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1.
ADP-ribosyl cyclase and NAD+ glycohydrolase (CD38, E.C.3.2.2.5) efficiently catalyze the exchange of the nicotinamidyl moiety of NAD+, nicotinamide adenine dinucleotide phosphate (NADP+) or nicotinamide mononucleotide (NMN+) with an alternative base. 4′-Pyridinyl drugs (amrinone, milrinone, dismerinone and pinacidil) were efficient alternative substrates (kcat/KM = 0.9-10 μM−1 s−1) in the exchange reaction with ADP-ribosyl cyclase. When CD38 was used as a catalyst the kcat/KM values for the exchange reaction were reduced two or more orders of magnitude (0.015-0.15 μM−1 s−1). The products of this reaction were novel dinucleotides. The values of the equilibrium constants for dinucleotide formation were determined for several drugs. These enzymes also efficiently catalyze the formation of novel mononucleotides in an exchange reaction with NMN+, kcat/KM = 0.05-0.4 μM−1 s−1. The kcat/KM values for the exchange reaction with NMN+ were generally similar (0.04-0.12 μM−1 s−1) with CD38 and ADP-ribosyl cyclase as catalysts. Several novel heterocyclic alternative substrates were identified as 2-isoquinolines, 1,6-naphthyridines and tricyclic bases. The kcat/KM values for the exchange reaction with these substrates varied over five orders of magnitude and approached the limit of diffusion with 1,6-naphthyridines. The exchange reaction could be used to synthesize novel mononucleotides or to identify novel reversible inhibitors of CD38. 相似文献
2.
The response of rapid light–response curves (RLCs) of variable fluorescence to changes in short- and long-term photoacclimation status was studied in an estuarine benthic diatom. The diatom Nitzschia palea was grown under low- (LL, 20 μmol m−2 s−1) and high-light (HL, 400 μmol m−2 s−1) conditions, with the purpose of characterising the effects of long-term photoacclimation on (i) steady-state light–response curves (LC) of relative electron transport rate, rETR, (ii) the response of RLCs to changes in ambient irradiance (E, the irradiance to which the sample is acclimated to immediately before the RLCs), (iii) the relationship of RLCs to LC parameters and non-photochemical quenching (NPQ). Photoacclimation to LL and HL conditions induced distinct light–response patterns of rETR and NPQ. Higher growth light resulted in rETR vs. E curves with lower initial slopes (α, 0.591 μmol−1 m2 s vs. 0.661 μmol−1 m2 s, for HL and LL, respectively) and markedly higher maximum rates (rETRm, 95.9 vs. 29.3), reached under higher E levels (higher light-saturation coefficient, Ek: 162.4 μmol m−2 s−1 vs. 44.3 μmol m−2 s−1). Acclimation to HL induced bi-phasic NPQ vs. E curves, with minimum values reached under low E levels (15–25 μmol m−2 s−1) and not on dark-acclimated samples. The response of RLCs to changes in ambient irradiance varied with the long-term photoacclimation status of the samples. The initial slope, αRLC, decreased monotonically with E in LL cultures, from 0.68 to 0.25 μmol−1 m2 s, while varied bi-phasically in HL-acclimated samples. Typically, αRLC of HL cultures increased under low E, reaching a maximum of 0.61 μmol−1 m2 s under 25–55 μmol m−2 s−1, and decreased gradually under higher E levels to 0.25 μmol−1 m2 s. RLC maximum rETR, rETRm,RLC, and saturation coefficient Ek,RLC, increased with E following a saturation-like pattern, with the HL cultures presenting markedly higher values for all the E range (maximum rETRm,RLC values were 108.6 and 33.4 for HL and LL cultures, respectively). An inverse relationship was consistently found between αRLC and NPQ, both on LL and HL cultures, causing strong correlations (P < 0.001 in all cases) between NPQ and the high light-induced decrease of αRLC, ΔαRLC. RLCs were confirmed to also provide information on the long-term photoacclimation status, as significant correlations (P < 0.001 both for HL and LL cultures) were verified between Ek and an index based on RLC parameters, Êk, both for LL and HL cultures. These results reinforce the usefulness of RLCs as a tool for inferring on the short- and long-term photoacclimation status of samples with different long-term light histories, through the estimation of LC parameters and the monitoring of NPQ levels. 相似文献
3.
Valentina Bosello-Travain Marcus Conrad Giorgio Cozza Alessandro Negro Silvia Quartesan Monica Rossetto Antonella Roveri Stefano Toppo Fulvio Ursini Mattia Zaccarin Matilde Maiorino 《Biochimica et Biophysica Acta (BBA)/General Subjects》2013
Background
Mammalian GPx7 is a monomeric glutathione peroxidase of the endoplasmic reticulum (ER), containing a Cys redox center (CysGPx). Although containing a peroxidatic Cys (CP) it lacks the resolving Cys (CR), that confers fast reactivity with thioredoxin (Trx) or related proteins to most other CysGPxs.Methods
Reducing substrate specificity and mechanism were addressed by steady-state kinetic analysis of wild type or mutated mouse GPx7. The enzymes were heterologously expressed as a synuclein fusion to overcome limited expression. Phospholipid hydroperoxide was the oxidizing substrate. Enzyme–substrate and protein–protein interaction were analyzed by molecular docking and surface plasmon resonance analysis.Results
Oxidation of the CP is fast (k+ 1 > 103 M− 1 s− 1), however the rate of reduction by GSH is slow (k′+ 2 = 12.6 M− 1 s− 1) even though molecular docking indicates a strong GSH–GPx7 interaction. Instead, the oxidized CP can be reduced at a fast rate by human protein disulfide isomerase (HsPDI) (k+ 1 > 103 M− 1 s− 1), but not by Trx. By surface plasmon resonance analysis, a KD = 5.2 μM was calculated for PDI–GPx7 complex. Participation of an alternative non-canonical CR in the peroxidatic reaction was ruled out. Specific activity measurements in the presence of physiological reducing substrate concentration, suggest substrate competition in vivo.Conclusions
GPx7 is an unusual CysGPx catalyzing the peroxidatic cycle by a one Cys mechanism in which GSH and PDI are alternative substrates.General significance
In the ER, the emerging physiological role of GPx7 is oxidation of PDI, modulated by the amount of GSH. 相似文献4.
Jutta Vlasits Paul G. FurtmüllerChrista Jakopitsch Marcel ZamockyChristian Obinger 《Biochimica et Biophysica Acta - Proteins and Proteomics》2010,1804(4):799-805
Catalase-peroxidases (KatGs) are unique bifunctional heme peroxidases that exhibit peroxidase and substantial catalase activities. Nevertheless, the reaction pathway of hydrogen peroxide dismutation, including the electronic structure of the redox intermediate that actually oxidizes H2O2, is not clearly defined. Several mutant proteins with diminished overall catalase but wild-type-like peroxidase activity have been described in the last years. However, understanding of decrease in overall catalatic activity needs discrimination between reduction and oxidation reactions of hydrogen peroxide. Here, by using sequential-mixing stopped-flow spectroscopy, we have investigated the kinetics of the transition of KatG compound I (produced by peroxoacetic acid) to its ferric state by trapping the latter as cyanide complex. Apparent bimolecular rate constants (pH 6.5, 20 °C) for wild-type KatG and the variants Trp122Phe (lacks KatG-typical distal adduct), Asp152Ser (controls substrate access to the heme cavity) and Glu253Gln (channel entrance) are reported to be 1.2 × 104 M− 1 s− 1, 30 M− 1 s− 1, 3.4 × 103 M− 1 s− 1, and 8.6 × 103 M− 1 s− 1, respectively. These findings are discussed with respect to steady-state kinetic data and proposed reaction mechanism(s) for KatG. Assets and drawbacks of the presented method are discussed. 相似文献
5.
The binding affinity of the two substrate–water molecules to the water-oxidizing Mn4CaO5 catalyst in photosystem II core complexes of the extremophilic red alga Cyanidioschyzon merolae was studied in the S2 and S3 states by the exchange of bound 16O-substrate against 18O-labeled water. The rate of this exchange was detected via the membrane-inlet mass spectrometric analysis of flash-induced oxygen evolution. For both redox states a fast and slow phase of water-exchange was resolved at the mixed labeled m/z 34 mass peak: kf = 52 ± 8 s− 1 and ks = 1.9 ± 0.3 s− 1 in the S2 state, and kf = 42 ± 2 s− 1 and kslow = 1.2 ± 0.3 s− 1 in S3, respectively. Overall these exchange rates are similar to those observed previously with preparations of other organisms. The most remarkable finding is a significantly slower exchange at the fast substrate–water site in the S2 state, which confirms beyond doubt that both substrate–water molecules are already bound in the S2 state. This leads to a very small change of the affinity for both the fast and the slowly exchanging substrates during the S2 → S3 transition. Implications for recent models for water-oxidation are briefly discussed. 相似文献
6.
Sanaullah Iqbal Thu-Ha Nguyen Tien Thanh Nguyen Thomas Maischberger Dietmar Haltrich 《Carbohydrate research》2010,345(10):1408-1416
Recombinant β-galactosidase from Lactobacillus plantarum WCFS1, homologously over-expressed in L. plantarum, was purified to apparent homogeneity using p-aminobenzyl 1-thio-β-d-galactopyranoside affinity chromatography and subsequently characterized. The enzyme is a heterodimer of the LacLM-family type, consisting of a small subunit of 35 kDa and a large subunit of 72 kDa. The optimum pH for hydrolysis of its preferred substrates o-nitrophenyl-β-d-galactopyranoside (oNPG) and lactose is 7.5 and 7.0, and optimum temperature for these reactions is 55 and 60 °C, respectively. The enzyme is most stable in the pH range of 6.5-8.0. The Km, kcat and kcat/Km values for oNPG and lactose are 0.9 mM, 92 s−1, 130 mM−1 s−1 and 29 mM, 98 s−1, 3.3 mM−1 s−1, respectively. The L. plantarum β-galactosidase possesses a high transgalactosylation activity and was used for the synthesis of prebiotic galacto-oligosaccharides (GOS). The resulting GOS mixture was analyzed in detail, and major components were identified by using high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) as well as capillary electrophoresis. The maximal GOS yield was 41% (w/w) of total sugars at 85% lactose conversion (600 mM initial lactose concentration). The enzyme showed a strong preference for the formation of β-(1→6) linkages in its transgalactosylation mode, while β-(1→3)-linked products were formed to a lesser extent, comprising ∼80% and 9%, respectively, of the newly formed glycosidic linkages in the oligosaccharide mixture at maximum GOS formation. The main individual products formed were β-d-Galp-(1→6)-d-Lac, accounting for 34% of total GOS, and β-d-Galp-(1→6)-d-Glc, making up 29% of total GOS. 相似文献
7.
Biological kinetic (bio-kinetic) study of the anaerobic stabilization pond treatment of palm oil mill effluent (POME) was carried out in a laboratory anaerobic bench scale reactor (ABSR). The reactor was operated at different feed flow-rates of 0.63, 0.76, 0.95, 1.27, 1.9 and 3.8 l of raw POME for a day. Chemical oxygen demand (COD) as influent substrates was selected for bio-kinetic study. The investigation showed that the growth yield (YG), specific biomass decay (b), maximum specific biomass growth rate (μmax), saturation constant (Ks) and critical retention time (Θc) were in the range of 0.990 g VSS/g CODremoved day, 0.024 day−1, 0.524 day−1, 203.433 g COD l−1 and 1.908 day, respectively. 相似文献
8.
Singh R Wiseman B Deemagarn T Jha V Switala J Loewen PC 《Archives of biochemistry and biophysics》2008,471(2):207-214
Catalase-peroxidases or KatGs from seven different organisms, including Archaeoglobus fulgidus,Bacillus stearothermophilus, Burkholderia pseudomallei, Escherichia coli, Mycobacterium tuberculosis, Rhodobacter capsulatus and Synechocystis PCC 6803, have been characterized to provide a comparative picture of their respective properties. Collectively, the enzymes exhibit similar turnover rates with the catalase and peroxidase reactions varying between 4900 and 15,900 s−1 and 8-25 s−1, respectively. The seven enzymes also exhibited similar pH optima for the peroxidase (4.25-5.0) and catalase reactions (5.75), and high sensitivity to azide and cyanide with IC50 values of 0.2-20 μM and 50-170 μM, respectively. The KMs of the enzymes for H2O2 in the catalase reaction were relatively invariant between 3 and 5 mM at pH 7.0, but increased to values ranging from 20 to 225 mM at pH 5, consistent with protonation of the distal histidine (pKa approximately 6.2) interfering with H2O2 binding to Cpd I. The catalatic kcat was 2- to 3-fold higher at pH 5 compared to pH 7, consistent with the uptake of a proton being involved in the reduction of Cpd I. The turnover rates for the INH lyase and isonicotinoyl-NAD synthase reactions, responsible for the activation of isoniazid as an anti-tubercular drug, were also similar across the seven enzymes, but considerably slower, at 0.5 and 0.002 s−1, respectively. Only the NADH oxidase reaction varied more widely between 10−4 and 10−2 s−1 with the fastest rate being exhibited by the enzyme from B. pseudomallei. 相似文献
9.
Celeste Aida S. Regino 《Inorganica chimica acta》2007,360(14):3971-3977
The effect of bicarbonate on the rates of the H2O2 oxidation of cysteine, gluthathione, and N-acetylcysteine to the corresponding disulfides was investigated. The relative oxidation rates at pH 8 for the different thiols are inversely related to the pKa values of the thiol groups, and the reactive nucleophiles are identified as the thiolate anions or their kinetic equivalents. The second-order rate constants at 25 °C for the reaction of the thiolate anions with hydrogen peroxide are 17 ± 2 M−1 s−1 for all three substrates. In the presence of bicarbonate (>25 mM), the observed rate of thiolate oxidation is increased by a factor of two or more, and the catalysis is proposed to be associated with the formation of peroxymonocarbonate from the equilibrium reaction of hydrogen peroxide with bicarbonate (via CO2). The calculated second-order rate constants for the direct reaction of the three thiolate anions with peroxymonocarbonate fall within the range of 900-2000 M−1 s−1. Further oxidation of disulfides by peroxymonocarbonate results in the formation of thiosulfonate and sulfonate products. These results strongly suggest that peroxymonocarbonate should be considered as a reactive oxygen species in aerobic metabolism with relevance in thiol oxidations. 相似文献
10.
Gustav Vaaje-Kolstad Geoffrey B. Fincher 《Archives of biochemistry and biophysics》2010,496(1):61-361
Three barley xyloglucan endotransglycosylases (HvXETs), known as xyloglucan xyloglucosyl transferases (EC 2.4.1.207), were subjected to kinetic and computational docking studies. The kcat·Km−1 values with the reduced [3H]-labelled XXXG, XXLG/XLXG and XLLG acceptor substrates were 0.02 × 10−2, 0.1 × 10−2 and 3.2 × 10−2 s−1 μM−1, while the Km constants were 10.6, 8.6 and 5.3 mM, obtained for HvXET3, HvXET4 and HvXET6, respectively. Docking of XLLG in acceptor-binding regions revealed that at least two conformational states were likely to participate in all isoforms. The assessments of kinetic and computational data indicated that the disposition of aromatic residues at the entrance to the active sites and the flexibility of proximal COOH-terminal loops could orient acceptors more or less favourably during binding, thus leading to tighter or weaker Km constants. The data suggested that binding of acceptors in HvXETs is guided by contributions from the conserved residues in the active sites and by the of neighbouring loops. 相似文献
11.
Brittany A. Kirk 《Journal of inorganic biochemistry》2010,104(2):207-210
In biological systems, enzymes often use metal ions, especially Mg2+, to catalyze phosphodiesterolysis, and model aqueous studies represent an important avenue of examining the contributions of these ions to catalysis. We have examined Mg2+ and Ca2+ catalyzed hydrolysis of the model phosphodiester thymidine-5′-p-nitrophenyl phosphate (T5PNP). At 25 °C, we find that, despite their different Lewis acidities, these ions have similar catalytic ability with second-order rate constants for attack of T5PNP by hydroxide (kOH) of 4.1 × 10−4 M−1s−1 and 3.7 × 10−4 M−1s−1 in the presence of 0.30 M Mg2+ and Ca2+, respectively, compared to 8.3 × 10−7 M−1s−1 in the absence of divalent metal ion. Examining the dependence of kOH on [M2+] at 50 °C indicates different kinetic mechanisms with Mg2+ utilizing a single ion mechanism and Ca2+ operating by parallel single and double ion mechanisms. Association of the metal ion(s) occurs prior to nucleophilic attack by hydroxide. Comparing the kOH values reveals a single Mg2+ catalyzes the reaction by 1800-fold whereas a single Ca2+ ion catalyzes the reaction by only 90-fold. The second Ca2+ provides an additional 10-fold catalysis, significantly reducing the catalytic disparity between Mg2+ and Ca2+. 相似文献
12.
Chao-Hsiung Lin Chih-Jen Yu Chu-Hsin Hsieh Ying-Feng Chang 《Analytical biochemistry》2009,385(2):224-3296
Fiber-optic biosensors have been studied intensively because they are very useful and important tools for monitoring biomolecular interactions. Here we describe a fluorescence detection fiber-optic biosensor (FD-FOB) using a sandwich assay to detect antibody-antigen interaction. In addition, the quantitative measurement of binding kinetics, including the association and dissociation rate constants for immunoglobulin G (IgG)/anti-mouse IgG, is achieved, indicating 0.38 × 106 M−1 s−1 for ka and 3.15 × 10−3 s−1 for kd. These constants are calculated from the fluorescence signals detected on fiber surface only where the excited evanescent wave can be generated. Thus, a confined fluorescence-detecting region is achieved to specifically determine the binding kinetics at the vicinity of the interface between sensing materials and uncladded fiber surface. With this FD-FOB, the mathematical deduction and experimental verification of the binding kinetics in a sandwich immunoassay provide a theoretical basis for measuring rate constants and equilibrium dissociation constants. A further measurement to study the interaction between human heart-type fatty acid-binding protein and its antibody gave the calculated kinetic constants ka, kd, and KD as 8.48 × 105 M−1 s−1, 1.7 × 10−3 s−1, and 2.0 nM, respectively. Our study is the first attempt to establish a theoretical basis for the florescence-sensitive immunoassay using a sandwich format. Moreover, we demonstrate that the FD-FOB as a high-throughput biosensor can provide an alternative to the chip-based biosensors to study real-time biomolecular interaction. 相似文献
13.
Further development of our recently published Glu(pNA)-containing peptides (Anal. Biochem. 428 (2012) 73–80) provided new fluorogenic substrates for the activated blood coagulation factor XIII. A first series was designed by incorporation of Glu(AMC) at the penultimate position from the N terminus. For the best derivative H-Tyr-Glu(AMC)-Val-Lys-Val-Ile-NH2, a moderate kcat/Km value of 34 s−1 M−1 was determined, which is more than 100-fold reduced compared with the previously reported Glu(pNA) substrates. Furthermore, two fluorescence resonance energy transfer (FRET) substrates were prepared by incorporation of an N-methyl-anthraniloyl fluorophore and a 2,4-dinitrophenyl quencher. Both substrates were excellently cleaved by FXIII-A2∗, which is generated from its zymogen by activation of thrombin in the presence of calcium ions. In the absence and presence of H-Gly-ethyl ester, kcat/Km values of 8010 and 8660 s−1 M−1, respectively, were found for the conversion of H-Lys(N(Me)Abz)-Glu(NH-(CH2)4-NH-Dnp)-Val-Lys-Val-Ile-Gly-NH2 (substrate 8). These values are more than 200-fold improved compared with the Glu(AMC) substrates. Substrate 8 is suitable for the measurement of FXIII-A2∗ activities in plasma samples as well as for in vitro measurements. Furthermore, it was used for the determination of the inhibitory potency of a newly synthesized chloromethyl ketone derivative, Cbz-Phe-Glu(CMK)-Val-Lys-Val-Ile-Gly-NH2, which was found to be a potent irreversible inhibitor of FXIII-A2∗. 相似文献
14.
Digestive physiology and characterization of digestive cathepsin L-like proteinase from the sugarcane weevil Sphenophorus levis 总被引:1,自引:0,他引:1
Soares-Costa A Dias AB Dellamano M de Paula FF Carmona AK Terra WR Henrique-Silva F 《Journal of insect physiology》2011,57(4):462-468
Sugarcane is an important crop that has recently become subject to attacks from the weevil Sphenophorus levis, which is not efficiently controlled with chemical insecticides. This demands the development of new control devices for which digestive physiology data are needed. In the present study, ion-exchange chromatography of S. levis whole midgut homogenates, together with enzyme assays with natural and synthetic substrates and specific inhibitors, demonstrated that a cysteine proteinase is a major proteinase, trypsin is a minor one and chymotrypsin is probably negligible. Amylase, maltase and the cysteine proteinase occur in the gut contents and decrease throughout the midgut; trypsin is constant in the entire midgut, whereas a membrane-bound aminopeptidase predominates in the posterior midgut. The cysteine proteinase was purified to homogeneity through ion-exchange chromatography. The purified enzyme had a mass of 37 kDa and was able to hydrolyze Z-Phe-Arg-MCA and Z-Leu-Arg-MCA with kcat/Km values of 20.0 ± 1.1 μM−1 s−1 and 30.0 ± 0.5 μM−1 s−1, respectively, but not Z-Arg-Arg-MCA. The combined results suggest that protein digestion starts in the anterior midgut under the action of a cathepsin L-like proteinase and ends on the surface of posterior midgut cells. All starch digestion takes place in anterior midgut. These data will be instrumental to developing S. levis-resistant sugarcane. 相似文献
15.
Rouslan V. Olkhov 《Analytical biochemistry》2009,385(2):234-7371
Bovine serum albumin antibodies (aBSA) have been screened from whole leporine anti serum on a biophotonic array. The array was initially printed with seed gold nanoparticles into a 96-spot configuration, and 130-nm gold nanoparticles were synthesised in situ on the surface of each spot. The gold nanoparticle surface was then functionalized with the proteins bovine serum albumin (BSA), fibrinogen, and immunoglobulin G (IgG) and with the amino acid glycine. The concentration of aBSA in the whole serum was determined using a kinetic analysis of the time-dependent light scattering from the nanoparticles. The aBSA-BSA kinetic parameters derived from the array are ka = (1.3 ± 0.3) × 105 M−1 s−1, kd = (4 ± 2) × 10−4 s−1, and KD = 3 nM, which compare favorably with those from continuous gold surfaces. The ultimate sensitivity of the array reader to the bulk refractive index (RI) is 1 × 10−4 refractive index units (RIU), corresponding to 1 μg ml−1 for aBSA. The nanoparticles appear to be more sensitive than the continuous gold surface to the aBSA binding event from whole serum, and this is interpreted in terms of the difference in RI contrast in the plasmon fields. 相似文献
16.
Kyong-Cheol Ko Ju Hee LeeJong Hyun Choi Jae Jun Song 《Biochemical and biophysical research communications》2013
Metagenomic resources representing ruminal bacteria were screened for novel exocellulases using a robotic, high-throughput screening system, the novel CelEx-BR12 gene was identified and the predicted CelEx-BR12 protein was characterized. The CelEx-BR12 gene had an open reading frame (ORF) of 1140 base pairs that encoded a 380-amino-acid-protein with a predicted molecular mass of 41.8 kDa. The amino acid sequence was 83% identical to that of a family 5 glycosyl hydrolase from Prevotella ruminicola 23. Codon-optimized CelEx-BR12 was overexpressed in Escherichia coli and purified using Ni–NTA affinity chromatography. The Michaelis–Menten constant (Km value) and maximal reaction velocity (Vmax values) for exocellulase activity were 12.92 μM and 1.55 × 10−4 μmol min−1, respectively, and the enzyme was optimally active at pH 5.0 and 37 °C. Multifunctional activities were observed against fluorogenic and natural glycosides, such as 4-methylumbelliferyl-β-d-cellobioside (0.3 U mg−1), CMC (105.9 U mg−1), birch wood xylan (132.3 U mg−1), oat spelt xylan (67.9 U mg−1), and 2-hydroxyethyl-cellulose (26.3 U mg−1). Based on these findings, we believe that CelEx-BR12 is an efficient multifunctional enzyme as endocellulase/exocellulase/xylanase activities that may prove useful for biotechnological applications. 相似文献
17.
Francisco J. FernandezDominique de Vries Esther Peña-Soler Miquel CollPhilipp Christen Heinz GehringM. Cristina Vega 《Biochimica et Biophysica Acta - Proteins and Proteomics》2012,1824(2):339-349
The joint substitution of three active-site residues in Escherichia colil-aspartate aminotransferase increases the ratio of l-cysteine sulfinate desulfinase to transaminase activity 105-fold. This change in reaction specificity results from combining a tyrosine-shift double mutation (Y214Q/R280Y) with a non-conservative substitution of a substrate-binding residue (I33Q). Tyr214 hydrogen bonds with O3 of the cofactor and is close to Arg374 which binds the α-carboxylate group of the substrate; Arg280 interacts with the distal carboxylate group of the substrate; and Ile33 is part of the hydrophobic patch near the entrance to the active site, presumably participating in the domain closure essential for the transamination reaction. In the triple-mutant enzyme, kcat′ for desulfination of l-cysteine sulfinate increased to 0.5 s− 1 (from 0.05 s− 1 in wild-type enzyme), whereas kcat′ for transamination of the same substrate was reduced from 510 s− 1 to 0.05 s− 1. Similarly, kcat′ for β-decarboxylation of l-aspartate increased from < 0.0001 s− 1 to 0.07 s− 1, whereas kcat′ for transamination was reduced from 530 s− 1 to 0.13 s− 1. l-Aspartate aminotransferase had thus been converted into an l-cysteine sulfinate desulfinase that catalyzes transamination and l-aspartate β-decarboxylation as side reactions. The X-ray structures of the engineered l-cysteine sulfinate desulfinase in its pyridoxal-5′-phosphate and pyridoxamine-5′-phosphate form or liganded with a covalent coenzyme-substrate adduct identified the subtle structural changes that suffice for generating desulfinase activity and concomitantly abolishing transaminase activity toward dicarboxylic amino acids. Apparently, the triple mutation impairs the domain closure thus favoring reprotonation of alternative acceptor sites in coenzyme-substrate intermediates by bulk water. 相似文献
18.
Florence J. MonlienLothar Helm Amira Abou-HamdanAndré E. Merbach 《Inorganica chimica acta》2002,331(1):257-269
Kinetic studies of X− exchange on [AuX4]− square-planar complexes (where X=Cl− and CN−) were performed at acidic pH in the case of chloride system and as a function of pH for the cyanide one. Chloride NMR study (330-365 K) gives a second-order rate law on [AuCl4]− with the kinetic parameters: (k2Au,Cl)298=0.56±0.03 s−1 mol−1 kg; ΔH2‡ Au,Cl=65.1±1 kJ mol−1; ΔS2‡ Au,Cl=−31.3±3 J mol−1 K−1 and ΔV2‡ Au,Cl=−14±2 cm3 mol−1. The variable pressure data clearly indicate the operation of an Ia or A mechanism for this exchange pathway. The proton exchange on HCN was determined by 13C NMR as a function of pH and the rate constant of the three reaction pathways involving H2O, OH− and CN− were determined: k0HCN,H=113±17 s−1, k1HCN,H=(2.9±0.7)×109 s−1 mol−1 kg and k2HCN,H=(0.6±0.2)×106 s−1 mol−1 kg at 298.1 K. The rate law of the cyanide exchange on [Au(CN)4]− was found to be second order with the following kinetic parameters: (k2Au,CN)298=6240±85 s−1 mol−1 kg, ΔH2‡ Au,CN=40.0±0.8 kJ mol−1, ΔS2‡ Au,CN=−37.8±3 J mol−1 K−1 and ΔV2‡ Au,CN=+2±1 cm3 mol−1. The rate constant observed varies about nine orders of magnitude depending on the pH and HCN does not act as a nucleophile. The observed rate constant of X− exchange on [AuX4]− are two or three orders of magnitude faster than the Pt(II) analogue. 相似文献
19.
Qurra-tul-Ann Afza Gardner Hooria Younas Muhammad Akhtar 《Biochimica et Biophysica Acta - Proteins and Proteomics》2013,1834(1):182-190
Human M-proinsulin was cleaved by trypsin at the R31R32–E33 and K64R65–G66 bonds (B/C and C/A junctions), showing the same cleavage specificity as exhibited by prohormone convertases 1 and 2 respectively. Buffalo/bovine M-proinsulin was also cleaved by trypsin at the K59R60–G61 bond but at the B/C junction cleavage occurred at the R31R32–E33 as well as the R31–R32E33 bond. Thus, the human isoform in the native state, with a 31 residue connecting C-peptide, seems to have a unique structure around the B/C and C/A junctions and cleavage at these sites is predominantly governed by the structure of the proinsulin itself. In the case of both the proinsulin species the cleavage at the B/C junction was preferred (65%) over that at the C/A junction (35%) supporting the earlier suggestion of the presence of some form of secondary structure at the C/A junction. Proinsulin and its derivatives, as natural substrates for trypsin, were used and mass spectrometric analysis showed that the kcat./Km values for the cleavage were most favourable for the scission of the bonds at the two junctions (1.02 ± 0.08 × 105 s− 1 M− 1) and the cleavage of the K29–T30 bond of M-insulin-RR (1.3 ± 0.07 × 105 s− 1 M− 1). However, the K29–T30 bond in M-insulin, insulin as well as M-proinsulin was shielded from attack by trypsin (kcat./Km values around 1000 s− 1 M− 1). Hence, as the biosynthetic path follows the sequence; proinsulin → insulin-RR → insulin, the K29–T30 bond becomes shielded, exposed then shielded again respectively. 相似文献
20.
Kinetic mechanism of the Ca2+-dependent switch-on and switch-off of cardiac troponin in myofibrils
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Solzin J Iorga B Sierakowski E Gomez Alcazar DP Ruess DF Kubacki T Zittrich S Blaudeck N Pfitzer G Stehle R 《Biophysical journal》2007,93(11):3917-3931
The kinetics of Ca2+-dependent conformational changes of human cardiac troponin (cTn) were studied on isolated cTn and within the sarcomeric environment of myofibrils. Human cTnC was selectively labeled on cysteine 84 with N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole and reconstituted with cTnI and cTnT to the cTn complex, which was incorporated into guinea pig cardiac myofibrils. These exchanged myofibrils, or the isolated cTn, were rapidly mixed in a stopped-flow apparatus with different [Ca2+] or the Ca2+-buffer 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid to determine the kinetics of the switch-on or switch-off, respectively, of cTn. Activation of myofibrils with high [Ca2+] (pCa 4.6) induced a biphasic fluorescence increase with rate constants of >2000 s−1 and ∼330 s−1, respectively. At low [Ca2+] (pCa 6.6), the slower rate was reduced to ∼25 s−1, but was still ∼50-fold higher than the rate constant of Ca2+-induced myofibrillar force development measured in a mechanical setup. Decreasing [Ca2+] from pCa 5.0-7.9 induced a fluorescence decay with a rate constant of 39 s−1, which was approximately fivefold faster than force relaxation. Modeling the data indicates two sequentially coupled conformational changes of cTnC in myofibrils: 1), rapid Ca2+-binding (kB ≈ 120 μM−1 s−1) and dissociation (kD ≈ 550 s−1); and 2), slower switch-on (kon = 390s−1) and switch-off (koff = 36s−1) kinetics. At high [Ca2+], ∼90% of cTnC is switched on. Both switch-on and switch-off kinetics of incorporated cTn were around fourfold faster than those of isolated cTn. In conclusion, the switch kinetics of cTn are sensitively changed by its structural integration in the sarcomere and directly rate-limit neither cardiac myofibrillar contraction nor relaxation. 相似文献