共查询到20条相似文献,搜索用时 15 毫秒
1.
Isocitrate dehydrogenase (IDH) catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate with NAD(P) as a cofactor in the tricarboxylic acid cycle. As a housekeeping protein in Helicobacter pylori, IDH was considered as a possible candidate for serological diagnostics and detection. Here, we identified a new icd gene encoding IDH from H. pylori strain SS1. The recombinant H. pylori isocitrate dehydrogenase (HpIDH) was cloned, expressed, and purified in E. coli system. The enzymatic characterization of HpIDH demonstrates its activity with k cat of 87 s?1, K m of 124 μM and k cat/K m of 7 × 105 M?1s?1 toward isocitrate, k cat of 80 s?1, K m of 176 μM and k cat/K m of 4.5 × 105 M?1s?1 toward NADP. The optimum pH of the enzyme activity is around 9.0, and the optimum temperature is around 50 °C. This current work is expected to help better understand the features of HpIDH and provide useful information for H. pylori serological diagnostics and detection. 相似文献
2.
Cloning,over-expression and characterization of a thermo-tolerant xylanase from Thermotoga thermarum
The xyn10B gene, encoding the endo-1,4-β-xylanase Xyn10B from Thermotoga thermarum, was cloned and expressed in Escherichia coli. The ORF of the xyn10B was 1,095 bp and encoded to mature peptide of 344 amino acids with a calculated MW of 40,531 Da. The recombinant xylanase was optimally active at 80 °C, pH 6.0 and retained approx. 60 % of its activity after 2 h at 75 °C. Apparent K m , k cat and k cat /K m values of the xylanase for beechwood xylan were 1.8 mg ml?1, 520 s?1 and 289 ml mg?1 s?1, respectively. The end products of the hydrolysis of beechwood xylan were mainly oligosaccharides but without xylose after 2 h hydrolysis. 相似文献
3.
Ka-Weng Ieong Gabriele Indrisiunaite Arjun Prabhakar Joseph D Puglisi Mns Ehrenberg 《Nucleic acids research》2021,49(5):2684
We used quench flow to study how N6-methylated adenosines (m6A) affect the accuracy ratio between kcat/Km (i.e. association rate constant (ka) times probability (Pp) of product formation after enzyme-substrate complex formation) for cognate and near-cognate substrate for mRNA reading by tRNAs and peptide release factors 1 and 2 (RFs) during translation with purified Escherichia coli components. We estimated kcat/Km for Glu-tRNAGlu, EF-Tu and GTP forming ternary complex (T3) reading cognate (GAA and Gm6AA) or near-cognate (GAU and Gm6AU) codons. ka decreased 10-fold by m6A introduction in cognate and near-cognate cases alike, while Pp for peptidyl transfer remained unaltered in cognate but increased 10-fold in near-cognate case leading to 10-fold amino acid substitution error increase. We estimated kcat/Km for ester bond hydrolysis of P-site bound peptidyl-tRNA by RF2 reading cognate (UAA and Um6AA) and near-cognate (UAG and Um6AG) stop codons to decrease 6-fold or 3-fold by m6A introduction, respectively. This 6-fold effect on UAA reading was also observed in a single-molecule termination assay. Thus, m6A reduces both sense and stop codon reading accuracy by decreasing cognate significantly more than near-cognate kcat/Km, in contrast to most error inducing agents and mutations, which increase near-cognate at unaltered cognate kcat/Km. 相似文献
4.
《Bioorganic & medicinal chemistry letters》2019,29(17):2452-2454
Protein tyrosine phosphatase (PTP) targeted, peptide based chemical probes are valuable tools for studying this important family of enzymes, despite the inherent difficulty of developing peptides targeted towards an individual PTP. Here, we have taken a rational approach to designing a SHP-2 targeted, fluorogenic peptide substrate based on information about the potential biological substrates of SHP-2. The fluorogenic, phosphotyrosine mimetic phosphocoumaryl aminopropionic acid (pCAP) provides a facile readout for monitoring PTP activity. By optimizing the amino acids surrounding the pCAP residue, we obtained a substrate with the sequence Ac-DDPI-pCAP-DVLD-NH2 and optimized kinetic parameters (kcat = 0.059 ± 0.008 s−1, Km = 220 ± 50 µM, kcat/Km of 270 M−1s−1). In comparison, the phosphorylated coumarin moiety alone is an exceedingly poor substrate for SHP-2, with a kcat value of 0.0038 ± 0.0003 s−1, a Km value of 1100 ± 100 µM and a kcat/Km of 3 M−1s−1. Furthermore, this optimized peptide has selectivity for SHP-2 over HePTP, MEG1 and PTPµ. The data presented here demonstrate that PTP-targeted peptide substrates can be obtained by optimizing the sequence of a pCAP containing peptide. 相似文献
5.
G. S. Dotsenko M. V. Semenova O. A. Sinitsyna S. W. A. Hinz J. Wery I. N. Zorov E. G. Kondratieva A. P. Sinitsyn 《Biochemistry. Biokhimii?a》2012,77(11):1303-1311
Genes of β-mannosidase 97 kDa, GH family 2 (bMann9), β-mannanase 48 kDa, GH family 5 (bMan2), and α-galactosidase 60 kDa, GH family 27 (aGal1) encoding galactomannan-degrading glycoside hydrolases of Myceliophthora thermophila C1 were successfully cloned, and the recombinant enzymes were purified to homogeneity and characterized. bMann9 displays only exo-mannosidase activity, the K m and k cat values are 0.4 mM and 15 sec?1 for p-nitrophenyl-β-D-mannopyranoside, and the optimal pH and temperature are 5.3 and 40°C, respectively. bMann2 is active towards galac-tomannans (GM) of various structures. The K m and k cat values are 1.3 mg/ml and 67 sec?1 for GM carob, and the optimal pH and temperature are 5.2 and 69°C, respectively. aGal1 is active towards p-nitrophenyl-α-D-galactopyranoside (PNPG) as well as GM of various structures. The K m and k cat values are 0.08 mM and 35 sec?1 for PNPG, and the optimal pH and temperature are 5.0 and 60°C, respectively. 相似文献
6.
Characterization of a d-psicose-producing enzyme, d-psicose 3-epimerase, from Clostridium sp. 总被引:1,自引:0,他引:1
Wanmeng Mu Wenli Zhang Dan Fang Leon Zhou Bo Jiang Tao Zhang 《Biotechnology letters》2013,35(9):1481-1486
The gene coding for d-psicose 3-epimerase (DPEase) from Clostridium sp. BNL1100 was cloned and expressed in Escherichia coli. The recombinant enzyme was purified by Ni-affinity chromatography. It was a metal-dependent enzyme and required Co2+ as optimum cofactor. It displayed catalytic activity maximally at pH 8.0 and 65 °C (as measured over 5 min). The optimum substrate was d-psicose, and the K m, turnover number (k cat), and catalytic efficiency (k cat/K m) for d-psicose were 227 mM, 32,185 min?1, and 141 min?1 mM?1, respectively. At pH 8.0 and 55 °C, 120 g d-psicose l?1 was produced from 500 g d-fructose l?1 after 5 h. 相似文献
7.
Marcia Y. Kondo Lilian C.G. Oliveira Debora N. Okamoto Marina R.T. de Araujo Claudia N. Duarte dos Santos Maria A. Juliano Luiz Juliano Iuri E. Gouvea 《Biochemical and biophysical research communications》2011,(4):640
Here we report the hydrolytic behavior of recombinant YFV NS2B/NS3 protease against FRET substrates mimicking the prime and non-prime region of the natural polyprotein cleavage sites. While the P2-P′1 motif is the main factor associated with the catalytic efficiency of Dengue (DV) and West Nile Virus (WNV) protease, we show that the kcat/Km of YFV NS2B/NS3 varied by more than two orders of magnitude, despite the presence of the same motif in all natural substrates. The catalytic significance of this homogeneity – a unique feature among worldwide prominent flavivirus – was kinetically analyzed using FRET peptides containing all possible combinations of two and three basic amino acids in tandem, and Arg and Lys residues produced distinct effects on kcat/Km. The parallel of our data with those obtained in vivo by Chambers et al. (1991) restrains the idea that these sites co-evolved with the NS2B/NS3 protease to promote highly efficient hydrolysis and supports the notion that secondary substrate interaction distant from cleavage sites are the main factor associated with the different hydrolytic rates on YFV NS2B-NS3pro natural substrates. 相似文献
8.
Gordana Kovačević Marija Blažić Bojana Draganić Raluca Ostafe Marija Gavrović-Jankulović Rainer Fischer Radivoje Prodanović 《Molecular biotechnology》2014,56(4):305-311
Aspergillus niger glucose oxidase (GOx) genes for wild-type (GenBank accession no. X16061, swiss-Prot; P13006) and M12 mutant (N2Y, K13E, T30 V, I94 V, K152R) were cloned into pPICZαA vector for expression in Pichia pastoris KM71H strain. The highest expression level of 17.5 U/mL of fermentation media was obtained in 0.5 % (v/v) methanol after 9 days of fermentation. The recombinant GOx was purified by cross-flow ultrafiltration using membranes of 30 kDa molecular cutoff and DEAE ion-exchange chromatography at pH 6.0. Purified wt GOx had k cat of 189.4 s?1 and K m of 28.26 mM while M12 GOx had k cat of 352.0 s?1 and K m of 13.33 mM for glucose at pH 5.5. Specificity constants k cat/K m of wt (6.70 mM?1 s?1) and M12 GOx (26.7 mM?1 s?1) expressed in P. pastoris KM71H were around three times higher than for the same enzymes previously expressed in Saccharomyces cerevisiae InvSc1 strain. The pH optimum and sugar specificity of M12 mutant of GOx remained similar to the wild-type form of the enzyme, while thermostability was slightly decreased. M12 GOx expressed in P. pastoris showed three times higher activity compared to the wt GOx toward redox mediators like N,N-dimethyl-nitroso-aniline used for glucose strips manufacturing. M12 mutant of GOx produced in P. pastoris KM71H could be useful for manufacturing of glucose biosensors and biofuel cells. 相似文献
9.
Zilong Li Ning Jiang Keqian Yang Jianting Zheng 《Extremophiles : life under extreme conditions》2016,20(2):149-156
Glucose-6-phosphate dehydrogenases (G6PDs) are important enzymes widely used in bioassay and biocatalysis. In this study, we reported the cloning, expression, and enzymatic characterization of G6PDs from the thermophilic bacterium Thermoanaerobacter tengcongensis MB4 (TtG6PD). SDS-PAGE showed that purified recombinant enzyme had an apparent subunit molecular weight of 60 kDa. Kinetics assay indicated that TtG6PD preferred NADP+ (k cat/K m = 2618 mM?1 s?1, k cat = 249 s?1, K m = 0.10 ± 0.01 mM) as cofactor, although NAD+ (k cat/K m = 138 mM?1 s?1, k cat = 604 s?1, K m = 4.37 ± 0.56 mM) could also be accepted. The K m values of glucose-6-phosphate were 0.27 ± 0.07 mM and 5.08 ± 0.68 mM with NADP+ and NAD+ as cofactors, respectively. The enzyme displayed its optimum activity at pH 6.8–9.0 for NADP+ and at pH 7.0–8.6 for NAD+ while the optimal temperature was 80 °C for NADP+ and 70 °C for NAD+. This was the first observation that the NADP+-linked optimal temperature of a dual coenzyme-specific G6PD was higher than the NAD+-linked and growth (75 °C) optimal temperature, which suggested G6PD might contribute to the thermal resistance of a bacterium. The potential of TtG6PD to measure the activity of another thermophilic enzyme was demonstrated by the coupled assays for a thermophilic glucokinase. 相似文献
10.
Zongqing Huang Xiaoshuang Liu Shaowei Zhang Ziduo Liu 《Journal of industrial microbiology & biotechnology》2014,41(1):65-74
A xylosidase gene, gsxyn, was cloned from the deep-sea thermophilic Geobacillus stearothermophilus, which consisted of 2,118 bp and encoded a protein of 705 amino acids with a calculated molecular mass of 79.8 kDa. The GSxyn of glycoside hydrolase family 52 (GH52) displayed its maximum activity at 70 °C and pH 5.5. The K m and k cat values of GSxyn for ρNPX were 0.48 mM and 36.64 s?1, respectively. Interestingly, a new exo-xylanase activity was introduced into GSxyn by mutating the tyrosine509 into glutamic acid, whereas the resultant enzyme variant, Y509E, retained the xylosidase activity. The optimum xylanase activity of theY509E mutant displayed at pH 6.5 and 50 °C, and retained approximately 45 % of its maximal activity at 55 °C, pH 6.5 for 60 min. The K m and k cat values of the xylanase activity of Y509E mutant for beechwood xylan were 5.10 mg/ml and 22.53 s?1, respectively. The optimum xylosidase activity of theY509E mutant displayed at pH 5.5 and 60 °C. The K m and k cat values of the xylosidase activity of Y509E mutant for ρNPX were 0.51 mM and 22.53 s?1, respectively. This report demonstrated that GH52 xylosidase has provided a platform for generating bifunctional enzymes for industrially significant and complex substrates, such as plant cell wall. 相似文献
11.
Matthew J. Grisewood Nathanael P. Gifford Robert J. Pantazes Ye Li Patrick C. Cirino Michael J. Janik Costas D. Maranas 《PloS one》2013,8(10)
OptZyme is a new computational procedure for designing improved enzymatic activity (i.e., kcat or kcat/KM) with a novel substrate. The key concept is to use transition state analogue compounds, which are known for many reactions, as proxies for the typically unknown transition state structures. Mutations that minimize the interaction energy of the enzyme with its transition state analogue, rather than with its substrate, are identified that lower the transition state formation energy barrier. Using Escherichia coli β-glucuronidase as a benchmark system, we confirm that KM correlates (R2 = 0.960) with the computed interaction energy between the enzyme and the para-nitrophenyl- β, D-glucuronide substrate, kcat/KM correlates (R2 = 0.864) with the interaction energy of the transition state analogue, 1,5-glucarolactone, and kcat correlates (R2 = 0.854) with a weighted combination of interaction energies with the substrate and transition state analogue. OptZyme is subsequently used to identify mutants with improved KM, kcat, and kcat/KM for a new substrate, para-nitrophenyl- β, D-galactoside. Differences between the three libraries reveal structural differences that underpin improving KM, kcat, or kcat/KM. Mutants predicted to enhance the activity for para-nitrophenyl- β, D-galactoside directly or indirectly create hydrogen bonds with the altered sugar ring conformation or its substituents, namely H162S, L361G, W549R, and N550S. 相似文献
12.
《Bioscience, biotechnology, and biochemistry》2013,77(4):910-912
We previously isolated from Nicotiana glutinosa leaves three distinct cDNA clones, NGR1, NGR2, and NGR3, encoding a wound-inducible RNase NW, and putative RNases NGR2 and NGR3, respectively. In this study, we produced RNases NW and NGR3 in Escherichia coli and purified them to homogeneity. RNase NGR3 had non-absolute specificity toward polynucleotides, although RNase NW preferentially cleaved polyinosinic acid (Poly I). Both RNases NW and NGR3 were more active toward diribonucleoside monophosphates ApG, CpU, and GpU. Furthermore, kinetic parameters for RNase NW (K m, 0.778 mM and k cat, 1938 min?1) and RNase NGR3 (K m, 0.548 mM and k cat, 408 min?1) were calculated using GpU as a substrate. 相似文献
13.
Mechanism of action of thrombin on fibrinogen. The reaction of thrombin with fibrinogen-like peptides containing 11, 14, and 16 residues 总被引:4,自引:0,他引:4
J W van Nispen T C Hageman H A Scheraga 《Archives of biochemistry and biophysics》1977,182(1):227-243
The following peptides were synthesized by classical methods in solution: Ac-Gly-Gly- Val-Arg-Gly-Pro-Arg-Val-Val-Glu-Arg-NHCH3 (A), Ac-Ala-Glu-Gly-Gly-Gly-Val- Arg-Gly-Pro-Arg-Val-Val-Glu-Arg-NHCH3 (B), and Ac-Phe-Leu-Ala-Glu-Gly-Gly- Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Glu-Arg-NHCH3 (C). The rates of hydrolysis of the Arg-Gly bond of these three peptides by thrombin were measured, and the values of were found to be 0.05 × 10?7 (A), 0.02 × 10?7 (B), and 1.6 × 10?7 (C) [(NIH units/ liter)s]?1. The value of for peptide C is less than 1% of that for fibrinogen [although the value of kcat itself, for peptide C (but not for A or B), is comparable to that for fibrinogen]. These results indicate that phenylanine and leucine at positions P9 and P8, respectively, play a key role in the reaction of thrombin with fibrinogen. The data also show that factors outside of the 16 residues of peptide C are important in determining the rate of hydrolysis of fibrogen by thrombin. 相似文献
14.
Douglas B. Jordan Kurt Wagschal Arabela A. Grigorescu Jay D. Braker 《Applied microbiology and biotechnology》2013,97(10):4415-4428
The hemicellulose xylan constitutes a major portion of plant biomass, a renewable feedstock available for conversion to biofuels and other bioproducts. β-xylosidase operates in the deconstruction of the polysaccharide to fermentable sugars. Glycoside hydrolase family 43 is recognized as a source of highly active β-xylosidases, some of which could have practical applications. The biochemical details of four GH43 β-xylosidases (those from Alkaliphilus metalliredigens QYMF, Bacillus pumilus, Bacillus subtilis subsp. subtilis str. 168, and Lactobacillus brevis ATCC 367) are examined here. Sedimentation equilibrium experiments indicate that the quaternary states of three of the enzymes are mixtures of monomers and homodimers (B. pumilus) or mixtures of homodimers and homotetramers (B. subtilis and L. brevis). k cat and k cat/K m values of the four enzymes are higher for xylobiose than for xylotriose, suggesting that the enzyme active sites comprise two subsites, as has been demonstrated by the X-ray structures of other GH43 β-xylosidases. The K i values for d-glucose (83.3–357 mM) and d-xylose (15.6–70.0 mM) of the four enzymes are moderately high. The four enzymes display good temperature (K t 0.5?~?45 °C) and pH stabilities (>4.6 to <10.3). At pH 6.0 and 25 °C, the enzyme from L. brevis ATCC 367 displays the highest reported k cat and k cat/K m on natural substrates xylobiose (407 s?1, 138 s?1?mM?1), xylotriose (235 s?1, 80.8 s?1?mM?1), and xylotetraose (146 s?1, 32.6 s?1?mM?1). 相似文献
15.
《Cell calcium》2018
Prokaryotic and eukaryotic Na+/Ca2+ exchangers (NCX) control Ca2+ homeostasis. NCX orthologs exhibit up to 104-fold differences in their turnover rates (kcat), whereas the ratios between the cytosolic (cyt) and extracellular (ext) Km values (Kint = KmCyt/KmExt) are highly asymmetric and alike (Kint ≤ 0.1) among NCXs. The structural determinants controlling a huge divergence in kcat at comparable Kint remain unclear, although 11 (out of 12) ion-coordinating residues are highly conserved among NCXs. The crystal structure of the archaeal NCX (NCX_Mj) was explored for testing the mutational effects of pore-allied and loop residues on kcat and Kint. Among 55 tested residues, 26 mutations affect either kcat or Kint, where two major groups can be distinguished. The first group of mutations (14 residues) affect kcat rather than Kint. The majority of these residues (10 out of 14) are located within the extracellular vestibule near the pore center. The second group of mutations (12 residues) affect Kint rather than kcat, whereas the majority of residues (9 out 12) are randomly dispersed within the extracellular vestibule. In conjunction with computational modeling-simulations and hydrogen-deuterium exchange mass-spectrometry (HDX-MS), the present mutational analysis highlights structural elements that differentially govern the intrinsic asymmetry and transport rates. The key residues, located at specific segments, can affect the characteristic features of local backbone dynamics and thus, the conformational flexibility of ion-transporting helices contributing to critical conformational transitions. The underlying mechanisms might have a physiological relevance for matching the response modes of NCX variants to cell-specific Ca2+ and Na+ signaling. 相似文献
16.
P. K. Chaurasia A. Yadav R. S. S. Yadav S. Yadava 《Applied Biochemistry and Microbiology》2013,49(6):592-599
A laccase from the culture filtrate of Phellinus linteus MTCC-1175 has been purified to homogeneity. The method involved concentration of the culture filtrate by ammonium sulphate precipitation and an anion exchange chromatography on DEAE-cellulose. The SDS-PAGE and native-PAGE gave single protein band indicating that the enzyme preparation was pure. The molecular mass of the enzyme determined from SDS-PAGE analysis was 70 kDa. Using 2.6-dimethoxyphenol, 2.2′[azino-bis-(3-ethylbonzthiazoline-6-sulphonic acid) diammonium salt] (ABTS) and 4-hydroxy-3,5-dimethoxybenzaldehyde azine as the substrates, the K m, k cat and k cat/K m values of the laccase were found to be 160 μM, 6.85 s?1, 4.28 × 104 M?1 s?1, 42 μM, 6.85 s?1, 16.3 × 104 M?1 s?1 and 92 μM, 6.85 s?1, 7.44 × 104 M?1 s?1, respectively. The pH and the temperature optima of the P. linteus MTCC-1175 laccase were 5.0 and 45°C, respectively. The activation energy for thermal denaturation of the enzyme was 38.20 kJ/mole/K. The enzyme was the most stable at pH 5.0 after 1 h reaction. In the presence of ABTS as the mediator, the enzyme transformed toluene, 3-nitrotoluene and 4-chlorotoluene to benzaldehyde, 3-nitrobenzaldehyde and 4-chlorobenzaldehyde, respectively. 相似文献
17.
《Bioscience, biotechnology, and biochemistry》2013,77(6):1344-1348
Aldehyde dehydrogenase ST0064, the closest paralog of previously characterized allosteric non-phosphorylating glyceraldehyde-3-phosphate (GAP) dehydrogenase (GAPN, ST2477) from a thermoacidophilic archaeon, Sulfolobus tokodaii, was expressed heterologously and characterized in detail. ST0064 showed remarkable activity toward succinate semialdehyde (SSA) (K m of 0.0029 mM and k cat of 30.0 s?1) with no allosteric regulation. Activity toward GAP was lower (K m of 4.6 mM and k cat of 4.77 s?1), and previously predicted succinyl-CoA reductase activity was not detected, suggesting that the enzyme functions practically as succinate semialdehyde dehydrogenase (SSADH). Phylogenetic analysis indicated that archaeal SSADHs and GAPNs are closely related within the aldehyde dehydrogenase superfamily, suggesting that they are of the same origin. 相似文献
18.
D-Lactate dehydrogenase (D-LDH) from Pediococcus pentosaceus ATCC 25745 was found to produce D-3-phenyllactic acid from phenylpyruvate. The optimum pH and temperature for enzyme activity were pH 5.5 and 45 °C. The Michaelis-Menten constant (K m), turnover number (k cat), and catalytic efficiency (k cat?K m) values for the substrate phenylpyruvate were estimated to be 1.73 mmol/L, 173 s?1, and 100 (mmol/L)?1 s?1 respectively. 相似文献
19.
Cobalt(II)-substituted carboxypeptidase A has been found to reversibly bind N3? and NCO?, but not NCS?, in the pH range 5–10, thus including the pH range of activity of the enzyme. The pH dependence of the anion binding constant is affected by two ionizations, which are assigned as those regulating kcat and KM. The electronic and 1H NMR spectra are consistent with a substantially pseudotetrahedral geometry of the anion derivatives. 相似文献
20.
Rebecca N. Adamek Roxanne V. Maniquis Sabaha Khakoo Michael D. Bridges Nicholas T. Salzameda 《Bioorganic & medicinal chemistry letters》2013,23(17):4848-4850
The West Nile Virus (WNV) has been a worldwide epidemic since the early 1990s. Currently there are no therapeutic treatments for WNV infections. One particular avenue of treatment is inhibition of the NS2B-NS3 protease, an enzyme that is crucial for WNV replication. In our effort to increase the number of NS2B-NS3 protease inhibitors, we report a novel FRET-based high throughput assay for the discovery of WNV NS2B-NS3 protease inhibitors. For this assay, a FRET-based peptide substrate was synthesized and kinetically characterized with the NS2B-NS3 protease. The new substrate exhibits a Km of 3.35 ± 0.31 μM, a kcat of 0.0717 ± 0.0016 s?1 and a kcat/Km of 21,400 ± 2000 M?1 s?1. 相似文献