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1.
Reductive titrations of a NAD-dependent type (type-D) and an O2-dependent type (type-O) of rat liver xanthine dehydrogenase showed that only the type-D enzyme formed a pronounced stable FAD semiquinone (FADH*). The FAD semiquinone was less stabilized in the presence of NAD. The Vmax value for xanthine-NAD activity of type-D enzyme was close to that for xanthine-O2 activity of type-O enzyme, while the Vmax value for xanthine-O2 activity of type-D enzyme was about one-fourth of that of type-O enzyme. The Km value for O2 of type-D enzyme was about five times as large as that of type-O enzyme. The absorbance spectrum of type-D enzyme during turnover with xanthine and O2 as substrates showed a considerable amount of FADH* formation, but that with xanthine and NAD as substrates showed only a negligible one. Low xanthine-O2 activity of type-D enzyme, as compared with that of type-O enzyme, seems to be explained by the conformational change occurring in conversion from type-O to type-D enzyme, which results in different reactivity of FAD to molecular oxygen and a higher fraction of FADH* during turnover. The binding of NAD may possibly increase the fraction of FADH2, resulting in a Vmax value of xanthine-NAD activity almost as high as that of xanthine-O2 activity of type-O enzyme. 相似文献
2.
M N Williams 《The Journal of biological chemistry》1975,250(1):322-330
When dihydrofolate reductase from a methotrexate-resistant strain of Escherichia coli B, MB 1428, is treated with approximately a 5 mol ratio of N-bromosuccinimide (NBS) to enzyme at pH 7.2 and assayed at the same pH, there is a 40% loss of activity due to the modification of 1 histidine residue and possibly 1 methionine residue before oxidation of tryptophan occurs. The initial modification is accompanied by a shift of the pH for maximal enzymatic activity from pH 7.2 to pH 5.5 Upon further treatment with N-bromosuccinimide, the activity is gradually reduced from 60 to 0% as tryptophan residues become oxidized. An NBS to enzyme mole ratio of approximately 20 results in 90% inactivation of the enzyme. When the enzyme is titrated with NBS in 6 M guanidine HCl, 5 mol of tryptophan react per mol of enzyme, a result in agreement with the total tryptophan content as determined by magnetic circular dichroism. The 40% NBS-inactivated sample posses full binding capacity for methotrexate and reduced triphosphopyridine nucleotide, and the Km values for dihydrofolate and TPNH are the same as for the native enzyme. After 90% inactivation, only half of the enzyme molecules bind methotrexate, and the dissociation constant for methotrexate is 40 nM as compared to 4 nM for native enzyme in solutions of 0.1 M ionic strength, pH 7.2 Also, TPNH is not bound as tightly to the modified enzyme-methotrexate complex as to the unmodified enzyme-methotrexate complex. Circular dichroism studies indicate the 90% NBS-inactivated enzyme has the same alpha helix content as the native enzyme but less beta structure, while the 40% inactivated enzyme is essentially the same as the native enzyme. Protection experiments were complicated by the fact that NBS reacts with the substrates and cofactors of the enzyme. Although protection of specific residues was not determined, it was clear that TPNH was partially protected from NBS reaction when bound to the enzyme, and the enzyme, and the enzyme was not inactivated by NBS until the TPNH had reacted. 相似文献
3.
酶的分子设计、改造与工程应用 总被引:4,自引:0,他引:4
酶工程的研究已经发展到分子水平 ,在体外通过基因工程、化学、物理等手段改造酶分子结构与功能 ,大幅提高了酶分子的进化效率和催化效率 ,生产有价值的非天然酶。对酶工程学若干“热点”和前沿课题的研究、应用进行了概述 ,分析了国际上酶工程研究及应用技术、手段、方法 ,包括体外分子进化、核酶和抗体酶的设计、酶分子的定向固定化技术、酶蛋白分子的化学修饰、融合酶、人工合成及模拟酶等技术 ,并展望了酶工程的技术进步和应用的新进展。 相似文献
4.
P M Anderson 《Biochemistry》1977,16(4):583-586
Carbamyl-phosphate synthetase from Escherichia coli is an allosteric enzyme which undergoes reversible association reactions in phosphate buffer. The positive allosteric effectors, ornithine, inosine 5'-monophosphate (IMP), and ammonia, facilitate oligomer formation, whereas uridine 5'-monophosphate (UMP), a negative effector, prevents or decreases oligomer formation. When the enzyme is immobilized by reaction with activated Sepharose, under conditions where the enzyme exists only as a monomer, nearly full catalytic activity is retained and the effects of ornithine, IMP, and UMP on the catalytic activity as a function of MgATP concentration are not significantly altered. Gel-filtration chromatography on Sephadex G-200 of catalytic quantities of the enzyme in the presence of all substrates showed that the elution volume was the same as that measured for the enzyme under conditions where it is known to exist in the monomer form. The specific activity of the enzyme does not increase when the concentration of the enzyme is increased 100-fold from a concentration at which the enzyme exists as monomer to a level at which the enzyme exists predominantly as oligomer. These results indicate that the monomer form of the enzyme is the principle active species and that oligomer formation is not directly related to enzyme activity or enzyme regulation. 相似文献
5.
Alanine:glyoxylate aminotransferase has been reported to be present as the apo enzyme in the peroxisomes and as the holo enzyme in the mitochondria in chick (white leghorn) embryonic liver. However, surprisingly, birds were found to be classified into two groups on the basis of intraperoxisomal forms of liver alanine:glyoxylate aminotransferase. In the peroxisomes, the enzyme was present as the holo form in group 1 (pigeon, sparrow, Java sparrow, Australian budgerigar, canary, goose, and duck), and as the apo form in group 2 (white leghorn, bantam, pheasant, and Japanese mannikin). In the mitochondria, the enzyme was present as the holo form in both groups. The peroxisomal holo enzyme was purified from pigeon liver, and the peroxisomal apo enzyme from chicken (white leghorn) liver. The pigeon holo enzyme was composed of two identical subunits with a molecular weight of about 45,000, whereas the chicken apo enzyme was a single peptide with the same molecular weight as the subunit of the pigeon enzyme. The peroxisomal holo enzyme of pigeon liver was not immunologically cross-reactive with the peroxisomal apo enzyme of chicken liver, the mitochondrial holo enzymes from pigeon and chicken liver, and mammalian alanine:glyoxylate aminotransferases 1 and 2. The mitochondrial holo enzymes from both pigeon and chicken liver had molecular weights of about 200,000 with four identical subunits and were cross-reactive with mammalian alanine:glyoxylate aminotransferase 2 but not with mammalian alanine:glyoxylate aminotransferase 1. 相似文献
6.
Anandamide (N-arachidonoylethanolamine) loses its cannabimimetic activity when it is hydrolyzed to arachidonic acid and ethanolamine by the catalysis of an enzyme referred to as anandamide amidohydrolase or fatty acid amide hydrolase. Cravatt's group and our group cloned cDNA of the enzyme from rat, human, mouse and pig, and the primary structures revealed that the enzymes belong to an amidase family characterized by the amidase signature sequence. The recombinant enzyme acted not only as an amidase for anandamide and oleamide, but also as an esterase for 2-arachidonoylglycerol. The reversibility of the enzymatic anandamide hydrolysis and synthesis was also confirmed with a purified recombinant enzyme. Several fatty acid derivatives like methyl arachidonyl fluorophosphonate potently inhibited the enzyme. The enzyme was distributed widely in mammalian organs such as liver, small intestine and brain. However, the anandamide hydrolyzing enzyme found in human megakaryoblastic cells was catalytically distinct from the previously known enzyme. 相似文献
7.
A new technique, the quantitative determination of total enzyme concentrations by specific immunoprecipitation with purified, radioiodinated antibodies, was used to investigate the presence and possible roles of inactive enzyme in the regulation of chalcone synthase. Dark-grown cell suspension cultures from parsley (Petroselinum hortense) contained neither catalytically active nor detectable amounts of immunoprecipitable chalcone synthase. Irradiation induced large increases and subsequent decreases of both. Significant differences in the peak positions and in the half-lives of active and total chalcone synthase indicated that induced cells contained inactive as well as active enzyme forms. The presence of inactive enzyme could be explained by two different modes of regulation, (i) simultaneous de novo synthesis of active and inactive enzyme (“Simultaneous Model”), or (ii) de novo synthesis of active enzyme only, with sequential steps of inactivation and degradation (“Sequential Model”). Both models were compatible with experimental results, as analyzed mathematically by investigating the relations between curves for rate of enzyme synthesis, enzyme activity, total enzyme, and half-lives of active and total enzyme. However, the “Simultaneous Model” postulated that de novo synthesis of inactive enzyme represented always the vast majority of total enzyme synthesis, while the Sequential Model integrated inactive enzyme with facility in a sequence of irreversible inactivation and degradation of active enzyme. Experiments with repeated induction indicated that cells containing large amounts of inactive enzyme increased enzyme activity by de novo synthesis rather than by activation of preexisting inactive enzyme. 相似文献
8.
Isolation and molecular kinetic properties of the angiotensin-converting enzyme from the human heart
An angiotensin-converting enzyme was isolated from human heart using N[-1(S)-carboxy-5-aminopentyl]glycyl-glycine as an affinity adsorbent. The isolation procedure resulted in an enzyme purified 1650-fold. The enzyme specific activity was 38.0 u./mg protein, Mr = 150 kD. The pH optimum for the angiotensin-converting enzyme towards Hip-His-Leu lies at 7.8, Km = 1.2 mM. The enzyme was inhibited by the substrate (Ks' = 14 mM). The enzyme effectively catalyzed the hydrolysis of angiotensin I (Km = 10 microM; kcat = 250 s-1). NaCl, CaCl2 as well as Na2SO4 in the absence of Cl- activated the enzyme, whereas CH3COONa and NaNO3 did not influence the enzyme activity. It was found that the bradykinin-potentiating factor inhibited the cardiac angiotensin-converting enzyme with IC50 = 4.0 X 10(-8) M. 相似文献
9.
Phenylalanine hydroxylase, important in phenylalanine metabolism in mammals, is regulated through short-term (activation) and long-term (induction) mechanisms. To help elucidate the structure-function relationships involved in the activation of this enzyme, we have isolated and characterized full-length cDNA clones to rat phenylalanine hydroxylase. Recombinant rat phenylalanine hydroxylase was placed into an expression vector in Escherichia coli. The enzyme has been purified to homogeneity and its physical and catalytic properties have been characterized. The molecular weight and the fluorescence emission spectrum of the recombinant enzyme were identical to those of the native enzyme. The recombinant enzyme could be activated by incubation with phenylalanine or lysolecithin or by phosphorylation, as is the rat liver enzyme. The extent of activation is the same as that for the native enzyme in each case except for phenylalanine, which activates the recombinant enzyme only 5- to 10-fold rather than the 15- to 30-fold activation observed with the native enzyme. The kinetic constants determined for the recombinant enzyme are also essentially the same as those reported for the native enzyme. We conclude that this enzyme is essentially identical to the native enzyme and should be very useful in the future study of this important hydroxylase. 相似文献
10.
Immunological comparison of the starch branching enzymes from potato tubers and maize kernels 总被引:5,自引:3,他引:2
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Starch branching enzyme was purified from potato (Solanum tuberosum L.) tubers as a single species of 79 kilodaltons and specific antibodies were prepared against both the native enzyme and against the gel-purified, denatured enzyme. The activity of potato branching enzyme could only be neutralized by antinative potato branching enzyme, whereas both types of antibodies reacted with denatured potato branching enzyme. Starch branching enzymes were also isolated from maize (Zea mays L.) kernels. All of the denatured forms of the maize enzyme reacted with antidenatured potato branching enzyme, whereas recognition by antinative potato branching enzyme was limited to maize branching enzymes I and IIb. Antibodies directed against the denatured potato enzyme were unable to neutralize the activity of any of the maize branching enzymes. Antinative potato branching enzyme fully inhibited the activity of maize branching enzyme I; the neutralized maize enzyme was identified as a 82 kilodalton protein. It is concluded that potato branching enzyme (Mr = 79,000) shares a high degree of similarity with maize branching enzyme I (Mr = 82,000), in the native as well as the denatured form. Cross-reactivity between potato branching enzyme and the other forms of maize branching enzyme was observed only after denaturation, which suggests mutual sequence similarities between these species. 相似文献
11.
In vivo as well as in vitro supply of sodium arsenate inhibited the 5-Amino levulinic acid dehydratase (5-aminolevulinate-hydrolyase EC 4.2.1.24, ALAD) activity in excised etiolated maize leaf segments during greening. The percent inhibition of enzyme activity by arsenate (As) was reduced by the supply of KNO3, but it was increased by the glutamine and GSH. Various inhibitors, such as, chloramphenicol, cycloheximide and LA, decreased the % inhibition of enzyme activity by As. The % inhibition of enzyme activity was also reduced by in vivo supply of DTNB. The enzyme activity was reduced substantially by in vitro inclusion of LA, both in the absence and presence of As. In vitro inclusion of DTNB and GSH inhibited the enzyme activity extracted from leaf segments treated without arsenate (-As enzyme) and caused respectively no effect and stimulatory effect on arsenate treated enzyme (+As enzyme). Increasing concentration of ALA during assay increased the activity of -As enzyme and +As enzyme to different extent, but double reciprocal plots for both the enzymes were biphasic and yielded distinct S0.5 values for the two enzymes (-As enzyme, 40 micromol/L and +As enzyme, 145 micromol/L) at lower concentration range of ALA only. It is suggested that As inhibits ALAD activity in greening maize leaf segments by affecting its thiol groups and/or binding of ALA to the enzyme. 相似文献
12.
Bolivar JM Cava F Mateo C Rocha-Martín J Guisán JM Berenguer J Fernandez-Lafuente R 《Applied microbiology and biotechnology》2008,80(1):49-58
The genome of Thermus thermophilus contains two genes encoding putative glutamate dehydrogenases. One of these genes (TTC1211) was cloned and overexpressed in Escherichia coli. The purified enzyme was a trimer that catalyzed the oxidation of glutamate to alpha-ketoglutarate and ammonia with either NAD+ or NADP+ as cofactors. The enzyme was also able to catalyze the inverse reductive reaction. The thermostability of the enzyme at neutral pH was very high even at 70 degrees C, but at acidic pH values, the dissociation of enzyme subunits produced the rapid enzyme inactivation even at 25 degrees C. The immobilization of the enzyme on glyoxyl agarose permitted to greatly increase the enzyme stability under all conditions studied. It was found that the multimeric structure of the enzyme was stabilized by the immobilization (enzyme subunits could be not desorbed from the support by boiling it in the presence of sodium dodecyl sulfate). This makes the enzyme very stable at pH 4 (e.g., the enzyme activity did not decrease after 12 h at 45 degrees C) and even improved the enzyme stability at neutral pH values. This immobilized enzyme can be of great interest as a biosensor or as a biocatalyst to regenerate both reduced and oxidized cofactors. 相似文献
13.
Brooks JL 《Plant physiology》1986,80(1):130-133
Tomato (Lycopersicon esculentum Mill) anionic peroxidase was found to catalyze oxidase reactions with NADH, glutathione, dithiothreitol, oxaloacetate, and hydroquinone as substrates with a mean activity 30% that of horseradish peroxidase; this is in contrast to the negligible activity of the tomato enzyme as compared to the horseradish enzyme in catalyzing an indoleacetic acid-oxidase reaction with only Mn2+ and a phenol as cofactors. Substitution of Ce3+ for Mn2+ produced an 18-fold larger response with the tomato enzyme than with the horseradish enzyme, suggesting a significant difference in the autocatalytic indoleacetic acid-oxidase reactions with these two enzymes. In attempting to compare enzyme activities with 2,4-dichlorophenol as a cofactor, it was found that reaction rates increased exponentially with both increasing cofactor concentration and increasing enzyme concentration. While the former response may be analogous to allosteric control of enzyme activity, the latter response is contrary to the principle that reaction rate is proportional to enzyme concentration, and additionally makes any comparison of enzyme activity difficult. 相似文献
14.
Ribonuclease T1 [EC 3.1.4.8] was coupled to a water-insoluble cross-linked polyacrylamide (Enzacryl AH) by the acid azide method. The immobilized enzyme exhibited about 45% and 77% of the original activity toward yeast RNA and 2', 3-cyclic GMP, respectively, as substrates. Although the specific activity was lowered by the coupling, the immobilized enzyme was found to be far more stable to heat and extremes of PH than the native enzyme. The immobilized enzyme was active toward RNA even above pH 9 (at 37 degree C) or above 60 degree C (at pH 7.5), where the native enzyme was inactive. The immobilized enzyme retained much of its activity as assayed at 37 degree C after incubation in the range of pH 1 to 10 at 37 degree C, or after heating at 100 degree C (at pH 7.5) under conditions where the native enzyme was inactivated to a considerable extent. The enzyme derivative could be repeatedly recovered and reused without much loss of activity. The active site glutamic acid-58 in the immobilized enzyme appeared to be nearly as reactive with iodoacetate as that in the native enzyme. 相似文献
15.
Malic enzymes of higher plants: characteristics, regulation, and physiological function 总被引:3,自引:3,他引:0
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Wedding RT 《Plant physiology》1989,90(2):367-371
The characteristics and distribution of the malic enzyme in plants is discussed as well as those features which appear to be limited to the plant NAD malic enzyme. Regulation of the malic enzyme as it relates to the physiological roles of this enzyme is also discussed. 相似文献
16.
平衡柱层析法测得每分子龙虾肌羧甲基化甘油醛-3磷酸脱氢酶能结合3.9分子NAD~+,而每分子光照酶则只能结合2分子NAD~+。 由蛋白荧光淬灭法得到,在25℃、pH7.0的磷酸盐缓冲液中,全酶、羧甲基酶及光照酶与NAD~+结合时均呈负协同性。 相似文献
17.
When enzyme molecules are distributed within a negatively charged matrix, the kinetics of the conversion of a negatively charged substrate into a product depends on the organization of fixed charges and bound enzyme molecules. Organization is taken to mean the existence of macroscopic heterogeneity in the distribution of fixed charge density, or of bound enzyme density, or of both. The degree of organization is quantitatively expressed by the monovariate moments of charge and enzyme distributions as well as by the bivariate moments of these two distributions. The overall reaction rate of the bound enzyme system may be expressed in terms of the monovariate moments of the charge density and of the bivariate moments of charge and enzyme densities. The monovariate moments of enzyme density do not affect the reaction rate. With respect to the situation where the fixed charges and enzyme molecules are randomly distributed in the matrix, the molecular organization, as expressed by these two types of moments, generates an increase or decrease of the overall reaction rate as well as a cooperativity of the kinetic response of the system. Thus both the alteration of the rate and the modulation of cooperativity are the consequence of a spatial organization of charges with respect to the enzyme molecules. The rate equations have been derived for different types of organization of fixed charges and enzyme molecules, namely, clustered charges and homogeneously distributed enzyme molecules, clustered enzyme molecules and homogeneously distributed charges, clusters of charges and clusters of enzymes that partly overlap, and clusters of enzymes and clusters of charges that are exactly superimposed. Computer simulations of these equations show how spatial molecular organization may modulate the overall reaction rate. 相似文献
18.
Nakakuki T Hayashi T Monma M Kawashima K Kainuma K 《Biotechnology and bioengineering》1983,25(4):1095-1107
Exomaltohexaohydrolase (E.C.3.2.1.98) was immobilized by radiocopolymerization of some synthetic monomers which were mixed in various combinations. Irradiation was carried out while the mixture of monomers and enzymes was frozen in petroleum ether-dry-ice bath. Recovery of the immobilized enzyme was 44-75%.The optimum pH of the enzyme slightly shifted to the acidic side. The pH stability was improved remarkably by immobilization. The enzyme was stable retaining more than 90% of its original activity in the range pH 4-11. The optimum reaction temperature of the enzyme increased about 2 degrees C. Heat stability was also improved by immobilization, and that the enzyme retained about 40% of its original activity after treatment at 75 degrees C for 15 min. The immobilized enzyme was stable to the repeated use of 20 cycles. The K(m) value of the enzyme for short-chain amylose was almost the same as that of native enzyme. When soluble starch was used as the substrate, the K(m), value of the enzyme was three times as large as that of native enzyme. Effects of various metal ions and inhibitors on the immobilized enzyme were also studied compared to the native enzyme. 相似文献
19.
The zinc-deficient enzyme binds the fluorescence probes for the enzyme substrate pocket (auramine O, 13-ethylberberine, chlorprothixene and acridine orange) more tightly than the native enzyme, whereas 1-anilinonaphthalene 8-sulphonic acid is bound with comparable affinity. The use of fluorescence probes as reporter ligands revealed that the formation of binary complexes between the zinc-deficient enzyme and aldehydes is possible (as with the native enzyme) and confirmed an increased affinity of coenzymes to the modified enzyme. The absence of catalytic zinc ions brings about a loss of the essential stabilization effect in simultaneous NADH and aldehyde binding to liver alcohol dehydrogenase. 2,2'-Bipyridine, which chelates the active-site zinc ion in the native enzyme, is bound rather loosely to the same site as aldehydes, auramine O and ethylberberine in the case of the zinc-depleted enzyme. The stopped-flow measurements showed that the pH dependence of auramine O and ethylberberine binding to native and zinc-depleted enzyme is essentially similar. These data are compatible with the presence of ionizable groups in the surroundings of the bound probes. This group might be either His-67, bound to the zinc ion, or the zinc-liganding water molecule in the case of the native enzyme (pK close to 9), or the free His-67 residue in the case of the zinc-deficient enzyme (pK about 8). 相似文献
20.
Glycogen debranching enzyme was partially purified from bovine brain using a substrate for measuring the amylo-1,6-glucosidase activity. Bovine cerebrum was homogenized, followed by cell-fractionation of the resulting homogenate. The enzyme activity was found mainly in the cytosolic fraction. The enzyme was purified 5,000-fold by ammonium sulfate precipitation, anion-exchange chromatography, gel-filtration, anion-exchange HPLC, and gel-permeation HPLC. The enzyme preparation had no alpha-glucosidase or alpha-amylase activities and degraded phosphorylase limit dextrin of glycogen with phosphorylase. The molecular weight of the enzyme was 190,000 and the optimal pH was 6.0. The brain enzyme differed from glycogen debranching enzyme of liver or muscle in its mode of action on dextrins with an alpha-1,6-glucosyl branch, indicating an amino acid sequence different from those of the latter two enzymes. It is likely that the enzyme is involved in the breakdown of brain glycogen in concert with phosphorylase as in the cases of liver and muscle, but that this proceeds in a somewhat different manner. The enzyme activity decreased in the presence of ATP, suggesting that the degradation of brain glycogen is controlled by the modification of the debranching enzyme activity as well as the phosphorylase. 相似文献