site directed mutagenesis as applied to biocatalysts

... either Asp376 or Asp396, the residues corresponding to Asp353 and Asp373 of IPU, respectively, appears to be properly positioned to act as a base in the hydrolytic reaction [17] Three Asp residues ... polygalacturonases (position equivalent to Asp372 of IPU) has been proposed to act as the acid (proton donor), while there are two candidates for a general base, two Asp residues of the polygalacturonases ... (Tosoh, Japan) was used, the recovery increased to 50% Ammonium sulfate was added directly to the dialysed crude enzyme to adjust it to 70% saturation and the supernatant was loaded on to the column...

Ngày tải lên: 19/02/2014, 16:20

... ACE2 has only one chloride-binding site (CL1) whereas ACE has two sites (one in each subdomain) In addition to this, the CL1 site is found in subdomain II, some distance from the active site, ... substrates (Fig 1A) To test this hypothesis, we used site- directed mutagenesis to replace the arginine with a glutamine residue (R273Q), i.e its counterpart in ACE This represents a positive to neutral ... Arg514 in ACE2 is displaced relative to Arg522 in ACE and is somewhat closer to the zinc-binding site The CL2 binding site is in close proximity to the catalytic site ˚ (10.4 A away from the zinc)...

Ngày tải lên: 20/02/2014, 01:20

... agmatinase, the Asn149Asp was markedly resistant to inhibition by arginine (Fig 4) Clearly, arginine was very poorly recognized as a substrate or inhibitor by the Asn149Asp variant The opposite ... ions was examined by sitedirected mutagenesis and kinetic studies Selection of target residues (Asp149, His120 and His145) was based on the roles assigned to the equivalent residues in arginase ... step was omitted and the assays were performed in the absence of added Mn2+, the His120Asn variant was found to be totally inactive, whereas half of full activity was expressed by the His145Asn...

Ngày tải lên: 20/02/2014, 02:21

... E1s was carried out as described previously [21,27] The E 1aS2 83C mutant was purified with mM dithiothreitol added to all buffers to prevent formation of disulfide bonds E2 and E3 were purified as ... protein ligase A, also essentially as described elsewhere [22,30] Enzyme assays The E1 component was assayed for catalytic activity by means of three different assays DCPIP assay This measures the ... properly as a lid for the active site, thereby granting easier access for the cofactor ThDP to its binding site The lower thermal stability of the mutant E1s and the increased Vmax in the DCPIP assay,...

Ngày tải lên: 20/02/2014, 23:20

... of B caldovelox arginase as a template Principal attention was given to the modeling of the active site The modelled structure was very similar to the template, with respect to the number and arrangement ... DISCUSSION By site- directed mutagenesis, a D153N mutant form of E coli agmatinase was obtained The mutant enzyme retained about 5% of wild-type activity and it was equally active when assayed in ... lines were computer-fitted to the appropriate equations Site- directed mutagenesis The D153N mutant form of E coli agmatinase was obtained by a two-step PCR [20], using the plasmid pKA5 containing...

Ngày tải lên: 21/02/2014, 01:21

... quantification of the aromatase expressed was used as an indicator of the transfection efficiency Whole cell aromatase activity and inhibition Aromatase activity was assessed in whole cells using ... reprotonation was not confirmed, as the mutation of K130 to Asn had no effect on the catalytic properties of the enzyme with androstenedione as a substrate According to our molecular model, reprotonation ... of cytochrome P450 reductase ´ (Kindly provided by V Luu-The, CHUL, Quebec), QuickChangeTM Site- Directed Mutagenesis kit from Stratagene (Montigny le Bretonneux, France), alkaline phosphatase...

Ngày tải lên: 21/02/2014, 15:20

... molecular docking on the above A Site- directed mutagenesis of aryl-alcohol oxidase model, site- directed mutagenesis and kinetic studies were used to identify the enzyme active site and evaluate the role ... substrate hydroxyl proton (alkoxide formation) by an active site base contributing to the transfer of a hydride from the substrate a-carbon to the flavin cofactor [40–46] Site- directed mutagenesis suggested ... Fungal strains and plasmids cDNA encoding P eryngii AAO with its own signal peptide was cloned into plasmid palcA, and the resulting vector (pALAAO) was used for site- directed mutagenesis, and transformation...

Ngày tải lên: 07/03/2014, 11:20

... the whole operator region For example, HbpR binding to the mutant operator behaved like the wildtype operator as far as the UAS C-1 region was concerned (Fig 4) Binding to the UAS C-2 region (in ... nucleotide motifs was characterized by site- directed mutagenesis and affinity binding by purified HbpR In several cases, DNaseI footprints were conducted to confirm the binding site contacts Additionally, ... measured at increasing concentrations of HbpR The percentage of free DNA was calculated from densitometric measurements of the radiolabeled bands as the density of the remaining unbound operator...

Ngày tải lên: 07/03/2014, 17:20

... acetylation of NAT Site- directed mutagenesis of the hamster NAT2 Tyr190 to Phe (Y190F), to Ile (Y190I) and to Ala (Y190A), was carried out using the pPH70D vector and QuickChange site- directed mutagenesis ... and 0.1 mm dithiothreitol] was added to PNPA (nal concentration 320 lm) to a total 500 lL volume [26] The reaction rate was determined at 25 C by measuring the linear increase in absorbance at ... active site, including Asp122 and the most closely associated inner sphere side chains Consequently, we propose that Y190 probably functions not only as a hydrogen bond donor to Asp122, but also as...

Ngày tải lên: 16/03/2014, 00:20

... serve as Ca2+-binding sites [19] The aim of the present study was to identify the exact Ca2+-binding sites of human TG2, by using sitedirected mutagenesis and targeting sites homologous to FXIIIa ... increased GTPase activity in the case of the GST–S4 mutant (130.7% ± 3.4% as compared with GST–WT as 100%), and greatly increased GTPase activity in the case of the GST–S5 mutant (353 ± 38% as ... transglutaminase catalytic triad of the active site decreased the binding of celiac autoantibodies to the enzyme In our experiments, celiac autoantibodies could bind to the C277S mutant and to the...

Ngày tải lên: 16/03/2014, 03:20

... Ser511 projecting into this region Table tACE and ACE2 residues subjected to site- directed mutagenesis For all residues subjected to PCR mutagenesis, the chloride-binding site at which they are ... residues Residues subjected to site- directed mutagenesis are shown in bold Cl) coordinating residues are shown in italic Cl) is unable to be bound by ACE2 at the CL2 site as a result of the side-chains ... active site (20.7 A), whereas the second site (CL2) is ˚ considerably closer, being 10.4 A from the zinc ion The N-domain of sACE has been shown to possess a CL2 site only, and so the enzyme has...

Ngày tải lên: 16/03/2014, 04:20

... b-glycosidases, as well as to predict the specificity of new Sfbgly50 mutants Materials and methods All reagents, unless otherwise specified, were purchased from Sigma or Merck Site- directed mutagenesis ... steric hindrance, whereas Q39 has a weak interaction with 6-OH [7] The purpose of the present study was to substitute residues Q39 and E451 of Sfbgly50, through site- directed mutagenesis, for residues ... tuning of the active site structure, in order to increase the catalytic activity of the mutant b-glycosidases However, the identification of these residues is not an easy task owing to the large number...

Ngày tải lên: 16/03/2014, 18:20

... cleavage by these proteases As soon as the protein molecule starts to unfold globally, however, the RNase A molecule becomes susceptible to these proteases, too The primary cleavage sites were found ... neurotoxin) belongs to the mammalian (nonsecretory or neurotoxin-type) ribonucleases 2, human RNase (P34096) belongs to the mammalian ribonucleases 4, human angiogenin (P03950) belongs to the ... members of the RNase A superfamily RNase A (P00656) from bovine pancreas, RNase (P07998) from human pancreas, and bs RNase (P00669) from bovine semen belong to the mammalian ribonucleases (pancreatic...

Ngày tải lên: 16/03/2014, 18:20

... Isomerase activity The rearrangement of HMKB to 2-acetolactate was used as an assay for the isomerase activity It has been shown [13,14] that the kinetic mechanism for the overall (reductoisomerase) ... proposal of Dumas et al [10] based on site- directed mutagenesis experiments Both metal ions are coordinated to the 594 R Tyagi et al inhibitor ⁄ product, as well as to several amino acid side-chains ... some reductase activity was observed When wildtype E coli KARI was denatured and refolded in the same manner, 25% (reductoisomerase) and 26% (reductase) activity was recovered The reductase activities...

Ngày tải lên: 16/03/2014, 18:20

... synthase wild-type and mutant enzymes after Ni2-nitrilotriacetic acid chromatography SDS/PAGE and staining by Coomassie-blue (I, marker proteins; II, AS- WT; III, AS- L86I; IV, AS- A204V; V, AS- E368D; ... glycosyltransferases (Eur J Biochem 270) 537 the nucleophile We were able to prove for the first time the suggested model by site- directed mutagenesis of arbutin synthase First the E368D mutein was generated ... important amino acids of glycosyltransferases, which is based on sequence alignment studies, is not satisfactory due to the results of the site- directed mutagenesis experiments presented here The...

Ngày tải lên: 17/03/2014, 09:20

... RgDAAO to a phenylalanine and a serine has been completed [11] After characterization of the corresponding mutants we were able to exclude any possibility that Y223 can act as an active -site base ... photoreduction was complete Disproportionation of the semiquinone was then followed until equilibration was reached (for up to 24 h) at 15 C Dissociation constants for ligands were measured spectrophotometrically ... ligand was plotted as a function of ligand concentration, after correction for any volume change Reagents Restriction enzymes and T4 DNA ligase were from Promega Life Sciences Site- directed mutagenesis...

Ngày tải lên: 17/03/2014, 17:20

... Perkin-Elmer (Boston, MA, USA) Site- directed mutagenesis Site- directed mutagenesis was carried out using the QuikChange Site- Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) The pMTNAEXST plasmid ... 2-PMPA were measured using HPLC and radioenzymatic assays as described in Experimental procedures N-acetyl-L-aspartyl-L-methionine was used as the substrate for wt rhGCPII, whereas NAAG was used for ... detailed analysis of the active site of glutamate carboxypeptidase II using sitedirected mutagenesis as a tool Amino acid residues important for substrate ⁄ inhibitor binding were determined from...

Ngày tải lên: 23/03/2014, 07:20

... transcription factor HAKN1 was expressed in Escherichia coli as a fusion with the maltose binding protein using vector pMALc2 The fusion protein was purified by affinity chromatography in amylose ... its EcoRI cohesive site was cloned into the BamHI site of pBluescript SK– Cleavage with HindIII and XbaI produces a 94-bp fragment that contains two HAKN1 binding sites in opposite orientations ... of HAKN1 to DNA shown by electrophoretic mobility shift assays When the oligonucleotide labeled at its XbaI site (at the opposite 3¢ end) was used, footprinting patterns were identical to those...

Ngày tải lên: 23/03/2014, 13:20

... are located at sites towards the inner (cytoplasmic) and outer (periplasmic) sides of the membrane-intrinsic domain of the enzyme (FrdCD) One site, the QP site (the proximal Q -site) , is located ... region of FrdB on the cytoplasmic 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 ... Initial mutagenesis studies suggested that 314 R A Rothery et al there may be two Q-sites present – a polar QB site (equivalent to the QP site) , and an apolar QA site (equivalent to the QD site) ...

Ngày tải lên: 23/03/2014, 13:20

... (Darmstadt, Germany) Site- directed mutagenesis Site- directed mutagenesis of (His)6PNAE was achieved using the QuikChangeTM in vitro Site- Directed Mutagenesis Kit, according to the manufacturer’s ... homology-modelling approach based on the precalculated alignment of the hydroxynitrile lyase to which PNAE has 43% identity The X-ray structure of hydroxynitrile lyase from H brasiliensis was used as a template ... His17 was changed to Ala, the specific activity significantly decreased, but still the kinetic data could be determined But in the case of His86, the activity was so low that it was not possible to...

Ngày tải lên: 24/03/2014, 04:21