共查询到20条相似文献,搜索用时 15 毫秒
1.
《Journal of Molecular Catalysis .B, Enzymatic》1999,6(1-2):99-110
α-Chymotrypsin (α-CT) activity was tested in aqueous media with the following cetyltrialkylammonium bromide surfactants in the series methyl, ethyl, propyl and butyl, different in the head group size, and for the sake of comparison also with the anionic sodium n-dodecyl sulfate and the zwitterionic myristyldimethylammonium propanesulfonate. N-glutaryl-l-phenylalanine p-nitroanilide hydrolysis rate was monitored at surfactant concentration above the critical micellar one. Only some cationic surfactants gave superactivity and the head group size had a major weight. The highest superactivity was measured in the presence of cetyltributylammonium bromide. The effect of both nature and concentration of three different buffers was also investigated. There is a dependence of enzyme superactivity on buffer type. Michaelis–Menten kinetics were found. The binding constants of substrate with micellar aggregates were determined in the used buffers and the effective improvement of reaction rate (at the same free substrate concentration in the medium) was calculated. kcat significantly increased while Km was little changed after correction to free substrate concentration. The ratio of kcat to Km was between 12 and 35 times higher than in pure buffer, depending on buffer and surfactant concentrations. The increase of α-CT activity (30%) was less important in the presence of 1×10−2 M tetrabutylammonium bromide, a very hydrophobic salt, unable to micellise. Fluorescence spectra showed differences of enzyme conformation in the presence of various surfactants. 相似文献
2.
Mikhailova AG Rumsh LD Dalgalarrondo M Chobert JM Haertle T 《Biochemistry. Biokhimii?a》2003,68(8):926-933
Temperature dependences of kinetic constants (k
cat and K
m) were studied for enzymatic hydrolysis of N-succinyl-L-alanyl-L-alanyl-L-prolyl-L-arginine-p-nitroanilide and N-succinyl-L-alanyl-L-alanyl-L-prolyl-L-lysine-p-nitroanilide by bovine cationic and rat anionic (wild-type and mutant) trypsins. The findings were compared with the corresponding literature data for hydrolysis of N-benzoyl-DL-arginine-p-nitroanilide by bovine cationic trypsin and natural trypsins of coldadapted fishes. The anionic and cationic trypsins were found to differ in organization of the S1 -substrate-binding pocket. The difference in the binding of lysine and arginine residues to this site (S1) was also displayed by opposite temperature dependences of hydrolysis constants for the corresponding substrates by the anionic and cationic trypsins. The data suggest that the effect of any factor on the binding of substrates (the K
m value) to the anionic and cationic trypsins and on the catalytic activity k
cat should be compared only with the corresponding data for the natural enzyme of the same type. Mutants of rat anionic trypsin at residues K188 or Y228 were prepared by site-directed mutagenesis as approximate models of natural psychrophilic trypsins. Substitution of the charged lysine residue in position 188 by hydrophobic phenylalanine residue shifted the pH optimum of the resulting mutant trypsin K188F from 8.0 to 9.0-10.0, similarly to the case of some natural psychrophilic trypsins, and also 1.5-fold increased its catalytic activity at low temperatures as compared to the wild-type enzyme. 相似文献
3.
The interaction of the cationic Gemini surfactant hexamethylene‐1,3‐bis (tetradecyldimethylammonium bromide) (14‐6‐14) with bovine serum albumin (BSA) has been investigated by fluorescence quenching spectra and three‐dimensional (3D) fluorescence spectra. The Stern–Volmer quenching constants KSV and the corresponding thermodynamic parameters ΔH, ΔG and ΔS have been estimated by the fluorescence quenching method. The results indicated that hydrophobic forces were the predominant intermolecular forces between BSA and the surfactant. Competitive experiments and the number of binding sites calculation show that 14‐6‐14 can be inserted in site‐II (in subdomain IIIA) of BSA. The effect of 14‐6‐14 on the conformation of BSA was evaluated by synchronous fluorescence spectroscopy and 3D fluorescence spectral methods. The results show that the conformation of BSA was changed dramatically in the presence of 14‐6‐14, by binding to the Trp and Try residues of BSA. The investigation provides interaction between BSA and 14‐6‐14 as a model for molecular design and industrial research. Copyright © 2011 John Wiley & Sons, Ltd. 相似文献
4.
In this study, we synthesized a zwitterionic DTNB derivative, 5-(2-aminoethyl)-dithio-2-nitrobenzoate (ADNB), and characterized its reactions with several cationic, anionic, and neutral thiols. Reactions with ADNB, unlike those with DTNB, are relatively insensitive to electrostatic environments and ionic strengths. At relatively low ionic strength, rate ratios,kADNB/kDTNB, varied from 0.22 for reactions with low-molecular-weight cationic thiols to 3.0 for those with low-molecular-weight anionic thiols. AkADNB/kDTNBratio of 200 for Cys-34 of BSA appears to reflect a very anionic environment.kADNB/kDTNBratios of 6 and 1, respectively, for canine and equine serum albumins, which have Glu-82 → Asp and Glu-82 → Ala substitutions suggest Glu-82 is the most important anionic residues affecting the reactivity of Cys-34 in BSA.kADNB/kDTNBratios appear to be useful for characterizing electrostatic environments of thiol groups in proteins. 相似文献
5.
Previous studies of the rate constants for the elementary steps of ATP hydrolysis by the soluble and membrane-bound forms of beef heart mitochondrial F1 supported the proposal that ATP is formed in high-affinity catalytic sites of the enzyme with little or no change in free energy and that the major requirement for energy in oxidative phosphorylation is for the release of product ATP.The affinity of the membrane-bound enzyme for ATP during NADH oxidation was calculated from the ratio of the rate constants for the forward binding step (k
+1) and the reverse dissociation step (k
–1).k
–1 was accelerated several orders of magnitude by NADH oxidation. In the presence of NADH and ADP an additional enhancement ofk
–1 was observed. These energy-dependent dissociations of ATP were sensitive to the uncoupler FCCP.k
+1 was affected little by NADH oxidation. The dissociation constant (K
d
ATP) increased many orders of magnitude during the transition from nonenergized to energized states. 相似文献
6.
Abstract The periplasmic enzyme β-lactamase was selectively released from Escherichia coli K12 by the amphiphilic quaternary ammonium compound tetradecyl betainate at certain concentration intervals. At low concentrations little enzyme was released, and at high concentrations enzyme inactivation occurred. Greater effects of tetradecyl betainate were seen both with respect to release and inactivation at higher pH. At intermediate concentrations of tetradecyl betainate high yields of β-lactamase were obtained with no detectable contribution of the cytoplasmic marker β-galactosidase. The highest yields of β-lactmase activity were obtained when high concentrations of salt were added 1 min after permeation of the bacteria with tetradecyl betainate. 相似文献
7.
Maw-Song Wang Richard D. Gandour James Rodgers John L. Haslam Richard L. Schowen 《Bioorganic chemistry》1975,4(4):392-406
The rate constants k12n for isomerization of the E1H isomer (pKa 8 in H2O) of ribonuclease-A to the E2H isomer (pKa = 6.1 in H2O), determined from proton-uptake measurements by the temperature-jump technique, in mixtures of protium and deuterium oxides (atom fraction of deuterium n), are described by the equation k12n = (733 ± 16)(1 − n + [0.46 ± 0.04]n)(1 − n + 0.69n)2sec−1 at 25°C. On the basis of the absolute magnitude of the rate constant, the magnitude of the solvent isotope effect and the proton inventory, it appears that the rate-determining step is proton transfer to a water molecule from the imidazolium form of a histidine residue, with a product-like activated complex resembling a hydronium ion. The subsequent motion of the protein structure to generate the new isomer (conformation change) must then occur in a time approaching a vibrational period. Alternative but less likely mechanisms include rate-limiting protein reorganization concerted with proton transfer to water, rate-limiting diffusion of hydronium ion away from the enzyme, or “solvation catalysis” of protein reorganization. 相似文献
8.
Effective hydrolysis of chitosan, the N-deacetylated product of chitin, remains challenging. Here, we report acid-promoted hydrolysis of chitosan in imidazolium based ionic liquids with good total reducing sugars (TRS) yield under mild conditions. TRS yield reached over 60% in the presence of about 6.0 wt% concentrated hydrochloric acid at 100 °C within 7 h. Kinetic modeling of a typical experimental data set suggested that the hydrolysis most likely followed a consecutive first-order reaction sequence, where k1 and k2, the rate constants for TRS formation and degradation, were determined to be 0.01372 and 0.00015 min−1, respectively. Our method may be useful to explore new applications of natural chitin resources. 相似文献
9.
Charge-pulse relaxation experiments of valinomycin-mediated Rb+ transport have been carried out in order to study the influence of membrane structure on carrier kinetics. From the experimental data the rate constants of association (kR) and dissociation (kD) of the ion-carrier complex as well as the rate constants of translocation of the complex (kMS) and of the free carrier (kS) could be obtained. The composition of the planar bilayer membrane was varied in a wide range. In a first series of experiments, membranes made from glycerolmonooleate dissolved in different n-alkanes (n-decane to n-hexadecane), as well as solvent-free membranes made from the same lipid by the Montal-Mueller technique were studied. The translocation rate constants kS and kMS were found to differ by less than a factor of two in the membranes of different solvent content. Much larger changes of the rate constants were observed if the structure of the fatty acid residue was varied. For instance, an increase in the number of double bonds in the C20 fatty acid from one to four resulted in an increase of kS by a factor of seven and in an increase of kMS by a factor of twenty-four. The stability constant K = kR/kD of the ion-carrier complex as well as the translocation rate constants kS and kMS were found to depend strongly on the nature of the polar headgroup of the lipid. The incorporation of cholesterol into glycerolmonooleate membranes reduced kR, kMS and kS up to seven-fold. 相似文献
10.
P.J. White 《The Journal of membrane biology》1998,164(3):275-281
The maxi cation channel from the plasma membrane of rye (Secale cereale L.) roots was studied following its incorporation into planar phosphatidylethanolamine bilayers. Current recordings were
made in the presence of 100-mm KCl containing quinine on both sides of the bilayer. Quinine produced voltage- and concentration-dependent blockade of the
channel, reducing its apparent unitary current and open probability. The voltage-dependence suggested that blockade was effected
from the cytoplasmic side by cationic quinine. Blockade was modelled using a kinetic scheme with two independent blocked states
termed B1 and B2 (B1⇆O⇄B2). Rate constants promoting fast kinetics (k
1
and k
−1
) were found to be several orders of magnitude greater than those promoting slow kinetics (k
2
and k
−2
). Analysis of the fast kinetics indicated that the rate constants for blockade of the open channel at the first site (k
1
) and its clearance (k
−1
) had voltage-dependencies (zδ
p
) of 0.41 and −0.71, respectively, and that the equilibrium dissociation constant for the binding site (K
d
(0)) was about 1 mm. Analysis of the slow kinetics indicated that the rate constants for blockade of the open channel at the second site (k
2
) and its clearance (k
−2
) had zδ
p
values of 0.12 and −1.27, respectively. The K
d
(0) value for the second binding site was about 10 mm.
Received: 20 January 1998/Revised: 1 May 1998 相似文献
11.
Kinetics of the steady-state oxidation of n–alkylferrocenes (alkyl = H, Me, Et, Bu and C5H11) by H2O2 to form the corresponding ferricenium cations catalyzed by horseradish peroxidase has been studied in micellar systems of
Triton X-100, CTAB, and SDS, mostly at pH 6.0 and 25 °C. The rate of oxidation of ferrocenes with longer alkyl radicals is
too slow to be measured. The reaction obeying the [RFc]:[H2O2] = 2 : 1 stoichiometry is strictly first-order in both HRP and RFc in a wide concentration range. The corresponding observed
second-order rate constants k, which refer to the interaction of the peroxidase compound II (HRP-II) with RFc, decrease with the elongation of the alkyl
substituent R, and this in turn is accompanied by an increase in the formal redox potentials E°′ in the same medium. Increasing the surfactant concentration lowers the rate constants k, the effect being due to the nonproductive binding of RFc to micelles rather than to enzyme inactivation. The micellar effects
are accounted for in terms of the Berezin pseudo-phase model of micellar catalysis applied to the interaction of enzyme with
organometallic substrates. The oxidation was found to occur primarily in the aqueous pseudo-phase and the calculated intrinsic
second-order rate constants k
w are (1.9 ± 0.5)×105, (2.7 ± 0.1)×104, and (5.9 ± 0.6)×103 M–1 s–1 for HFc, EtFc, and n–BuFc, respectively. The data obtained were used for estimating the self-exchange rate constants for the HRP-II/HRP couple
in terms of the Marcus formalism.
Received: 15 July 1996 / Accepted: 15 November 1996 相似文献
12.
The association of coenzyme A(CoASH) and glutathione (GSH) with the water-soluble polymers and their esterolytic reactivities were evaluated through the reaction with p-nitrophenyl acetate in the presence of cationic polymer micelles: partially laurylated poly(2-ethyl-1-vinylimidazole) and poly(4-vinylpyridine). The polymer micelles with high lauryl-group content (more than 12 mol%) markedly accelerated the reaction at very low concentrations of the polymer. Other polymers with no or small lauryl-group content only slightly enhanced the association and the reaction rate. From the rate-polymer concentration profiles, the association constants (K) and the rate constants for thiol coenzymes bound to the polymer (k′a,bound) were determined: for polymers with more than 12 mol % lauryl-group content, KCoASH = 1110–2270 M?1, KGSH = 170–503M?1, k′a,bound at pH 8.65 = 142–341M?1 sec?1. k′a,bound were 20–340 times larger than that observed in the absence of the polymer. The logarithm of k′a,bound was found to be correlated well with the polymer hydrophobicity, indicating that the hydrophobic environment of the polymer activated the bound thiol anions. On the other hand, the polymer hydrophobicity did not correlate with the association constant. 相似文献
13.
The maximum at 220 nm in the circular dichroism spectrum of native collagen solution changed to a negative value after heat denaturation or collagenase hydrolysis. The enzyme induced rate of CD change at 220 nm was shown to be first order in collagen concentration. The specific rate constant k is actually a combined rate constant kfast and kslow in which the ratio kf/ks is 4.1. The initial rates were linear with respect to enzyme concentration, and the Km was found to be 5.5 × 10?7 M. The rate of ultraviolet hyperchromicity at 220 nm on collagen hydrolysis was determined. The kfast was the same as that obtained by CD. The kf/ks ratio was 4.6. Both methods may be readily used to assay for collagenase activity. 相似文献
14.
Anastassia Kanavarioti Sherwood Chang Diann J. Alberas 《Journal of molecular evolution》1990,31(6):462-469
Summary Selected imidazolide-activated nucleotides have been subjected to hydrolysis under conditions similar to those that favor their template-directed oligomerization. Rate constants of hydrolysis of the P–N bond in guanosine 5-monophosphate 2-methylimidazolide (2-MeImpG) and in guanosine 5-monophosphate imidazolide (ImpG), kh, have been determined in the presence/absence of magnesium ion as a function of temperature and polycytidylate [poly(C)] concentration. Using the rate constant of hydrolysis of 2-MeImpG and the rate constant of elongation, i.e., the reaction of an oligoguanylate with 2-MeImpG in the presence of poly(C) acting as template, the limiting concentration of 2-MeImpG necessary for oligonucleotide elongation to compete with hydrolysis can be calculated. The limiting concentration is defined as the initial concentration of monomer that results in its equal consumption by hydrolysis and by elongation. These limiting concentrations of 2-MeImpG are found to be 1.7 mM at 37°C and 0.36 mM at 1°C. Boundary conditions in the form of limiting concentration of activated nucleotide may be used to evaluate a prebiotic model for chemical synthesis of biopolymers. For instance, the limiting concentration of monomer can be used as a basis of comparison among catalytic, but nonenzymatic, RNA-type systems.We also determined the rate constant of dimerization of 2-MeImpG, k2=0.45±0.06 M–1 h–1 in the absence of poly(C), and 0.45±0.06k20.97±0.13 M–1 h–1 in its presence at 37°C and pH 7.95. This dimerization, as well as the trimerization of 2-MeImpG, which represent the first steps in the oligomerization reaction, are markedly slower than the elongation of longer oligoguanylates, (pG)
n
n>6. This means that in the presence of low concentrations of 2-MeImpG (1.7 mM) the system directs the elongation of longer oligomers more efficiently than the formation of short oligomers such as dimers and trimers. These results will be discussed as a possible example of chemical selection in template-directed reactions of nucleotides. 相似文献
15.
Eugenia Reynoso R. Daniel Cacciari Carlos A. Suchetti Hernn A. Montejano M. Alicia Biasutti 《Luminescence》2019,34(3):324-333
Photosensitized oxidation of bovine serum albumin (BSA), by using perinaphtenone as a sensitizer, has been studied at pH 7.4 and 11. The selected sensitizer does not present ground‐state complexation with BSA and ensures that the mechanism is mediated by O2(1△g). Strong dependence between BSA–O2(1△g) photo‐oxidation and the pH of the medium has been found. The relative oxygen uptake rate (v? △ O2 ) and the total quenching rate constant (kt) values are higher at pH 11 than pH 7.4. The enhancement in the alkaline condition is due to conformational changes in the protein and the reactivity of tyrosinate anion with O2(1△g). Even when the tendency with the pH in the presence of sodium dodecyl sulfate (SDS) micelles is similar to that observed in homogeneous media, an increment on the kt value is detected. This effect may be attributable to the strong interaction of BSA–SDS, which leads to the protein unfolding and could leave more exposed photo‐oxidizable amino acids. A protective effect against the O2(1△g)‐mediated photo‐oxidation was observed in reverse micelles (RMs) of sodium bis(2‐ethylhexyl)sulfosuccinate (AOT) by comparing the kt values obtained at W = 10 with respect to the one obtain in homogeneous media. The latter could be mainly explained by the modification in the solvent polarity. Also, another important observation was found, the internal pH inside RMs of AOT sensed through tyrosine absorption was independent of the one used for the formation of the water pool. Hence, the kt values observed at both pH, are quite similar. 相似文献
16.
《Bioscience, biotechnology, and biochemistry》2013,77(4):946-950
The kinetics of glucoamylase-catalyzed hydrolysis of starch granules from six different botanical sources (rice, wheat, maize, cassava, sweet potato, and potato) was studied by the use of an electrochemical glucose sensor. A higher rate of hydrolysis was obtained as a smaller size of starch granules was used. The adsorbed amount of glucoamylase on the granule surface per unit area did not vary very much with the type of starch granules examined, while the catalytic constants of the adsorbed enzyme (k 0) were determined to be 23.3±4.4, 14.8±6.0, 6.2±1.8, 7.1±4.1, 4.6±3.0, and 1.6±0.6 s?1 for rice, wheat, maize, cassava, sweet potato, and potato respectively, showing that k 0 was largely influenced by the type of starch granules. A comparison of the k 0-values in relation to the crystalline structure of the starch granules suggested that k 0 increases as the crystalline structure becomes dense. 相似文献
17.
根据过渡态理论设计和合成了能诱导产生催化选择性水解布洛芬甲酯的催化抗体的四面体硫酸盐半抗原,并与牛血清白蛋白(BSA)偶联制备成免疫源,通过免疫手段成功筛选出具有加速选择性水解生成S-布洛芬的特异性催化抗体.其Kcat,app/Kuncat,app达1.6x104.进一步地将催化抗体运用到W/O微乳体系(反胶束)中进行布洛芬酯的选择性水解研究,其动力学研究证明其催化过程同样遵循Michaelis.Menten方程.考察了pH值和温度对催化初速度影响,Wo(体系中水和琥珀酸二辛酯磺酸钠(AOT)的摩尔比)对催化初速度影响呈现为钟罩型,最适的Wo.为21. 相似文献
18.
Andrew R. Draganski Rashmi S. Tiwari Kasi V. Sundaresan Thomas J. Nack Yumin You Richard D. Ludescher 《Food biophysics》2010,5(4):337-345
The properties of amorphous solid proteins influence the texture and stability of low-moisture foods, the shelf-life of pharmaceuticals,
and the viability of seeds and spores. We have investigated the relationship between molecular mobility and oxygen permeability
in dry food protein films—bovine α-lactalbumin (α-La), bovine β-lactoblobulin (β-Lg), bovine serum albumin (BSA), soy 11S
globulin, and porcine gelatin—using phosphorescence from the triplet probe erythrosin B. Measurements of the phosphorescence
decay in the absence (nitrogen) and presence (air) of oxygen versus temperature provide estimates of the non-radiative decay
rate for matrix-induced quenching (k
TS0) and oxygen quenching (k
Q[O2]) of the triplet state. Since the oxygen quenching constant is the product of the oxygen solubility ([O2]) and a term (k
Q) proportional to the oxygen diffusion coefficient, it is a measure of the oxygen permeability through the films. For all
proteins except gelatin, Arrhenius plots of k
TS0 reveal a gradual increase of apparent activation energy across a broad temperature range starting at ∼50 °C; this suggests
that there is a steady increase in the available modes of molecular motion with increasing temperature within the protein
matrix. Arrhenius plots for k
Q[O2] were linear for all proteins with activation energies ranging from 24 to 29 kJ/mol. The magnitude of the oxygen quenching
constants varied in the different proteins; the rates were approximately 10-fold higher in α-La, β-Lg, and BSA than in 11S
glycinin and gelatin. Although the rate of oxygen permeability was not directly affected by the increased mobility of the
protein matrix, plots of k
Q[O2] versus k
TS0 were linear over nearly three orders of magnitude in the protein films, suggesting that the matrix mobility plays a specific
role in modulating oxygen permeability. This effect may reflect differences in matrix-free volume that directly influence
both mobility and oxygen solubility. 相似文献
19.
The kinetics of t-[3H]butylbicycloorthobenzoate (TBOB) binding to the convulsant sites of the γ-aminobutyric acidA (GABAA) receptor-ionophore complex were examined in synaptosomal membrane preparations of rat brain. On and off rates of TBOB binding were accelerated by 1 μM GABA and decelerated by 1 μM bicuculline methochloride, a GABAA antagonist. The presence of GABA and bicuculline methochloride created rapid and slow phases of dissociation, respectively. The three groups of rate constants distinguished for the dissociation of 4 nM and 30 nM [3H]TBOB represent multiaffinity states of the convulsant sites depending on the presence of GABA or bicuculline methochloride. Apparent association rate constants do not obey the equation kapp=koff±kon [TBOB] without assuming interconvertibility of the kinetic states during binding. Avermectin B1a (AVM B1a), a chloride channel opening agent, accelerated the association and dissociation of TBOB and resulted in a biphasic effect on TBOB binding, i.e., enhancement at low concentrations (EC50, 7.8 nM) followed by displacement at high concentrations (IC50 6.3 μM) of AVM B1a. AVM B1a resulted in similar biphasic effects on t- [35S]butylbicyclophosphorothionate binding. DIDS, an isothiocyanatostilbene derivative with irreversible anion channel blocking effect, selectively inhibited basal [3H]TBOB binding (IC50 125 μM DIDS) leaving the enhancement by AVM B1a unaffected. 相似文献
20.
《Bioorganic & medicinal chemistry letters》2014,24(14):3046-3049
Several triplex-forming oligonucleotides (TFOs) partially modified with 2′-O-(2-aminoethyl)- or 2′-O-(2-guanidinoethyl)-nucleotides were synthesized and their association rate constants (kon) with double-stranded DNA were estimated by UV spectrophotometry. Introduction of cationic modifications in the 5′-region of the TFOs significantly increased the kon values compared to that of natural TFO, while no enhancement in the rate of triplex DNA formation was observed when the modifications were in the middle and at the 3′-region. The kon value of a TFO with three adjacent cationic modifications at the 5′-region was found to be 3.4 times larger than that of a natural one. These results provide useful information for overcoming the inherent sluggishness of triplex DNA formation. 相似文献