site directed mutagenesis and yeast reverse 2 hybrid guided selections to investigate the mechan
Tài liệu Báo cáo khoa học: nsights into the reaction mechanism of glycosyl hydrolase family 49 Site-directed mutagenesis and substrate preference of isopullulanase doc
... 4B) Together with the observations from the site- directed mutagenesis, Trp4 02 of IPU appears to be a residue participating in subsite )1 The residues that form potential subsites )1 and )2 are ... pattern was investigated using TLC The W402F enzyme liberated isopanose from pullulan, and isomaltose and glucose from panose, and the action patterns of the wildtype and the W402F enzymes were ... Primers used for the mutant constructions For PCR mutagenesis (W21F, W32F, W240F and W402F), the same sequences on the opposite strand were also used as described in Materials and methods Lower...
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Tài liệu Báo cáo khoa học: Insights into the interaction of human arginase II with substrate and manganese ions by site-directed mutagenesis and kinetic studies Alteration of substrate specificity by replacement of Asn149 with Asp docx
... Mn2+A and Mn2+B, respectively [9 ,24 ] However, the volume of the active site cleft was found to be larger for arginase II Moreover, the D2 32 (Od1)-Mn2+B FEBS Journal 27 2 (20 05) 4540–4548 ª 20 05 ... inhibitor of the enzyme [9] As indicated by the crystal structure, Mn2+A and Mn2+B are, respectively, coordinated by His 120 and His145, which are equivalent to the histidines at position 101 and 126 ... binding sites The estimated Kd values were 1.8 ± 0 .2 · 10)8 m for the wild-type and His145Asn enzymes and 16 .2 ± 0.5 · 10)8 m for the His 120 Asn variant, whereas the Vmax values were nearly equal to...
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Tài liệu Báo cáo Y học: Insights into the reaction mechanism of Escherichia coli agmatinase by site-directed mutagenesis and molecular modelling ppt
... with the sequences of 1CEV and 1RLA and pasted into the structural alignment The alignment was then corrected by hand Since the sequences of the two arginases and agmatinase greately differ in the ... given to the modeling of the active site The modelled structure was very similar to the template, with respect to the number and arrangement of structural elements (a-helix and b-sheets), and one ... by the conservation of the whole topology of the active site, would explain the essentially unaltered interaction of the D153N variant with the metallic cofactor In the modelled structure, the...
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Báo cáo khoa học: Investigation of the substrate specificity of a b-glycosidase from Spodoptera frugiperda using site-directed mutagenesis and bioenergetics analysis pdf
... [A39fuc]à 2. 2 )9.7 11.8 11.1 8 .2 1.4 )1 .2 4.5 )14.6 4.1 1.1 3.1 1.4 )1 .2 ± ± ± ± ± ± ± 0.1 0.1 0.1 0.1 0 .2 0.1 0 .2 ± ± ± ± ± ± ± 0 .2 0 .2 0.1 0 .2 0 .2 0.1 0 .2 Interactions involving residue 451 In the ... 0.00061 0.0000 82 0.051 0.00 92 0.00067 0.45 1.48 1.7 2. 5 3.1 1.61 6.0 2. 3 2. 4 0.098 0.0079 0.00 024 0.000 026 0.0 321 0.0015 0.00 029 0.35 0.0050 0.00014 0.00093 0.00 028 5 0.0377 0.00038 0.00031 2. 0 3.7 1.7 ... possible to isolate the DGà corresponding to energy of the hydrogen bond between residue 39 3-OH (/3), which was added to the energy of /4e /4a interactions was the and and Results and Discussion The...
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Báo cáo khoa học: Site-directed mutagenesis and footprinting analysis of the interaction of the sunflower KNOX protein HAKN1 with DNA ppt
... more markedly These results agree with the fact that mutations of these two nucleotides abolish binding of HAKN1 to DNA On FEBS Journal 27 2 (20 05) 190 20 2 ª 20 04 FEBS the other hand, because nucleotides ... pattern towards the 3¢ end of the top strand and the 5¢ end of the bottom strand (Fig 4B) This agrees with the presence of a larger residue that makes stable contacts with this region of DNA The ... represent the top and bottom strands of the binding site, respectively A portion of the same fragment digested with defined restriction enzymes was used as a standard to calculate the position of the...
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Báo cáo khoa học: The phosphate site of trehalose phosphorylase from Schizophyllum communeprobed by site-directed mutagenesis and chemical rescue studies pptx
... phosphorylase from 20 21 22 23 24 25 26 27 28 29 30 C Goedl and B Nidetzky Schizophyllum commune examined in steady-state kinetic studies with deoxy and deoxyuoro substrate analogues and inhibitors Biochem ... Propargylamine 2, 2 ,2- Triuoro ethylamine 10.6 10.6 9 .2 8 .2 5.7 46.5 65.0 25 .4 75.8 79.5 )0.64 )0.15 )1.38 )0.43 0 .27 0.61 10 24 0.9 14 6.7 16 39 1.5 23 (24 )b 11 ( 122 )b a V [E] measured in the absence and ... from the NE and NH2 atoms of the guanidine side chain of Arg535 to two oxygen atoms of phosphate dominate the contacts between the enzyme and the phosphate group [3,6] The side chain of Lys540 and...
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Tài liệu Báo cáo khoa học: Identification of critical active-site residues in angiotensin-converting enzyme-2 (ACE2) by site-directed mutagenesis docx
... Journal 27 2 (20 05) 35 12 3 520 ª 20 05 FEBS Fig Schematic view of the active site of ACE2 and tACE Binding interactions of the inhibitor (A) MLN-4760 at the active site of ACE2 and (B) lisinopril at the ... al (20 02) Substrate-based design of the first class of angiotensin-converting enzyme-related 3 520 J L Guy et al 22 23 24 25 26 27 28 29 30 31 carboxypeptidase (ACE2) inhibitors J Am Chem Soc 124 , ... substrate FEBS Journal 27 2 (20 05) 35 12 3 520 ª 20 05 FEBS binding (Arg273 in ACE2 and Gln281, Lys511, Tyr 520 in tACE, which bind the C-terminal carboxy group of the respective inhibitors) and catalysis...
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Báo cáo khoa học: Characterization of HbpR binding by site-directed mutagenesis of its DNA-binding site and by deletion of the effector domain pot
... (in which the mutations had been placed), was impaired both at the 3¢ end of the central regions R 22 and R23, whereas the region R21 and the 5¢ part of R 22 were again protected, as for the wild-type ... DA-XylR, DA-DmpR and DA-TouR [21 ,25 ,27 ] Contrary to XylR, DmpR and TouR derivatives deleted of their A-domain, which activated transcription in the absence of inducer [25 ,26 ,29 ], a similar HbpR ... Meer JR (20 02) Identification and physical characterization of the HbpR binding sites of the hbpC and hbpD promoters J Bacteriol 184, 29 14– 29 24 20 Jaspers MC, Sturme M & van der Meer JR (20 01) Unusual...
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Báo cáo khoa học: Probing the catalytic potential of the hamster arylamine N-acetyltransferase 2 catalytic triad by site-directed mutagenesis of the proximal conserved residue, Tyr190 pot
... with [2- 3H] acetyl-coenzyme A (AcCoA) in the absence of amine [21 ] and the active site Cys68 or Cys69 has been further identied through thiol-specic modication and site- directed mutagenesis [22 24] ... close to D 122 (I 120 , V 121 , A 123 and G 124 ), and residue L69, close to C68, and residue L108, close to H107, were affected Changed chemical shifts of F 125 and F1 92, which form the edge -to- face aromatic ... cavity to shift (Fig S2A) Such shifting is caused by changes in the atoms chemical environment, and the affected residues included those proximal to Y190, such as H107, D 122 , F 125 and F1 92, the...
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Báo cáo khoa học: Functional significance of five noncanonical Ca2+-binding sites of human transglutaminase 2 characterized by site-directed mutagenesis potx
... similarities to one of the TG3 Ca2+-binding sequences In the case of S2 and S3, we generated two separate mutants (S2A and S2B mutants, and S3A and S3B mutants), as here the suspected Ca2+-binding sites ... S2B –5 –10 190 S5 W S2A 20 0 S4 21 0 22 0 23 0 Wavelength (nm) Fig CD spectra of recombinant TG2 proteins 24 0 25 0 As the transglutaminase activity of TG2 is Ca2+dependent, decreased activity of the ... celiac autoantibodies to TG2 is influenced by the presence or absence of Ca2+ in the case of guinea pig TG2 [27 ] Therefore, we also examined the effect of Ca2+ and GDP on the binding of celiac autoantibodies...
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Báo cáo khoa học: Residues affecting the chloride regulation and substrate selectivity of the angiotensin-converting enzymes (ACE and ACE2) identified by site-directed mutagenesis pot
... R 522 Q R186QR489QR 522 Q 16.67 7.14 3.79 7.13 3. 62 2. 72 100.0 42. 8 22 .7 42. 8 21 .7 16.3 Wild-type R169Q W271A K481Q R514Q R169QK481QR514Q 1.98 0.10 0.10 0. 42 1.03 1.05 100.0 5 .2 5.3 21 .0 52. 0 53 .2 ... binds the chloride ion at the CL2 site [22 ] The ionic interaction of this residue with the chloride ion is therefore essential to the chloride sensitivity mediated by the CL2 site Although the CL2 ... Journal 27 5 (20 08) 6033–60 42 ª 20 08 The Authors Journal compilation ª 20 08 FEBS CL1 site A tACE variants W279 /27 1 R489/K481 R 522 Q R489Q W279A R186Q WT Q281/R273 Mock A Chloride regulation of ACE and...
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Báo cáo khoa học: Probing suggested catalytic domains of glycosyltransferases by site-directed mutagenesis Tobias Hefner and Joachim Stockigt ¨ ppt
... (GENE:AT2G2 326 0) (A thaliana, O 221 82) , AT II: putative glucosyltransferase (GENE:AT2G2 325 0) (A thaliana, O 221 83), AT III: putative glucosyltransferase (GENE:AT2G2 321 0) (A thaliana, O 221 86), DB: ... 3.5 28 .9 2. 1 1.0 0.6 1.8 0.8 0.8 0.9 < 0.03 0.6 100 7.3 3.5 2. 1 6 .2 2.8 2. 8 3.1 < 0.1 2. 1 1.0 2. 5 3.4 5.6 1.7 2. 8 2. 6 1.6 > 40 1.7 Ó FEBS 20 03 538 T Hefner and J Stockigt (Eur J Biochem 27 0) ... L86I A204V E368D E368A H360R H360K H360E 440 4 52 706 411 398 – – – 46.5 38.3 12. 3 1.9 6 .2 – – – 20 2.9 180.4 139.1 17.8 24 .0 0 .22 0.17 0.05 24 .1 20 .4 13.1 0.9 1.1 n.d n.d n.d 2. 59 1.84 1.30 0. 12 0.08...
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Báo cáo khoa học: Defining the QP-site of Escherichia coli fumarate reductase by site-directed mutagenesis, fluorescence quench titrations and EPR spectroscopy doc
... ˚ A of the menaquinone (MQ) observed at the QP -site in the structure of FrdABCD: T205, F206, Q 225 and K 228 from FrdB; R28, E29, W86, L89 and A93 from FrdC; and W14, F17, G18, H80, R81 and H84 ... FrdC-A32V FrdC-F38M FrdC-W86R FrdD-H80K FrdD-H84K ND 2. 5 39.0 20 .0 25 .0 2. 5 2. 5 ND 20 .0 3.0 ND 3.54 3.13 1.44 3 .26 2. 97 3 .26 ND 3.61 3.68 NDa 3.48 3 .21 1.39 2. 74 2. 96 3.14 2. 37 3.15 3.09 NDa 3.60 2. 49 ... side of the membrane The other site, the ˚ QD site (the distal Q -site) is located approximately 25 A from the QP site on the opposite (periplasmic) side of the membrane [3,10] The relatively large...
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Báo cáo Y học: Potential active-site residues in polyneuridine aldehyde esterase, a central enzyme of indole alkaloid biosynthesis, by modelling and site-directed mutagenesis ppt
... (lgÆmL)1) Wild-type C257A C213S/G152Q C170A C132A C20A H17A H86A S87A* D216A* H244A* 36 46 33 34.5 108 ND 27 3 ND ND ND ND 32. 4 21 .5 22 .5 4.9 8.1 ND 0 .2 0. 024 ND ND ND 146.7 122 .5 98.56 20 .58 73.0 ND ... M+-1), 335 (17, M+-CH2OH), 24 9 [71, 365-C(CH2OH)CO2CH2CH3], 23 5 (15), 1 82 (24 ), 168 (100), 156 (21 ), 143( 12) , 129 (15), 115 (21 ) Molecular mass determination PNAE was found to migrate on SDS/PAGE ... group The locations of the mutated residues in the model and the kinetic results support the notion that the enzyme has a very similar topology to HbHNL However, to gain more insight into the mechanism...
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Báo cáo khoa học: Site-directed mutagenesis, kinetic and inhibition studies of aspartate ammonia lyase from Bacillus sp. YM55-1 pptx
... responsible for binding the amino group of are predicted to be Thr101, Asn1 42 and His188 [23 ] To investigate the importance of these residues to the mechanism of AspB, eight single site- directed mutants ... Lys 324 and Asn 326 The substrate-binding model suggests that residues Thr187, Lys 324 and Asn 326 are responsible for binding the C1 carboxylate group of [23 ] To investigate the importance of these ... proposed to function as the general acid catalyst that protonates the leaving C2 amino group (Scheme 2) [21 ] In view of the crystallographic observations with FumC [25 ] and FEBS Journal 27 6 (20 09) 29 943007...
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Báo cáo khoa học: Tracking interactions that stabilize the dimer structure of starch phosphorylase from Corynebacterium callunae Roles of Arg234 and Arg242 revealed by sequence analysis and site-directed mutagenesis doc
... 10 and % for the R234A and R242A mutant, respectively The situation is different for sulfate, which stabilizes wild-type StP and the R234A mutant to approximately the same extent In the R242A ... R234A R242A ± 3/n.a 3.8 ± 0.4/n.a 2. 60 ± 0.06 /20 ± 2. 00 ± 0. 02/ 12 ± 0.5 4.45 ± 0.05/stableb 2. 93 ± 0. 02/ stableb + phosphatea 5 .2 (3.45 3.55 2. 27 ± ± ± ± 0.2c/stableb,c 0.03d/stableb,d) 0.02d/stableb,d ... affects the tertiary and/ or quarternary interactions involving Arg234 and Arg2 42 thus leading to a greater stability For the interpretation of the kinetic stabilities of the R234A and R242A mutants,...
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Aerobic Uptake of Cholesterol by Ergosterol Auxotrophic Strains in Candidaglabrata & Random and Site-Directed Mutagenesis of ERG25 in Saccharomyces cerevisiae
... 5’-GGCCAAAACATCATTGTTCATGTAACTGTACC-3’ 21 F-ERG25seq2 R-ERG25seq2 F-ERG25seq3 R-ERG25seq3 F-ERG25seq4 R-ERG25seq4 F-ERG25seqA R-ERG25seqA F-ERG25seqB R-ERG25seqB F-ERG25seqC R-ERG25seqC F-ERG25seqD R-ERG25seqD 5’-CCGTTCTTCTATCTCATTTCTTGGTCGAGGCC-3’ ... 53˚C 0:30 72 C 2: 00 Repeat 2- 4 (x25) 72 C 5:00 4˚C ∞ 2. 7 .2 Site- Directed Mutagenesis Strategene’s QuickChange II XL Site- Directed Mutagenesis Kit was used to perform site- directed mutagenesis ... pJWP182A From Site- directed mutagenesis p 426 ADH; erg25; H 62 changed to A (EcoRI/SalI) p 426 ADH; erg25; F67 changed to A (EcoRI/SalI) p 426 ADH; erg25; Q88 changed to A (EcoRI/SalI) p 426 ADH; erg25;...
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Tài liệu Báo cáo khoa học: Site-directed mutagenesis of a loop at the active site of E1 (a2b2) of the pyruvate dehydrogenase complex A possible common sequence motif docx
... 1 .23 0.96 1 .21 1.1 1 .2 291 11.1 63 8.8 2. 1 49 56 66 427 24 6 64 13 63 186 25 17 19 0.46 0.35 1 .25 0.91 0.97 0. 62 0.45 1.39 0.98 1.30 23 21 0.56 21 5.5 5 .2 13 2. 9 3.0 2. 7 20 17 22 32 42 177 121 ... E1aR267A E1aD276A E1aY281A E1aR282A E1aS283C E1aY281A/R282A/S283A E1aY281A/R282A E1aY281S/R282S Km (lM) kcat/Km (M)1Æs)1 · 103) kcat (s)1) Km (lM) kcat/Km (M)1Æs)1 · 103) 0.48 0.30 1. 62 0.71 0. 82 ... E1aY281A E1aR282A E1aS283C E1aY281A/R282A/S283A E1aY281A/R282A E1aY281S/R282S 0. 128 0.133 0. 325 0.319 0 .26 8 0 .23 0 0.156 0.4 52 0 .27 9 0.41 ± ± ± ± ± ± ± ± ± ± 0.004 0.001 0.0 02 0.005 0.003 0.007 0.004...
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Tài liệu Báo cáo Y học: Study of substrate specificity of human aromatase by site directed mutagenesis pdf
... ± 24 73 ± 21 27 ± NC2 NC2 23 88 ± NC2 ND 24 43 ± NC3 4107 ± ± ± ± ± ± ± 30 51 28 12 16a 41 ± 10 104a 176 ± 63 25 1c 386 ± 1 52 1461 301b 623 302a 1465 7 82 106 159 133 49 81 69 ± ± ± ± ± ± 48 20 48 25 ... NC2 NC2 68 ± NC2 ND 29 0 ± NC3 635 ± 38 100 ± 23 824 100 ± 27 71 50 113 ± 43 76 ± 30 605a 657 56 ± 19 87 ± 21 51a 84 7a 47 20 a 28 7 109 34 91 89 67 3041 ± NC1 NC1 NC1 NC1 NC2 1 727 ± 26 71 ± NC1 324 0 ... genes [26 ] and the study of their catalytic differences [27 ,28 ] also highlight the need to understand which residues of the enzyme are involved in the substrate specificity In addition to human and...
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Báo cáo khoa học: Site-directed mutagenesis of selected residues at the active site of aryl-alcohol oxidase, an H2O2-producing ligninolytic enzyme pot
... alcohol 2, 4-Hexadien-1-ol 6 32 ± 158 30 ± 47 ± 22 7 ± 105 15 ± 65 ± 24 27 ± 1 42 ± 523 0 ± 615 540 ± 27 114 ± 21 0 ± 94 ± 119 ± 127 0 ± 55 639 ± 68 25 ± 39 ± 29 3 ± 8±1 28 ± 53 ± 90 ± 1700 ± 89 4 92 ± 26 ... (Tyr201, Tyr 222 and Tyr385) [53], d-amino acid oxidase (Tyr55, Tyr 224 and Tyr 228 ) [54], and vanillyl-alcohol oxidase (Tyr108, Tyr187, Phe 424 and Tyr503) [55] The last of these is related to AAO, ... domains, the FAD-binding domain (bottom part) and the substratebinding domain (top part), and one cofactor molecule with the adenine moiety buried in the FAD domain, and the flavin moiety expanding to...
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