site specific mutagenesis of ache

... to the active site (Fig 1), as the main cleavage sites Cleavage of the E1a-subunit activated the enzyme, as determined by a decarboxylation assay of the free E1 in the presence of an artificial ... PLOT software Temperature-dependence of catalytic activity The dependence of the catalytic activity of E1 on temperature was investigated using the DCPIP assay over a temperature range of 15 ... be noted that Tyr281 and Arg282 are located at the mouth of the funnel-shaped active site of E1, at the bottom of which lies ˚ the ThDP cofactor some 20–25 A from the protein ÔsurfaceÕ [14] Thus...

Ngày tải lên: 20/02/2014, 23:20

... 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 of some selected residues ... 50% of them clustered together in front of the rectus (re)-face of the isoalloxazine ring of the FAD cofactor This substrate location is shown in Fig 2A, which includes the 10 molecules of veratryl ... hydroxyl group in Ca, representative of the range of AAO substrates [9,19,33] Most of these alcohols docked in front of the re-side of the isoalloxazine ring of FAD [34], with the benzylic ˚ carbon...

Ngày tải lên: 07/03/2014, 11:20

... figures Sequence numbers refer to the locations of the transcriptional start site of hbpC (B) Alignments of the sequences of three HbpR-binding sites within the hbp gene region [19] The lane ‘cons-1’ ... binding site for HbpR and to examine the necessity of the sensing A-domain of HbpR for DNA binding The role of individual nucleotides and nucleotide motifs was characterized by site- directed mutagenesis ... of the hbpC promoter in Escherichia coli Results Mutagenesis of the HbpR-binding sites Sequence alignment of the UASs found in the HbpR operators in the hbpC and hbpD promoters defined a set of...

Ngày tải lên: 07/03/2014, 17:20

... elucidation of the catalytic mechanism of these enzymes would allow a more comprehensive understanding of the origin of substrate specicity and structure function relationships Previously, studies of ... effects, resulting in losses of activity of 88% and 95%, respectively, relative to wild-type enzyme Presteady-state kinetics of NAT acetylation The rate of acetylation of NATs was determined with ... In the case of Y190F, the value of log (k2 Kmacetyl) rose as a function of pH until a plateau was reached above pH 7.5 The data were t into a one-pKa model with a pKaacetyl value of 5.16 0.05,...

Ngày tải lên: 16/03/2014, 00:20

... attempted to understand these roles by mutagenesis of E coli KARI FEBS Journal 272 (2005) 593–602 ª 2005 FEBS Mutagenesis of the active site of E coli KARI Most of the mutations abolish the overall ... properties of the acetohydroxy acid isomeroreductase of Salmonella typhimurium J Biol Chem 244, 1118–1127 601 Mutagenesis of the active site of E coli KARI Primerano DA & Burns RO (1983) Role of acetohydroxy ... organization of this enzyme is substantially different from that of the spinach enzyme with the active site constructed from two monomers of a dodecamer In contrast, the active site of spinach...

Ngày tải lên: 16/03/2014, 18:20

... consisted of μl of diluted cDNA, 0.3 μM of forward and reverse primers (1 μl each), 10 μl of the 2X QuantiTect™ SYBR® green master mix, 0.1 μl of HK-UNG (Epicentre, Madison, WI), and μl of RNase-free ... observed in the HP content of the tissues between ACL-X and control joints for any of the regions tested Error bars indicate standard error of the mean Values are μg of HP/mg of tissue wet weight Differential ... observed in the GAG content of the tissues between ACL-X and control joints for any of the regions tested Error bars indicate standard error of the mean Values are μg of GAG/mg of tissue wet weight...

Ngày tải lên: 20/06/2014, 00:20

... conjugation of QD's to Akt-PH-EGFP via intein mediated protein splicing (a) Schematic representation of site- specific intein-mediated conjugation of QD's to target protein (b) Co-localization of QDot585 ... appearance of the band in the agarose gel is due to the fact that the size of the protein-QD conjugates varies greatly as a result of the Page of (page number not for citation purposes) Journal of Nanobiotechnology ... Briefly, we genetically tagged EGFP fusions of the PH domains of Akt and Btk with the N-terminus half of a split intein (IN) The complementary C-terminus half of the intein (IC) was biotinylated and...

Ngày tải lên: 11/08/2014, 00:22

... of QDs to the C-terminus of the PH domain of Akt (B) Schematic representation of site specific Ssp DnaB mini intein-mediated conjugation of QDs to the N-terminus of mem-EGFP homology domain of ... N-terminus of target proteins This expands the possibilities of the intein-based system allowing for the first time in vivo site specific conjugation of QDs and other nanostructures to the N terminus of ... http://www.jnanobiotechnology.com/content/9/1/37 Page of 14 Figure In vivo conjugation of QDs to the C- or N-terminus of target proteins via intein mediated protein splicing (A) Schematic representation of site- specific Ssp DnaE...

Ngày tải lên: 11/08/2014, 00:23

... scheme of random mutagenesis “insert” creation 27 2.2 Diagrammatic scheme of random mutagenesis “vector” creation .29 3.1 GC profile of wild type Candida glabrata 39 3.2 GC profile of ... 40 3.3 GC profile of ERG7 in Candida glabrata 41 3.4 GC profile of ERG11 in Candida glabrata 42 3.5 GC profile of ERG25 in Candida glabrata 43 3.6 GC profile of ERG27 in ... Luria-Bertani (LB) media with the addition of 60 µg/ml of ampicillin for selection LB media consisted of 10 g of pancreatic digest of casein, g of yeast extract, and 10 g/L of sodium chloride (46) dissolved...

Ngày tải lên: 24/08/2014, 12:11

... length of 0.54m needs to be established in order to set a baseline of analysis Considering that the most of the energy gathered from the wind profile in the area of Roswell are at wind speeds of ... energy on account of their radial position Figure 11 shows the mid-section of the optimized blade generates almost an identical amount of energy as that of the Phase VI outer portion of the blade ... harvested For a better analysis of this information a plot of the angles of attack in every segment of the blade is shown in Figure 13, which shows that the angles of attack in the airfoils go from...

Ngày tải lên: 09/09/2015, 10:17

... a-(1fi4)-linked glucose units of pullulan (Fig 4A) IPU is an anomer-inverting enzyme Comparison of the active sites of the model of IPU and Dex49A The active site structures of the model of IPU (Fig 4A) and ... Trp425 of Dex49A, which is the corresponding residue of Trp402 of IPU, is reportedly located in the vicinity of the active site and could form a binding site for a glucosyl unit in subsite )1 ... than the residues in subsites +1 and +2 [17] Comparison of the active sites of the model of IPU and Dex49A clearly shows that residues located in the negatively numbered subsites are highly conserved...

Ngày tải lên: 19/02/2014, 16:20

... 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 active site of tACE Hydrogen bonds ... inhibitor This study provides the first investigation of the importance of key active -site residues of ACE2 through site- directed mutagenesis, with the aim of providing practical evidence for their role ... result of replacing this single residue could not have 3517 Critical active -site residues of ACE2 been predicted simply by analysis of the active -site structure Further to this, the role of His505...

Ngày tải lên: 20/02/2014, 01:20

... arginase I [9] Differences in the binding of the a-carboxylate and a-amino groups of BEC were also ascribed to the larger volume of the active site cleft of arginase II, which allows more water-mediated ... differences between the active sites of this enzyme and arginase I In spite of the relatively low agmatinase activity of the Asn149Asp variant, it is clear that the interactions of arginase II with l-arginine ... by replacement of this residue with aspartate To the best of our knowledge, this is the first report in which the substrate specificity of arginase was altered by using sitedirected mutagenesis Experimental...

Ngày tải lên: 20/02/2014, 02:21

... positions of other Fig Scheme of the binuclear manganese cluster and the localization of the side-chains of Asp153 and Asn153 in the modelled structures of wild-type and D153N mutant forms of E coli ... Upon replacement of Asp153 with asparagine, the whole topology of the active site was found to be conserved in agmatinase In the modelled active site structure, the side chains of Asp153 and Asn153 ... revealed 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,...

Ngày tải lên: 21/02/2014, 01:21

... relationships of human aromatase (Eur J Biochem 269) 1401 Fig Hypothesis on the position of MR20814 inside the active site The position of MR20814 inside the active site correspond to: an orientation of ... that an increase of the hydrophobic character of this part of the human active site modified the aromatization efficiency of androstenedione but increased the inhibition potency of MR 20814 and ... stabilization of the steroid (D-ring) and particularly the role of D476 Positioning of the steroid inside the active site will differ according to its C(17) group and modification of the polarity of these...

Ngày tải lên: 21/02/2014, 15:20

... Ca2+-binding sites of TG2 ´ly R Kira et al Fig Multiple sequence alignment of Ca2+-binding sites of transglutaminases Sequence alignments of Ca2+-binding sites of TG2 compared with the other members of ... binding site of wild typeTG2 0.0 Weak binding sites of wild type TG2 Weak binding sites of S1 TG2 0.0 0.00 –0.1 –0.3 n = 0.52 ± 0.63 Kd = 0.1 ± 0.03 µM –0.4 kcal·mol–1 of injectant –0.2 kcal·mol–1 of ... confirmed that each of the five mutagenized sites contributes to Ca2+ binding of TG2 It was also observed that disruption of one site by mutation leads to weaker ⁄ loss of binding to other sites, and this...

Ngày tải lên: 16/03/2014, 03:20

... projection of their side-chains into the location of this site Consequently, ACE2 only binds a chloride ion at one CL1 site [24] Previous mutagenesis studies of sACE have shown that the CL2 site residue ... site- mediated regulation of tACE and ACE2 activities are unknown In addition, although Arg1098 has been identified as essential to chloride regulation of the CL2 6034 site of sACE, the roles of ... CL2 sites of ACE and ACE2 were changed by site- directed mutagenesis, and the effects on chloride sensitivity, substrate selectivity and inhibitor potency were observed Results PCR mutagenesis of...

Ngày tải lên: 16/03/2014, 04:20

... specificity through mutagenesis [10–12,22] This suggests that simultaneous mutations of other residues, perhaps even outside the active site, are required for a fine tuning of the active site structure, ... 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 presenting hydrogen bond-forming ... fitted to a Michaelis–Menten equation by using the ENZFITTER software (Elsevier-Biosoft, Cambridge, UK) Calculation of the energy of noncovalent interactions in the ESà complex Different Sfbgly50...

Ngày tải lên: 16/03/2014, 18:20

... is the signal of the substrate before the addition of enzyme, Fend is the signal after cleavage of all substrate, and [E] is the concentration of RNase A CD spectroscopy CD spectra of RNase A and ... rate constants of this RNase A variant correspond to those of wild-type RNase A (Fig 4), indicating that the local unfolding of the Ala20 loop is independent of the global unfolding of the RNase ... of the concept of the unfolding region Acknowledgements We are grateful to Professor R T Raines (University of Wisconsin, Madison, WI, USA) for the gift of the plasmid pBXR, to Professor G Vriend...

Ngày tải lên: 16/03/2014, 18:20

... of the site- directed mutagenesis experiments presented here The question concerning the mechanism of one of the basic reactions in cells, the transfer of a sugar moiety during the formation of ... nucleotide recognition sites Exchange of Lys86 against the neutral Ile resulted in only a slight increase of the Km-value and approximately 18% decrease of Vmax Replacement of Ala204 to Val caused ... synthase exhibits the His-Glu site (Fig 2), proposed as a catalytic domain of general importance for the mechanism of sugar transfer The significance of this HisGlu site we also explored by an alignment...

Ngày tải lên: 17/03/2014, 09:20