induced tyrosine phosphorylation of Ras-GTPase activating protein-associated protein p62 and phospholipase C gamma 1 was dramatically reduced in the cells expressing DEL1011 and DEL1011+F992. On the other hand, tyrosine phosphorylation of Shc, complex formation of Shc-Grb2/Ash, and activation of microtubule-associated protein kinase were still fully induced upon EGF stimulation without binding of Shc or Grb2/Ash to the mutant receptor. Thus, tyrosine phosphorylation of Shc may play a crucial role for activating Ras and generating mitotic signals by the activated EGFR mutant. 相似文献
12.
Synthetic peptides as substrates and inhibitors of a retroviral protease. 总被引:13,自引:2,他引:13
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M Kotler R A Katz W Danho J Leis A M Skalka 《Proceedings of the National Academy of Sciences of the United States of America》1988,85(12):4185-4189
Processing of the gag and pol gene precursor proteins of retroviruses is essential for infectivity and is directed by a viral protease that is itself included in one of these precursors. We demonstrate here that small synthetic peptides can be used as both model substrates and inhibitors to investigate the specificity and molecular parameters of the reaction. The results indicate that a peptide that extends five amino acids but not three amino acids in both directions from a known cleavage site is accurately hydrolyzed by the protease of avian sarcoma-leukosis virus. Substitutions of the amino acids to either side of the peptide bond to be cleaved affect the ability of the peptide (as well as a larger precursor protein) to serve as a substrate. The specificity is more stringent for the amino acid that will become the carboxyl end after cleavage. Some substitutions produced peptides that were not cleaved but could act as inhibitors of cleavage of a susceptible peptide. Thus, small model substrates may be used to explore both the binding and catalytic properties of these important proteases. 相似文献
13.
Effect of amino acid substitutions on the catalytic and regulatory properties of aspartate transcarbamoylase. 总被引:5,自引:3,他引:5
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E A Robey S R Wente D W Markby A Flint Y R Yang H K Schachman 《Proceedings of the National Academy of Sciences of the United States of America》1986,83(16):5934-5938
Although intensive investigations have been conducted on the allosteric enzyme, aspartate transcarbamoylase, which catalyzes the first committed reaction in the biosynthesis of pyrimidines in Escherichia coli, little is known about the role of individual amino acid residues in catalysis or regulation. Two inactive enzymes produced by random mutagenesis have been characterized previously but the loss of activity is probably attributable to changes in the folding of the chains stemming from the introduction of charged and bulky residues (Asp for Gly-128 and Phe for Ser-52). Site-directed mutagenesis of pyrB, which encodes the catalytic chains of the enzyme, was used to probe the functional roles of several amino acids by making more conservative substitutions. Replacement of Lys-84 by either Gln or Arg leads to virtually inactive enzymes, confirming chemical studies indicating that Lys-84 is essential for catalysis. In contrast, substitution of Gln for Lys-83 has only a slight effect on enzyme activity, whereas chemical modification causes considerable inactivation. Gln-133, which has been shown by x-ray crystallography to reside near the contact region between the catalytic and regulatory chains, was replaced by Ala. This substitution has little effect on catalytic activity but leads to a marked increase in cooperativity. The Gln-83 mutant, in contrast, exhibits much less cooperativity. Since a histidine residue may be involved in catalysis and His-134 has been shown by x-ray diffraction studies to be in close proximity to the site of binding of a bisubstrate analog, His-134 was replaced by Ala, yielding a mutant with only 5% wild-type activity, considerable cooperativity, and lower affinity for aspartate and carbamoylphosphate. All of the mutants, unlike those in which charged or bulky residues replaced small side chains, bind the bisubstrate analog, which promotes the characteristic "swelling" of the enzymes indicative of the allosteric transition. 相似文献
14.
Human aspartic protease memapsin 2 cleaves the beta-secretase site of beta-amyloid precursor protein 总被引:3,自引:0,他引:3
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Lin X Koelsch G Wu S Downs D Dashti A Tang J 《Proceedings of the National Academy of Sciences of the United States of America》2000,97(4):1456-1460
The cDNAs of two new human membrane-associated aspartic proteases, memapsin 1 and memapsin 2, have been cloned and sequenced. The deduced amino acid sequences show that each contains the typical pre, pro, and aspartic protease regions, but each also has a C-terminal extension of over 80 residues, which includes a single transmembrane domain and a C-terminal cytosolic domain. Memapsin 2 mRNA is abundant in human brain. The protease domain of memapsin 2 cDNA was expressed in Escherichia coli and was purified. Recombinant memapsin 2 specifically hydrolyzed peptides derived from the beta-secretase site of both the wild-type and Swedish mutant beta-amyloid precursor protein (APP) with over 60-fold increase of catalytic efficiency for the latter. Expression of APP and memapsin 2 in HeLa cells showed that memapsin 2 cleaved the beta-secretase site of APP intracellularly. These and other results suggest that memapsin 2 fits all of the criteria of beta-secretase, which catalyzes the rate-limiting step of the in vivo production of the beta-amyloid (Abeta) peptide leading to the progression of Alzheimer's disease. Recombinant memapsin 2 also cleaved a peptide derived from the processing site of presenilin 1, albeit with poor kinetic efficiency. Alignment of cleavage site sequences of peptides indicates that the specificity of memapsin 2 resides mainly at the S(1)' subsite, which prefers small side chains such as Ala, Ser, and Asp. 相似文献
15.
A single active site residue directs oxygenation stereospecificity in lipoxygenases: stereocontrol is linked to the position of oxygenation
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Coffa G Brash AR 《Proceedings of the National Academy of Sciences of the United States of America》2004,101(44):15579-15584
Lipoxygenases are a class of dioxygenases that form hydroperoxy fatty acids with distinct positional and stereo configurations. Several amino acid residues influencing regiospecificity have been identified, whereas the basis of stereocontrol is not understood. We have now identified a single residue in the lipoxygenase catalytic domain that is important for stereocontrol; it is conserved as an Ala in S lipoxygenases and a Gly in R lipoxygenases. Our results with mutation of the conserved Ala to Gly in two S lipoxygenases (mouse 8S-LOX and human 15-LOX-2) and the corresponding Gly-Ala substitution in two R lipoxygenases (human 12R-LOX and coral 8R-LOX) reveal that the basis for R or S stereo-control also involves a switch in the position of oxygenation on the substrate. After the initial hydrogen abstraction, antarafacial oxygenation at one end or the other of the activated pair of double bonds (pentadiene) gives, for example, 8S or 12R product. The Ala residue promotes oxygenation on the reactive pentadiene at the end deep in the substrate binding pocket and S stereochemistry of the product hydroperoxide, and a Gly residue promotes oxygenation at the proximal end of the reactive pentadiene resulting in R stereochemistry. A model of lipoxygenase reaction specificity is proposed in which product regiochemistry and stereochemistry are determined by fixed relationships between substrate orientation, hydrogen abstraction, and the Gly or Ala residue we have identified. 相似文献
16.
Different sites of acivicin binding and inactivation of gamma-glutamyl transpeptidases. 总被引:2,自引:0,他引:2
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T K Smith Y Ikeda J Fujii N Taniguchi A Meister 《Proceedings of the National Academy of Sciences of the United States of America》1995,92(6):2360-2364
Acivicin is a potent inhibitor of gamma-glutamyl transpeptidase (EC 2.3.2.2), an enzyme of importance in glutathione metabolism. Acivicin inhibition and binding are prevented by gamma-glutamyl substrates and analogs (e.g., serine plus borate), consistent with the previous postulate that acivicin and substrates bind to the same enzyme site. Inactivation of rat kidney transpeptidase by acivicin leads to its binding as an ester to Thr-523. The pig enzyme, which has Ala-523 in place of Thr-523, is inhibited by acivicin with esterification at Ser-405. The human enzyme has Thr-524 (corresponding to Thr-523 in rat); its inactivation leads to esterification of Ser-406 (corresponding to Ser-405 in rat and pig). Hydroxylamine treatment of the acivicin-inactivated enzymes restores activity and releases the acivicin-derived threo-beta-hydroxyglutamate moiety. The findings indicate that there are significant structural differences between the active site region of the rat enzyme and the active site regions of the human and pig. Human mutant enzymes in which Thr-524 and Ser-406 were replaced by Ala, separately and together, are enzymatically active, indicating that these amino acid residues are not required for catalysis. However, esterification of these residues (and of another near the active site) effectively blocks the active site or hinders its function. Acivicin can bind at enzyme sites that are close to that at which gamma-glutamylation occurs; it may bind at the latter site and then be transesterified to another enzyme site. 相似文献
17.
Irene M. Voynick Joseph S. Fruton 《Proceedings of the National Academy of Sciences of the United States of America》1971,68(2):257-259
Examination of the kinetic parameters for the hydrolysis, by acid proteinases, of a single peptide bond (between p-nitro-L-phenylalanyl and L-phenylalanyl) in a series of oligopeptides has shown that secondary interactions are important factors in determining the catalytic efficiency. Comparison of the action of highly purified pepsinlike enzymes (Rhizopus proteinase, Mucor proteinase, rennin) with that of swine pepsin A indicates significant differences among them, either in the binding of the substrate (as estimated by K(m)), or in the catalytic efficiency (as measured by k(cat)), or both. It may be concluded from these data that, in their action on oligopeptide substrates, the specificity of proteinases operating by a similar catalytic mechanism cannot be explained solely in terms of the amino acid residues flanking the sensitive peptide bond; in addition, the specificity includes significant contributions from secondary interactions arising from complementary relations between parts of the substrate and of the enzyme at a distance from the catalytic site. Data are also presented for the effect of urea (about 1 M) on the kinetic parameters of several acid proteinases; under the conditions of these studies, the binding of the substrate is affected to a much lesser degree than is the catalytic efficiency. 相似文献
18.
Identification of residues that control specific binding of the Shc phosphotyrosine-binding domain to phosphotyrosine sites. 总被引:9,自引:1,他引:9
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P van der Geer S Wiley G D Gish V K Lai R Stephens M F White D Kaplan T Pawson 《Proceedings of the National Academy of Sciences of the United States of America》1996,93(3):963-968
The Shc adaptor protein contains two phosphotyrosine [Tyr(P)]binding modules--an N-terminal Tyr(P) binding (PTB) domain and a C-terminal Src homology 2 (SH2) domain. We have compared the ability of the Shc PTB domain to bind the receptors for nerve growth factor and insulin, both of which contain juxtamembrane Asn-Pro-Xaa-Tyr(P) motifs implicated in PTB binding. The Shc PTB domain binds with high affinity to a phosphopeptide corresponding to the nerve growth factor receptor Tyr-490 autophosphorylation site. Analysis of individual residues within this motif indicates that the Asn at position -3 [with respect to Tyr(P)], in addition to Tyr(P), is critical for PTB binding, while the Pro at position -2 plays a less significant role. A hydrophobic amino acid 5 residues N-terminal to the Tyr(P) is also essential for high-affinity binding. In contrast, the Shc PTB domain does not bind stably to the Asn-Pro-Xaa-Tyr(P) site at Tyr-960 in the activated insulin receptor, which has a polar residue (Ser) at position -5. Substitution of this Ser at position -5 with Ile markedly increased binding of the insulin receptor Tyr-960 phosphopeptide to the PTB domain. These results suggest that while the Shc PTB domain recognizes a core sequence of Asn-Pro-Xaa-Tyr(P), its binding affinity is modulated by more N-terminal residues in the ligand, which therefore contribute to the specificity of PTB-receptor interactions. An analysis of residues in the Shc PTB domain required for binding to Tyr(P) sites identified a specific and evolutionarily conserved Arg (Arg-175) that is uniquely important for ligand binding and is potentially involved in Tyr(P) recognition. 相似文献
19.
Src homology 2 domain as a specificity determinant in the c-Abl-mediated tyrosine phosphorylation of the RNA polymerase II carboxyl-terminal repeated domain. 总被引:10,自引:1,他引:10
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J Duyster R Baskaran J Y Wang 《Proceedings of the National Academy of Sciences of the United States of America》1995,92(5):1555-1559
The Src-homology (SH) 2 domain, found in a variety of proteins, has a binding site for phosphotyrosine-containing peptides. In adaptor proteins such as Grb2, the SH2 domain plays an important role in the assembly of signal transducer complexes. Many nonreceptor tyrosine kinases--e.g., Abl and Src--also contain SH2 domains. Without a functional SH2 domain, these tyrosine kinases retain catalytic activity but lose their biological function. This result suggests that the SH2 domain may be involved in the selection of biologically relevant substrates. We have previously shown that the carboxyl-terminal repeated domain (CTD) of the mammalian RNA polymerase II is a substrate for the Abl but not the Src tyrosine kinase. This specificity is conferred in part by the SH2 domain. The Abl SH2 domain binds the tyrosine-phosphorylated [Tyr(P)] CTD and is required for the processive and stoichiometric phosphorylation of the 52 tyrosines in the CTD. Mutation of the Abl SH2 or exchanging it with that of Src, which does not bind the Tyr(P)-CTD, abolished processivity and reduced the CTD kinase activity without any effect on autophosphorylation or the phosphorylation of nonspecific substrates. These results demonstrate that the SH2 domain of the Abl tyrosine kinase plays an active role in catalysis and suggests that SH2 domain and the tyrosine kinase domain may act in concert to confer substrate specificity. 相似文献
20.
Crystal structures of two tropinone reductases: Different reaction stereospecificities in the same protein fold 总被引:4,自引:0,他引:4
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Keiji Nakajima Atsuko Yamashita Hiroyuki Akama Toru Nakatsu Hiroaki Kato Takashi Hashimoto Junichi Oda Yasuyuki Yamada 《Proceedings of the National Academy of Sciences of the United States of America》1998,95(9):4876-4881
A pair of tropinone reductases (TRs) share 64% of the same amino acid residues and belong to the short-chain dehydrogenase/reductase family. In the synthesis of tropane alkaloids in several medicinal plants, the TRs reduce a carbonyl group of an alkaloid intermediate, tropinone, to hydroxy groups with different diastereomeric configurations. To clarify the structural basis for their different reaction stereospecificities, we determined the crystal structures of the two enzymes at 2.4- and 2.3-Å resolutions. The overall folding of the two enzymes was almost identical. The conservation was not confined within the core domains that are conserved within the protein family but extended outside the core domain where each family member has its characteristic structure. The binding sites for the cofactor and the positions of the active site residues were well conserved between the two TRs. The substrate binding site was composed mostly of hydrophobic amino acids in both TRs, but the presence of different charged residues conferred different electrostatic environments on the two enzymes. A modeling study indicated that these charged residues play a major role in controlling the binding orientation of tropinone within the substrate binding site, thereby determining the stereospecificity of the reaction product. The results obtained herein raise the possibility that in certain cases different stereospecificities can be acquired in enzymes by changing a few amino acid residues within substrate binding sites. 相似文献