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1.
Cholesterol oxidase stereospecifically isomerizes cholest-5-en-3-one to cholest-4-en-3-one. When the base catalyst for isomerization, Glu361, is mutated to Asp, the rate of deprotonation of cholest-5-en-3-one is not affected, but protonation of the dienolic intermediate becomes rate-limiting. This may be a consequence of the large distance between the catalytic base and carbon-6 of the intermediate in the mutant enzyme. 相似文献
2.
Cholesterol oxidase catalyzes the oxidation and isomerization of cholesterol to cholest-4-en-3-one via cholest-5-en-3-one. It has been proposed that His447 acts as the general base catalyst for oxidation, and that the resulting imidazolium ion formed acts as an electrophile for isomerization. In this work, we undertook an assessment of the proposed dual roles of His447 in the oxidation and isomerization reactions. To test its role, we constructed five mutants, H447Q, H447N, H447E, H447D, and H447K, that introduce hydrogen bond donors and acceptors and carboxylate bases at this position, and a sixth mutant, E361Q, to test the interplay between His447 and Glu361. These mutants were characterized using steady-state kinetics and deuterium substrate and solvent isotope effects. For those mutants that catalyze either oxidation of cholesterol or isomerization of cholest-5-en-3-one, the Km's vary no more than 3-fold relative to wild type. H447K is inactive in both oxidation (> 100,000-fold reduced) and isomerization assays (> 10,000-fold reduced). H447E and H447D do not catalyze oxidation (> 100,000-fold reduced), but do catalyze isomerization, 10(4) times slower than wild type. The k(cat) for H447Q is 120-fold lower than wild type for oxidation, and the same as wild type for isomerization. The k(cat) for H447N is 4400-fold lower than wild type for oxidation, and is 30-fold lower than wild type for isomerization. E361Q does not catalyze isomerization (> 10,000-fold reduced), and the k(cat) for oxidation is 30-fold lower than wild type. The substrate deuterium kinetic isotope effects for the wild-type and mutant-catalyzed oxidation reactions suggest that mutation of His447 to an amide results in a change of the rate-determining step from hydride transfer to hydroxyl deprotonation. The deuterium solvent and substrate kinetic isotope effects for isomerization indicate that an amide at position 447 is an effective electrophile to catalyze formation of a dienolic intermediate. Moreover, consideration of kinetic and structural results together suggests that a hydrogen bonding network involving His447, Glu361 and Asn485, Wat541, and substrate serves to position the substrate and coordinate general base and electrophilic catalysis. That is, in addition to its previously demonstrated role as base for deprotonation of carbon-4 during isomerization, Glu361 has a structural role and may act as a general base during oxidation. The His447, Asn485, Glu361, and Wat541 residues are conserved in other GMC oxidoreductases. Observation of this catalytic tetrad in flavoproteins of unknown function may be diagnostic for an ability to oxidize unactivated alcohols. 相似文献
3.
The stoichiometry of the reaction catalyzed by skin sulfhydryl oxidase was investigated. Dithiothreitol (DTT) was used as the substrate for skin sulfhydryl oxidase. The consumption of DTT, consumption of oxygen, and production of hydrogen peroxide were measured during the enzyme reaction. The molar ratio of DTT:O2:H2O2 in the enzyme reaction was 1:1.02:0.89. Correspondingly, the stoichiometry of the enzyme reaction was calculated to be [formula: see text] 相似文献
4.
M Yamashita M Toyama H Ono I Fujii N Hirayama Y Murooka 《Canadian Metallurgical Quarterly》1998,11(11):1075-1081
Site-directed mutagenesis was used to identify key amino acid residues of the cholesterol oxidase from Streptomyces sp., which catalyzes the oxidation of cholesterol and the isomerization of 5-cholesten-3-one. Eight mutant enzymes were constructed and the following amino acid substitutions were identified: N318A, N318H, E356A, E356D, H441A, H441N, N480A and N480Q. Mutants N318A and N318H retained both oxidation and isomerization activities. The mutant E356D retained oxidation but not isomerization activity. On the other hand, mutants N480A and N480Q showed no oxidation activity but retained their isomerization activities. The two catalytic reactions, oxidation and isomerization, in cholesterol oxidase were thus successfully separated. When the H441A or H441N mutation was introduced, both the oxidase and isomerase activities were completely lost. The H441, E356 and N480 residues thus appear to participate in the catalysis of cholesterol oxidase, whereas N318 does not. An analysis of the products of these mutant enzymes suggested that the previously proposed 6-hydroxylation reaction by cholesterol oxidase is actually autooxidation from 5-cholesten-3-one. Kinetic studies of the purified wild-type and mutant enzymes showed that the k(cat)/Km values for oxidation in E356D and for isomerization in N480A increased six- and threefold, respectively, over those in the wild-type. These mutational effects and the reaction mechanisms are discussed in terms of the three-dimensional structure of the enzyme constructed on the basis of homology modeling. 相似文献
5.
An aqueous primary standard is needed for measuring cholesterol by enzymatic procedures. Sodium deoxycholate solubilizes cholesterol in water. Crystalline cholesterol (1.00, 3.00, 4.00, and 5.00 g/L), added to a solution containing 150 g of this compound per liter of a 9 g/L saline solution, was measured by a cholesterol oxidase procedure, with a centrifugal analyzer. The solubilizer did not interfere. When compared to an isopropanol-based commercial cholesterol standard, the cholesterol standard in solubilizer showed excellent correlation (r = 0.986; m = 0.989). Day-to-day variation for the mixture during nine days was small (CV, 2.9% at 100 g/L, 3.7% at 3.00 g/L, and 1.8% at 4.00 g/L). Linearity was maintained up to 5.00 g/L. The cholesterol concentration in four reference sera so analyzed maintained CV's of less than 4%. The viscosity of the mixture was similar to that of serum. The standard mixtures, stored at room temperature for 360 days, remained stable. The solubilized cholesterol standard is shown to be suitable for use in the enzymatic procedure. 相似文献
6.
The investigation of the effect of oxidized lipoproteins on platelet activity is important for the understanding of the plague formation under atherosclerosis. In the present work, we examined the influence of low density lipoproteins (LDL) on ADP-induced platelet aggregation in the platelet rich plasma. In was demonstrated that mixing of plasma and LDL was accompanied by the decrease of ADP-induced aggregation parameters as compared to control (mixing with buffer). After 1 h incubation, platelet ADP-aggregation in the sample containing oxidized LDL (oxLDL) exceeded the ADP-aggregation in the control sample. The dependence of the aggregation parameters on the incubation time and on the degree of LDL oxidation were obtained. No difference in the cholesterol and phospholipid content was observed between cells incubated with buffer, native or oxidized LDL. Therefore, the possible oxLDL-induced accumulation of cholesterol in platelet membranes is excluded as a reason for the increased cell aggregation. 相似文献
7.
The kinetic properties of Mg(2+)-ATPase (EC 3.6.1.3) from myometrium cell plasma membranes have been studied. Under conditions of enzyme saturation with ATP (0.5-1.0 mM) or Mg2+ (1.0-5.0 mM) the initial maximal rates of the Mg(2+)-dependent enzymatic ATP hydrolysis, V0 ATP and V0 Mg, are 27.4 +/- 3.3 and 25.2 +/- 4.1 mumol Pi/hour/mg of protein, respectively. The apparent Michaelis constant, Km, for ATP and of the apparent activation constant, K alpha, for Mg2+ are equal to 28.1 +/- 2.6 and 107.0 +/- 26.0 microM, respectively. The bivalent metal ions used at 1.0 mM suppress the Mg(2+)-dependent hydrolysis of ATP whose efficiency decreases in the following order: Cu2+ > Zn2+ = Ni2+ > Mn2+ > Ca2+ > Co2+. Alkalinization of the incubation medium from pH 6.0 to pH 8.0 stimulates the Mg(2+)-dependent hydrolysis of ATP. It has been found that Mg(2+)-ATPase has the properties of an H(+)-sensitive enzymatic sensor which is characterized by a linear dependence between the initial maximal rate of the reaction, V0, and the pH value. The feasible role of plasma membrane Mg(2+)-ATPase in some reactions responsible for the control of proton and Ca2+ homeostasis in myometrium cells has been investigated. 相似文献
8.
Glycerol dehydrogenase catalyzes the reversible NAD(+)-dependent oxidation of glycerol to form dihydroxyacetone. Initial velocity, product, and dead-end inhibition studies performed for the forward and reverse reactions support an ordered kinetic mechanism with NAD+ binding first and NADH released last. A monovalent cation is required for enzymatic activity and glycerol binding, with K+ having the highest activity as measured by V. The pH dependence of the kinetic parameters V and V/Kglycerol, as well as the temperature dependence of the V pH profile, suggested that an enzymic carboxylate group functions as a base in catalysis. The pH dependence of the primary deuterium kinetic isotope effect shows that DV/Kglycerol increases from a pH-independent value of 1.15 at high pH values to a pH-independent value of 2.44 at low pH values. DV exhibits a similar pH dependence, increasing from a pH-independent value of 2.57 at high pH values to a pH independent value of 4.88 at low pH values. A chemical mechanism for enzymatic glycerol oxidation is proposed based on the data. 相似文献
9.
The pH dependence of the chemical shifts of the 31P resonances of enzyme-bound substrates 2-phosphoglycerate (PGA) and phosphoenolpyruvate (PEP) were measured to obtain further insight into the catalytic mechanism of yeast enolase. The 31P resonances of PGA and PEP bound to the enolase-Mg complex are individually observed by NMR. The Keq,internal = 1.5 favoring PEP was measured. A pH dependence of the 31P chemical shifts gives pKa values of 5.82 and 6.16 for bound PGA and PEP, respectively, indicating that both ligands bind predominantly with their phosphate groups as the dianionic species and their ionization has been altered. The phosphoryl group of PGA has been suggested as playing a role in catalysis [Nowak, T., Mildvan, A. S., and Kenyon, G. L. (1973) Biochemistry 12, 1690-1701]. The pH dependence of the kinetic parameters for Mg-enolase shows a single break in the plot of pKm, PGA vs pH at pH 6.27 with a pH independence above pH 7. This is consistent with the trianion of PGA preferably binding to the enzyme. The kcat profile gives pKA values of 5.94 and 8.35, and kcat/Km profiles give pKA values of 5.85, 6.25, and 8.39. Activation studies with Mg2+ show a pH independence for the activator constant (Ka), but a pH-dependent inhibition at higher concentrations of Mg2+. The log kcat and kcat/Ka profiles from Mg2+ activation give pKA values of about 5.9 and 8.4. These results confirm the importance of residues with pKA values of about 5.9 and 8.4 (His and Lys residues?) but do not support a function for the phosphoryl group of the substrate. The pH dependence of the Ki,Mg2+ gives pKA fits of 5. 95, 7.13, and 8.35. Data from cation inhibition suggest that the phosphate of the substrate and a His residue on enolase may bind the inhibitory Mg2+. 相似文献
10.
The experimental results on the interconversion of dihydroxyacetone phosphate and D-glyceraldehyde 3-phosphate catalyzed by triosephosphate isomerase that are presented in the previous five papers are here collected and analyzed according to the theory presented in the first paper (Albery, W.J., Knowles, J.R. (1976), Biochemistry 15, the first of eight papers in a series in this issue). The rate constants and fractionation factors so derived allow the construction of theGibbs free-energy profile for this enzyme-catalyzed reaction. 相似文献
11.
W Richmond 《Canadian Metallurgical Quarterly》1976,22(10):1579-1588
Idescribe an assay for total cholesterol in serum, with use of the AutoAnalyzer ii (Technicon), in which cholesterol esters are saponified by alkali, cholesterol is held in aqueous micellar solution with a surfactant (Triton X-100) and oxidized by cholesterol oxidase, and the hydrogen peroxide produced is measured by chelation with Ti4+ and xylenol orange. An assay for free cholesterol in serum, based on similar principles, is also described, and the two can be run simultaneously on a dual-channel AutoAnalyzer II. Standard solutions of cholesterol in isopropanol have poorer carryover characteristics than sera, and therefore do not reach the same continuous-flow steady state as sera of equivalent concentrations. Consequent potential calibration errors are avoided by using micellar solutions of cholesterol containing albumin for standardization. The formation of cholesterol peroxide in solutions of cholesterol in isopropanol is demonstrated, and this constitutes another potential source of error in the calibration of enzymic cholesterol assays. In analyzing patients' sera, results of the total cholesterol assay correlate well with those of a mechanized method in which cholesterol esterase and cholesterol oxidase are used; an automated Abell method, calibrated with solutions of cholesterol in isopropanol, gave slightly higher values. Determinations of the ratio of free cholesterol to total cholesterol by our automated cholesterol exidase assays given values that agree well with published results in which digitonin precipitation is used. 相似文献
12.
Clostridial phosphotransacetylase catalyzes acyl group transfer between coenzyme A (CoA) and inorganic phosphate and also the arsenolysis of acetyl-coenzyme A (AcCoA) to yield acetate and CoA-SH. The enzyme mobility on sodium dodecyl sulfate electrophoresis corresponds to a molecular weight of 70 000. Kinetics of both forward and reverse reactions are of the ternary type as previously reported and product inhibition data are consistent with a random binding scheme. One essential sulfhydryl group per 70 000 daltons was inactivated in a pseudo-first-order process by either N-ethylmaleimide or 5,5'-dithiobis (nitrobenzoic acid). Reduction of the rate of this inactivation by 50% in the presence of AcCoA or acetyl phosphate concentrations near their kinetic K values demonstrates binding of these acyl donors in simple enzyme-substrate complexes. Moreover, pulse-chase experiments show these binary complexes to be functional and also show that they do not dissociate rapidly compared with their rates of catalytic turnover. Incubation of the enzyme with 14C-labeled acyl donors failed to produce labeled protein after passage through Sephadex. This was true despite efforts to mimic "substrate synergism" with desulfo-CoA or to compensate for unfavorable equilibria by means of CoA traps. Very slow isotope exchange reactions of 32Pi into acetyl phosphate and [3H]CoA into AcCoA were at first observed. As in the cases of several other enzymes recently reexamined, these were shown on careful inspection to be artifacts of contamination by second substrates. Attempts to detect exchange reactions between acetyl phosphate and Pi, even in the presence of the CoA analogue, desulfo-CoA, were also unsuccessful. Therefore, no evidence for an acyl-enzyme could be detected. Furthermore, our data allow us to develop arguments which, we believe, indicate that an acyl-enzyme intermediate is extremely improbable in the reaction catalyzed by phosphotransacetylase. 相似文献
13.
从高炉风口喷吹烧结烟气是一种有别于传统烟气脱硝工艺的新思路。为探究烧结烟气喷入风口后,焦炭催化CO-NO还原反应的动力学过程,实验以CO、NO气体为原料,以焦炭为催化剂,将不同流量配比的CO和NO混合气体在炉温为850~975℃时通入炉内,在出口处测量CO和NO反应后体积分数。实验结果表明,焦炭对CO还原NO反应有明显的催化作用;反应温度不变,CO和NO的流量比增大时,NO的还原率会增大;CO和NO的流量比不变,温度增大时,NO的还原率也会增大;炉温850、900、925、950、975℃时,反应速率常数分别为0.56、0.88、0.78、1.28、1.38 m^(3)/(mol·s),通过拟合动力学参数得到反应的活化能E;为84.1 kJ/mol。 相似文献
14.
Seven patients with a form of membranoproliferative glomerulonephritis distinct in its glomerular ultrastructure from other forms are described. The use of silver impregnated electron micrographs revealed contiguous subepithelial and subendothelial deposits associated with basement membrane disruption, replication and layering of lamina densalike material. By light and fluorescence microscopy the appearance was distinctive but not diagnostic. Immunohistology consistently showed abundant C3 and properdin in a granular pattern while immunoglobulins and Clq were variably present. Low serum C3 concentrations were observed at some time in each patient, often accompanied by low levels of properdin, whereas the concentrations of Clq and C4 were normal. The patients were indistinguishable in their clinical course from those with other types of MPGN. 相似文献
15.
LL Kochkina VZ Akhverdian LL Kiselev VV Zinov'ev EG Malygin 《Canadian Metallurgical Quarterly》1976,10(2):437-444
The rates of tryptophanyl-tRNA formation catalyzed by beef pancreas tryptophanyl-tRNA synthetase were measured in a concentration range of each substrate (tryptophan, ATP and yeast tRNATrp) and also in the presence of various concentrations of substrate analogues (tryptamine and alpha,beta-methylene analogue of ATP) concentrations. The data obtained were compared with the kinetic equations which described various possible mechanisms of the reaction. The comparison of the mechanisms was based on the calculation of relative probabilities of each hypothesis the efficiency of which was demonstrated earlier. The calculations have shown that two mechanisms according to which the intermediate enzyme-aminoacyl-adenylate complex formation involves the enzyme-aminoacyl-tRNA complex are the most probable ones. 相似文献
16.
The effects of temperature, dielectric permeability and ionic strength on the activity of purified Ca2+, Mg(2+)-ATPase solubilized from myometrial sarcolemma have been studied under saturation of the enzyme with Ca2+, Mg2+ and ATP. The values of activation energy calculated from Arrhenius plots for both ATP hydrolase reactions catalysed by solubilized and reconstituted into azolectin liposomes Ca2+, Mg(2+)-ATPase and Mg2+, ATP-dependent Ca2+ transport by the reconstituted enzyme were 56.4 +/- 1.5, 68.0 +/- 5.1 and 63.1 +/- 2.9 kJ/mol, respectively. Analysis of experimental data in terms of the Laidler-Scatchard and Bronsted-Bjerrum theories revealed that the separation of the reaction products--the chelate MgADP complex--from the active site of the enzyme bearing one unity positive charge is the limiting step of the Ca2+, Mg(2+)-dependent enzymatic ATP-hydrolysis under conditions of substrate saturation. The values of the electrostatic components of the free energy, enthalpy and entropy of activation of the ATP hydrolase reaction were 46.6 +/- 0.3 kJ/mol, -(20.5 +/- 0.4) kJ/mol and -(214.2 +/- 4.3) J/(mol.degrees K), respectively. The nonelectrostatic component of activation enthalpy was 76.9 kJ/mol. The results obtained suggest that changes in polarity of the incubation medium markedly affect the activity of transport Ca2+, Mg(2+)-ATPase solubilized from smooth muscle cell plasma membranes and that the electrostatic interactions between the enzyme active site and specific reagents (MgADP, in particular) significantly contribute to the energetics of the ATP hydrolase reaction. 相似文献
17.
18.
To characterize the calcium (Ca2+)-releasing effects of histamine and GTP gamma S, the drug-induced tension developments were measured in beta-escin-treated skinned longitudinal smooth muscle of guinea pig ileum. Intracellular Ca2+ stores were loaded with Ca2+ by incubating the muscle for 10 min in a Ca(2+)-containing solution. Histamine (10-100 microM), applied after Ca(2+)-loading, produced a transient rise in tension. The effect of histamine was not preserved after treatment with 20 mM caffeine, a Ca(2+)-store releaser. The effect of histamine was potentiated by GTP; inhibited by GDP beta S, an antagonist of GTP for binding to G-proteins; or heparin, an antagonist of inositol 1,4,5-trisphosphate (IP3) for binding to its receptor; and mimicked by IP3. When GTP gamma S (20 microM) was applied and continued to be present for 15 min, a transient rise in tension followed by a small, sustained rise in tension was elicited. The effect of GTP gamma S was completely inhibited by GDP beta S. The initial, transient component of the biphasic GTP gamma S response was abolished or markedly inhibited after treatment with caffeine, heparin or the calcium ionophore A23187. The present results suggest that histamine and GTP gamma S cause a release of Ca2+ from caffeine-sensitive stores which is mediated by IP3 formed through a G-protein-coupled mechanism. The GTP gamma S-induced Ca2+ release is not considered to involve such an IP3-independent process as described in chemically-skinned arterial muscle. 相似文献
19.
20.
Monoamine oxidase activity was measured in isolated rat liver mitochondria using the radiochemical assay with [14C]tyramine as substrate. With toluene as the extracting solvent the apparent activity in the resting state (State 4) was much higher than in the active state (State 3) in agreement with Smith and Reid (Smith, G.S. and Reid, R.A. (1978) Biochem. J. 176, 1011-1014). However, with ethyl acetate or diethyl ether as extracting solvents, the activity in both states was almost identical and several times higher than that measured with toluene. p-Hydroxyphenylacetaldehyde, p-hydroxyphenylacetalcohol and p-hydroxyphenylacetic acid were identified as final reaction products, the latter one being hardly extractable with toluene. It is concluded that monoamine oxidase activity is not influenced by the respiratory state of mitochondria and that differences found by Smith and Reid are due to different extractability of secondary reaction products. NADPH-dependent aldehyde reductase was tentatively identified in rat liver mitochondria, its specific activity amounting to about one fourth of that in the cytosol. 相似文献