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1.
Thermal stability of the hemagglutinin-neuraminidase from Sendai virus evidences two folding domains
The domain structure of hemagglutinin-neuraminidase from Sendai virus (cHN) was investigated by studying the thermal stability in the 20-100 degrees C range. Differential scanning calorimetry evidences two conformational transitions. The first transition is apparently a reversible two-state process, with Tm 48.3 degrees C, and is shifted to 50.1 degrees C in the presence of the substrate analogue 2,3-dehydro-2-deoxy-N-acetyl neuraminic acid, meaning that the substrate binding domain is involved in the transition. The second transition, with apparent Tm 53.2 degrees C, is accompanied by irreversible loss of enzymatic activity of the protein, and the presence of the substrate analogue does not affect the Tm. The data indicate that cHN is composed of two independent folding domains, and that only one domain is involved in the binding of the substrate. Our results suggest that the paramyxovirus neuraminidases have the folding properties of a two-domain protein. 相似文献
2.
NADP+-linked isocitrate dehydrogenase (E.C.1.1.1.42) has been purified to homogeneity from germinating pea seeds. The enzyme is a tetrameric protein (mol wt, about 146,000) made up of apparently identical monomers (subunit mol wt, about 36,000). Thermal inactivation of purified enzyme at 45 degrees and 50 degrees C shows simple first order kinetics. The enzyme shows optimum activity at pH range 7.5-8. Effect of substrate [S] on enzyme activity at different pH (6.5-8) suggests that the proton behaves formally as an "uncompetitive inhibitor". A basic group of the enzyme (site) is protonated in this pH range in the presence of substrate only, with a pKa equal to 6.78. On successive dialysis against EDTA and phosphate buffer, pH 7.8 at 0 degrees C, yields an enzymatically inactive protein showing kinetics of thermal inactivation identical to the untreated (native) enzyme. Maximum enzyme activity is observed in presence of Mn2+ and Mg2+ ions (3.75 mM). Addition of Zn2+, Cd2+, Co2+ and Ca2+ ions brings about partial recovery. Other metal ions Fe2+, Cu2+ and Ni2+ are ineffective. 相似文献
3.
Pyruvate kinase, activated by fructose-1,6-biphosphate from Salmonella typhimurium LT-2, has been isolated and purified to homogeneity. The enzyme, similar to that from Escherichia coli, is a tetramer with an approximate molecular weight of 240,000. The native enzyme shows optimum pH 6.8 (T = 30 degrees C). The enzymatic reaction does not require K+ ions; while Mg2+ or Mn2+ are essential for its activity. The non-activated enzyme shows sigmoid kinetics to phosphoenolpyruvate with a Hill coefficient of 2.73; the activated enzyme becomes michaelian with KSADP y KSPEP 0.25 and 0.08 mM, respectively. Both substrates excess and ATP cause enzyme inhibition. In agreement with the experimental results a steady-state random-ordered hybrid Bi-Bi mechanism with two dead-end complexes is proposed. 相似文献
4.
The kinetics of refolding of ribonuclease A were monitored by the return of catalytic activity and inhibitor binding at -15 degrees C in 35% methanol cryosolvent at pH* 3.0 and 6.0. Catalytic activity was measured with cytidine 2',3'-cyclic monophosphate as substrate; inhibitor binding was determined with the competitive inhibitor cytidine 2'-monophosphate. Biphasic kinetics were observed at pH* 3.0 for both return of catalytic activity and inhibitor binding. At pH* 6.0 the rate of return of catalytic activity was monophasic, whereas that of inhibitor binding was biphasic. For both inhibitor binding and catalytic activity one of the observed rates was pH-dependent. Full return of catalytic activity was obtained at the completion of the refolding process. The observations are interpreted in terms of two parallel pathways of refolding for slow-refolding ribonuclease, with several native-like, partially folded intermediate states on the minor slow-refolding pathway. Of particular note is the presence of at least one such species that has inhibitor-binding capacity but not catalytic activity. This may be rationalized in terms of the known native structure. In addition, an intermediate is postulated which has the incorrect Pro-93 conformation and only partial catalytic activity (42% of the native). The slowest observed transient is attributed to the isomerization of this proline residue and return of full catalytic activity. 相似文献
5.
Unaltered catabolism of desialylated low-density lipoprotein in the pig and in cultured rat hepatocytes.
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A D Attie D B Weinstein H H Freeze R C Pittman D Steinberg 《The Biochemical journal》1979,180(3):647-654
Removal of the terminal sialic acid residues from many serum glycoproteins results in exposure of their penultimate galactose residues and rapid clearance from circulation by the liver. Low-density lipoprotein is a glycoprotein containing 21 galactose and 9 sialic acid residues per particle. Studies in this laboratory and others have shown that both the liver and extrahepatic tissues contribute to the degradation of low-density lipoprotein. This study was undertaken to determine whether desialylation of pig low-density lipoprotein alters its removal from circulation. Low-density lipoprotein was incubated at 37 degrees C with an agarose-bound neuraminidase, proteinase-free, from Clostridium perfringens. After 18 h at pH 5.0, 70% of the sialic acid residues were removed. The desialylated 131I-labelled and native 125I-labelled low-density lipoproteins were simultaneously injected into a pig, and their disappearance from plasma was followed for 96 h. The turnovers of the two were identical. In contrast, neuraminidase-treated fetuin was cleared about 200-fold faster than native fetuin. Studies were also performed in cultured rat hepatocytes. Rates of degradation of native and neuraminidase-treated low-density lipoprotein were similar, whereas asialo-fetuin was degraded at six to ten times the rate of native fetuin. Thus desialylation does not appear to alter low-density-lipoprotein catabolism by hepatic or extrahepatic cells. 相似文献
6.
R C Bateman Y A Kim C Slaughter L B Hersh 《The Journal of biological chemistry》1990,265(15):8365-8368
Neutral endopeptidase 24.11 is rapidly inactivated by N-bromoacetyl-D-leucylglycine in a reaction which follows first-order kinetics at pH 8 and 37 degrees C. The concentration dependence of inactivation revealed saturation kinetics with an apparent Ki of 10 mM and kappa inact of 0.4 min-1 at saturating inhibitor concentration. Enzyme can be protected from inactivation by either the substrate Leu5-enkephalin or the competitive inhibitors Phe-Gly or Phe-Ala. Inactivation of enzyme by N-bromo-[14C]acetyl-D-leucylglycine proceeds with the incorporation of a stoichiometric amount of labeled inhibitor. Tryptic digestion of the radioactively labeled enzyme followed by high performance liquid chromatography allowed the isolation of a modified peptide with the sequence T-D-V-H-S-P-G-N-F-R in which histidine (His704) is the modified residue. Site-directed mutagenesis was used to generate a mutant form of the enzyme in which histidine 704 was converted to a glutamine residue. This mutant enzyme retained less than 0.1% of the activity of the native enzyme. These results demonstrate that His704 is at the active site of neutral endopeptidase 24.11 and suggest a catalytic role for this residue. 相似文献
7.
Regulation of osteogenesis by fetuin. 总被引:5,自引:0,他引:5
C Binkert M Demetriou B Sukhu M Szweras H C Tenenbaum J W Dennis 《The Journal of biological chemistry》1999,274(40):28514-28520
8.
M D Fideu M J Herranz M Ruíz-Amil M L Pérez 《Comparative biochemistry and physiology. B, Comparative biochemistry》1985,81(4):1067-1071
Glycolytic enzyme phosphofructokinase (PFK) from sea-bass liver shows inhibition for ATP4- and MG-ATP2-, and ATP4- is a competitive inhibitor with respect to MG-ATP2-. Free Mg2+ behaves as a mixed inhibitor on the kinetic with respect to the true enzyme substrate Mg-ATP2-, and eliminates the inhibition effect of this substrate. The kinetics with respect to Mg-ATP2- at non-inhibiting concentrations is not visibly affected by temperature of pH variation. The inhibiting effect of Mg-ATP2- is more marked at 22 and 10 degrees C (of three assayed temperatures 22, 15 and 10 degrees C and at physiological pH 6.8) as opposed to the maximum activity pH (8.0). 相似文献
9.
The inactivation of a metalloproteinase from Pseudomonas fluorescens Biotype I with EDTA was investigated at 22 degrees C and 37 degrees C. At 22 degrees C proteolytic activity decreases linearly with time and an inactive apoenzyme is obtained by dialysis. Proteolytic activity can be restored with several metal-ions, Ca2+, Zn2+, Mg2+, Sr2+ and co2+ give the best results. Activity and substrate specificity are influenced by the metal-ions. Reactivation depends on the concentration of the metal-ions, optimum concentration is 1 mM for Ca2+ and 50 microM for Zn2+. The isoelectric point of the apoenzyme is around 8.0, this is about 0.3 pH-units lower than the isoelectric point of the native proteinase. At 37 degrees C inactivation follows first order kinetics and is irreversible because of autolysis as shown by a gel filtration-experiment. 相似文献
10.
Marko Golicnik Goran Sinko Vera Simeon-Rudolf Zoran Grubic Jure Stojan 《Archives of biochemistry and biophysics》2002,398(1):23-31
The action of a potent tricyclic cholinesterase inhibitor ethopropazine on the hydrolysis of acetylthiocholine and butyrylthiocholine by purified horse serum butyrylcholinesterase (EC 3.1.1.8) was investigated at 25 and 37 degrees C. The enzyme activities were measured on a stopped-flow apparatus and the analysis of experimental data was done by applying a six-parameter model for substrate hydrolysis. The model, which was introduced to explain the kinetics of Drosophila melanogaster acetylcholinesterase [Stojan et al. (1998) FEBS Lett. 440, 85-88], is defined with two dissociation constants and four rate constants and can describe both cooperative phenomena, apparent activation at low substrate concentrations and substrate inhibition by excess of substrate. For the analysis of the data in the presence of ethopropazine at two temperatures, we have enlarged the reaction scheme to allow primarily its competition with the substrate at the peripheral site, but the competition at the acylation site was not excluded. The proposed reaction scheme revealed, upon analysis, competitive effects of ethopropazine at both sites; at 25 degrees C, three enzyme-inhibitor dissociation constants could be evaluated; at 37 degrees C, only two constants could be evaluated. Although the model considers both cooperative phenomena, it appears that decreased enzyme sensitivity at higher temperature, predominantly for the ligands at the peripheral binding site, makes the determination of some expected enzyme substrate and/or inhibitor complexes technically impossible. The same reason might also account for one of the paradoxes in cholinesterases: activities at 25 degrees C at low substrate concentrations are higher than at 37 degrees C. Positioning of ethopropazine in the active-site gorge by molecular dynamics simulations shows that A328, W82, D70, and Y332 amino acid residues stabilize binding of the inhibitor. 相似文献
11.
Butyrylcholinesterase purified from human serum as 6600-fold was heated at 37°, 40°, 45°, and 50°C for 24 hr. It was observed that the enzyme heated at 45°C for 24 hr converted to a stabilized form and followed Michaelis-Menten kinetics, whereas the enzyme samples, heated at the other temperatures for 24 hr, shown negative cooperativity with respect to its substrate, butyrylthiocholine. Even the sample heated at 45°C for 12 hr shown negative cooperativity. On the contrary to the heated enzyme at 40°C for 24 hr, the heated enzyme at 45°C for 24 hr could not be reactivated when it was kept at 4°C for 24 hr. In the kinetic studies, it was found that substrate analogs choline and benzoylcholine inhibited both the native enzyme and the enzyme heated at 45°C for 24 hr competitively, whereas succinylcholine was the partial competitive inhibitor of native enzyme but the pure competitive inhibitor of the heated enzyme. 相似文献
12.
Further Characterization of a Myelin-Associated Neuraminidase: Properties and Substrate Specificity 总被引:3,自引:2,他引:1
A neuraminidase activity in myelin isolated from adult rat brains was examined. The enzyme activity in myelin was first compared with that in microsomes using N-acetylneuramin(alpha 2----3)lactitol (NL) as a substrate. In contrast to the microsomal neuraminidase which exhibited a sharp pH dependency for its activity, the myelin enzyme gave a very shallow pH activity curve over a range between 3.6 and 5.9. The myelin enzyme was more stable to heat denaturation (65 degrees C) than the microsomal enzyme. Inhibition studies with a competitive inhibitor, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid, showed the Ki value for the myelin neuraminidase to be about one-fifth of that for the microsomal enzyme (1.3 X 10(-6) M versus 6.3 X 10(-6) M). The apparent Km values for the myelin and the microsomal enzyme were 1.3 X 10(-4) M and 4.3 X 10(-4) M, respectively. An enzyme preparation that was practically devoid of myelin lipids was then prepared and its substrate specificity examined. The "delipidated enzyme" could hydrolyze fetuin, NL, and ganglioside substrates, including GM1 and GM2. When the delipidated enzyme was exposed to high temperature (55 degrees C) or low pH (pH 2.54), the neuraminidase activities toward NL and GM3 decreased at nearly the same rate. Both fetuin and 2,3-dehydro-2-deoxy-N-acetylneuraminic acid inhibited NL and GM3 hydrolysis. With 2,3-dehydro-2-deoxy-N-acetylneuraminic acid, inhibition of NL was greater than that of GM3; however, the Ki values for each substrate were almost identical. GM3 and GM1 also competitively inhibited the hydrolysis of NL and NL similarly inhibited GM3 hydrolysis by the enzyme. These results indicate that rat brain myelin has intrinsic neuraminidase activities toward nonganglioside as well as ganglioside substrates, and that these two enzyme activities are likely catalyzed by a single enzyme entity. 相似文献
13.
Effects of solubilisation on some properties of the membrane-bound respiratory enzyme D-amino acid dehydrogenase of Escherichia coli 总被引:3,自引:0,他引:3
Solubilisation, delipidation and partial purification of the membrane-bound enzyme D-amino acid dehydrogenase of Escherichia coli K12 produced significant changes in several of its properties. Solubilised enzyme showed a broader substrate specificity, increased affinity for at least three substrates, and a lower pH optimum with D-alanine as substrate. Solubilised enzyme was more heat-labile than native enzyme, particularly at 37 degrees C, and re-binding to envelope preparations restored protection against heat denaturation. Activity of delipidated enzyme could be increased by addition of pure phospholipids. Native enzyme showed biphasic Arrhenius kinetics associated with phase changes of membrane lipids. 相似文献
14.
R Bala?a-Fouce D Ordo?ez-Escudero F Sanz-Sánchez A Garrido-Pertierra 《Comparative biochemistry and physiology. B, Comparative biochemistry》1986,83(1):221-225
S-Adenosylmethionine decarboxylase (EC 4.1.1.50) has been partially purified from rabbit liver by ammonium sulphate fractionation and gel filtration and anion exchange chromatographies. Sodium dodecylsulphate-polyacrylamide disc gel electrophoresis analysis showed an approximate dimeric subunit mol. wt of 34,000. The enzyme showed a pH optimum at 7.5 (in phosphate buffer) and did not require bivalent cations for catalysis. The enzyme showed sigmoid kinetics to S-adenosylmethionine with a Hill coefficient of 1.7, which became michaelian with Km 70 microM in the presence of 2.5 mM putrescine. Methylglyoxal bis(guanylhydrazone) was an effective inhibitor of the enzyme, but phenylated derivatives of this compound as phenylglyoxal bis(guanylhydrazone) and diphenylglyoxal bis-(guanylhydrazone) inhibited less well. 相似文献
15.
Irreversible inhibition of butyrylcholinesterase by soman was studied in the presence of the substrate (o-nitrophenyl butyrate). Inhibition was found of the competitive complexing type. Study at different temperatures and pressures showed that the behavior of the enzyme differs from that of the inhibitor-free enzyme. In the absence of inhibitor, enzyme kinetics displayed a non-linear temperature dependence with a break at 21 degrees C. In the presence of a non-inhibitor structural analog of soman (pinacolyl dimethylphosphinate and methyl dimethylphosphinate), the Arrhenius plot break is slightly shifted (18 degrees C). On the other hand, in the presence of soman this break is abolished. The pressure-dependence of the substrate hydrolysis revealed also differences between the native enzyme and the enzyme in the presence of soman: the sign and magnitude of the apparent activation volume (delta V not equal to) were different for the two reactions. Beyond 300 bar, in the presence of soman, a plateau (delta V not equal to approx. 0) was observed over a large pressure range depending on temperature. Such a behavior with respect to temperature and pressure can reflect a soman-induced enzyme conformational state. Thus, temperature and pressure perturbations of the kinetics allow to complete the inhibition scheme of butyrylcholinesterase by soman. Our data suggest that upon soman binding, the enzyme undergoes a long-lived soman-induced-fit conformational change preceding the phosphonylation step. However, an alternative hypothesis according to which the enzyme processes a secondary soman-binding site cannot be ruled out. 相似文献
16.
T Bellini C Pasti M C Manfrinato M Tomasi F Dallocchio 《Biochemical and biophysical research communications》1999,262(2):401-405
The 3D structure of paramyxovirus hemagglutinin-neuraminidase has not yet been resolved; however, a theoretical model has been built by using influenza virus and bacterial neuraminidases as template [V. C. Epa (1997) Proteins Struct. Funct. Gen. 29, 264-281]. Two common features of the catalytic mechanism of the neuraminidases of known 3D structure are the anomeric specificity and the involvement of a tyrosine residue in the stabilization of the transition state. These key features have been investigated on the water-soluble ectodomain of the hemagglutinin-neuraminidase from Sendai virus (cHN). The anomeric specificity of the hydrolysis of the substrate by cHN has been investigated by NMR spectroscopy. The immediate product of the reaction was the alpha-anomer, meaning that cHN belongs between glycohydrolases retaining anomeric configuration like influenza virus neuraminidase. Measurements of the UV difference spectrum upon binding of the substrate analogue 2,3-dehydro 2-deossi N-acetyl neuraminic acid indicate the ionization of a tyrosine residue and decreased polarity in the environment of a tryptophan residue. Functional significance of the spectral data was derived from the known structure of influenza neuraminidase, where a tyrosinate ion is involved in the stabilization of the transition-state carbonium ion, and a tryptophan residue is involved in the binding of the acetyl moiety of the substrate. The data give experimental support to the 3D model of paramyxovirus neuraminidase. 相似文献
17.
Effects of phthalic anhydride modification on horseradish peroxidase stability and activity 总被引:5,自引:0,他引:5
Phthalic anhydride (PA) modification stabilizes horseradish peroxidase (HRP) by reversal of the positive charge on two of HRP's six lysine residues. Native and PA-HRP had half-inactivation temperatures of 51 and 65 degrees C and half-lives at 65 degrees C of 4 and 17 min, respectively. PA-HRP was more resistant to dimethylformamide at room temperature and tetrahydrofuran at 60 degrees C and to unfolding by heat, guanidine chloride, EDTA, and the reducing agent tris(2-carboxyethyl)phosphine hydrochloride. Binding of the hydrophobic probe Nile Red to the native enzyme and to PA-HRP was similar. The kinetics of both HRPs with the substrates ABTS, ferrocyanide, ferulic acid, and indole-3-propionic acid were measured, as was binding of the inhibitor benzhydroxamic acid. Small improvements in the catalytic properties were detected. 相似文献
18.
Substrate and dilution effects on the inhibition of acetylcholinesterase by carbamates 总被引:3,自引:2,他引:1
1. The kinetics of acetylcholinesterase (EC 3.1.1.7) activity and its inhibition by eserine or by Sevin (1-naphthyl N-methylcarbamate) have been studied over the substrate concentration range 5x10(-8) to 2.5x10(-2)m. 2. Equations are given for inhibition as a function of time, substrate and inhibitor concentrations, and the relevant parameters determined at 25 degrees and 37 degrees . 3. The observed and calculated effects of time, dilution, substrate addition and enzyme concentration were in good agreement and consistent with a steady-state carbamylation by eserine or by Sevin in the presence of excess of inhibitor. 4. The quantitative destruction of either inhibitor at high enzyme concentrations implied by the carbamylation hypothesis has been confirmed experimentally. 5. The importance and possibility of allowing quantitatively for dilution and substrate effects when estimating carbamate inhibition are demonstrated. 相似文献
19.
It was demonstrated that the relations between substrate and product concentrations for a reaction catalyzed by michaelian enzyme incorporated in a multienzyme system can be graphically represented by a diverging set of straight lines intersecting in one point, the flux velocity being treated as a parameter. A competitive inhibitor shifts the intersection point along the line of equilibrium state. The relations between the concentrations of more than two reagents are represented by a set of equivelocity surfaces. The relations between substrate and product concentrations for a kinetically cooperative reaction conforming to the graphical representation by the second--order curves were analyzed. The stability criterion was obtained for a multienzyme system with the first enzyme allosterically regulated by products of subsequent reactions. 相似文献
20.
Substrate-dependent inhibition kinetics of an active site-directed inhibitor of ADAMTS-4 (Aggrecanase 1) 总被引:1,自引:0,他引:1
Wittwer AJ Hills RL Keith RH Munie GE Arner EC Anglin CP Malfait AM Tortorella MD 《Biochemistry》2007,46(21):6393-6401
ADAMTS-4 (aggrecanase-1) is implicated in the breakdown of articular cartilage and is an attractive target for therapeutic intervention in arthritis. Cleavage of the native substrate, aggrecan, occurs through exosite interactions and peptide sequence recognition. Although expected to be competitive with aggrecan, the hydroxamic acid, SC81956, demonstrated noncompetitive inhibition kinetics with a Ki of 23 nM. The IC50 of SC81956 did not change when aggrecan was varied from 12.8 to 200 nM (0.2-3.3 times the apparent aggrecan Km of 61 nM) but was shifted as expected for a competitive inhibitor when increasing levels of a low molecular weight peptide substrate were added to a fluorogenic peptide assay system. These observations are consistent with a model for aggrecan cleavage where substrate initially binds at an exosite, followed by binding of the appropriate peptide sequence at the active site. A peptide-competitive inhibitor could bind both free enzyme and initial substrate-enzyme exosite complex but would be excluded by the final Michaelis complex. Noncompetitive appearing kinetics for such inhibitors is predicted as long as the equilibrium between the two forms of enzyme-substrate complex significantly favors the initial exosite complex. In support, hydrolysis of a low molecular weight peptide substrate and its inhibition by SC81956 were unaffected by aggrecan concentrations substantially above the Km. These observations suggest that the apparent Km for aggrecan cleavage predominately reflects the exosite interaction. Consequently, the efficacy of active-site inhibitors of ADAMTS-4 will not be limited by competition with native substrate as predicted from the Km determined by traditional kinetic models. 相似文献