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氨基酰化酶中金属锌离子的功能作用 总被引:1,自引:0,他引:1
氨基酰化酶是含锌金属酶。该酶每摩尔蛋白中含2摩尔Zn(Ⅱ)离子。金属鳌合剂与酶作用,通过竞争螯合Zn(Ⅱ)离子使酶活力下降。残余活力与残留金属含量呈正相关。竞争螯合的结果,生成不含金属的脱辅基酶蛋白,并导致酶活力的丧失。脱辅基酶由于加入Zn(Ⅱ)离子而恢复其活力。实验表明金属锌离子是氨基酰化酶催化活力所必需。与Zn(Ⅱ)离子相似,Co(Ⅱ)离子也可与脱辅基酶相结合并使之复活。 在190—240nm区域内对比了天然酶、脱辅基酶蛋白与Co(Ⅱ)置换氨基酰化酶的圆二色谱。远紫外圆二色谱表明,与天然酶相比,在脱辅基酶中由于金属离子的丧失导致主链构象发生变化,其中α螺旋增加约7%。因而锌离子(钴离子)对蛋白主链的反应最适构象有一定的稳定作用。脱辅基酶与Co(Ⅱ)离子结合,酶的主链构象恢复至与天然酶几近相同。可认为这是促使酶复活的内在因素。 相似文献
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锌离子对氨基酰化酶构象及其稳定性的影响 总被引:3,自引:0,他引:3
天然氨基酰化酶和脱谷氨基酰化酶无论在二级结构(用CD和FTIR监测)还是三级结构上(以荧光发射光谱监测)都有明显的差异,表明了脱锌后酶的有序度降低;当比较天然和脱锌氨基酸化酶对去圬剂的稳定性时,结果表明脱锌后酶的构象的稳定性明显降低.因此可以认为锌离子对维持酶分子活性部位的特定构象以及构象的稳定性具有重要的作用. 相似文献
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Water Concentration and Activity Effects on Aminoacylase in Aqueous/Organic One-Liquid-Phase Systems
Aminoacylase has been employed as a model system to study its catalytic properties at low water concentrations/water activities with different water-miscible organic cosolvents. Cosolvents assayed were alcohols and polyols with pure logarithm of the partition coefficient (log P) values, on the standard water/octanol system, ranging between -5.2 and 0.24.
Experimental hydrolysis equilibrium constants (Kapp), at a constant water concentration, decreased with the fall in log P of the cosolvent, as well as with reduction of the water concentration/water activity, as would be expected. The enzyme hydrolytic and synthetic activities, measured at a constant water concentration/water activity value, followed a sigmoidal dependence on log P of the cosolvent employed when the water concentration or water activity values were lower than 50% (w/w) or 0.66, respectively. This became a hyperbolic relationship at higher water concentration/water activity values. A linear relationship between the logarithm of the limiting water activity necessary to maintain enzyme activity and log P was obtained. Both hydrolytic and synthetic activities were suppressed for water activities higher than 0.66 and cosolvents with log P lower than -1.6. 相似文献
Experimental hydrolysis equilibrium constants (Kapp), at a constant water concentration, decreased with the fall in log P of the cosolvent, as well as with reduction of the water concentration/water activity, as would be expected. The enzyme hydrolytic and synthetic activities, measured at a constant water concentration/water activity value, followed a sigmoidal dependence on log P of the cosolvent employed when the water concentration or water activity values were lower than 50% (w/w) or 0.66, respectively. This became a hyperbolic relationship at higher water concentration/water activity values. A linear relationship between the logarithm of the limiting water activity necessary to maintain enzyme activity and log P was obtained. Both hydrolytic and synthetic activities were suppressed for water activities higher than 0.66 and cosolvents with log P lower than -1.6. 相似文献
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Fatty acid-amino acid conjugates (FACs) have been identified in Lepidopteran larvae as elicitors of plant defenses. Plant responses include the production of primary defense compounds and induction of secondary defense strategies including attraction of parasitoid wasps. These elicitors are present despite fitness costs, suggesting that they are important for the larvae’s survival. In order to exploit FAC-mediated plant defense responses in agricultural settings, an understanding of FAC purpose and metabolism is crucial. To clarify their role, enzymes involved in this metabolism are being investigated. In this work a previously undiscovered FAC hydrolase was purified from Heliothis virescens frass by liquid chromatography and PAGE techniques and was identified as an aminoacylase-like protein (L-ACY-1) using MALDI-ToF/ToF and Edman sequencing. The full length gene was cloned and expressed in Escherichia coli and a polyclonal antibody against L-ACY-1 was made. L-ACY-1 was confirmed to be responsible for FAC hydrolysis activity through inhibition of N-linolenoyl-l-glutamine hydrolysis by titration with the polyclonal anti-L-ACY-1 antibody. L-ACY-1 activity is dependent on a divalent cation. This is the first time an aminoacylase has been described from an insect. L-ACY-1 appears to play a vastly different role in insects than ACYs do in mammals and may be involved in maintaining glutamine supplies for gut tissue metabolism. Identification of L-ACY-1, a FAC hydrolase, clarifies a previously uncharacterized portion of FAC metabolism. 相似文献
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本文研究了不同浓度盐酸胍和脲溶液对猪肾氨基酰化酶活性和构象的影响。研究结果表明,在低浓度的胍和脲溶液中(小于2mol/L),酶分子的整体构象变化的程度与活力变化的程度基本是平行的;而在高浓度的胍和脲溶液中(2mol/L以上),失活程度稍大于构象变化的程度。这些结果与分子量和亚基组成基本相同,但不含金属配基的肌酸激酶的结果,以及小分子量的胰凝乳蛋白酶和牛胰核糖核酸酶的结果相比较来看,可以认为配基锌离子的存在对酶分子的活性部位区域构象的稳定作用有一定的贡献,致使氨基酰化酶的活性部位的构象状态不象后三种酶那样脆弱。同时,我们还发现锌离子的存在对酶分子整体构象的稳定性上贡献很小。 相似文献
7.
A class of metalloenzymes, known as zinc hydrolases, catalyze a variety of hydrolytic reactions on many different substrates in important metabolic pathways. Deacetylation is an example of one of the types of reactions catalyzed by zinc hydrolases. The biological importance of the reactions catalyzed by many zinc hydrolases, including zinc-dependent deacetylases, has made these enzymes pharmaceutical targets for the development of inhibitors and, therefore, a clear understanding of the mechanisms of these enzymes is warranted. This review focuses on the current understanding of the mechanisms catalyzed by various zinc-dependent deacetylases and, in particular, the reaction mechanism catalyzed by the enzyme UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase, also known as LpxC. In general, the zinc-water functions as the nucleophile with zinc stabilization of the tetrahedral intermediate and general-acid-base catalysis (GABC) provided by enzyme residue(s). Two types of GABC mechanisms have been identified, one that uses a single bifunctional GABC and another that uses a GABC pair. 相似文献
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Purification, Characterization, and Localization of Aspartoacylase from Bovine Brain 总被引:5,自引:3,他引:2
Rajinder Kaul Jose Casanova Anne B. Johnson Peter Tang Reuben Matalon 《Journal of neurochemistry》1991,56(1):129-135
Canavan disease, an autosomal recessive disorder, is characterized biochemically by N-acetylaspartic aciduria and aspartoacylase (N-acyl-L-aspartate amidohydrolase; EC 3.5.1.15) deficiency. However, the role of aspartoacylase and N-acetylaspartic acid in brain metabolism is unknown. Aspartoacylase has been purified to apparent homogeneity with a specific activity of approximately 19,000-20,000 nmol of aspartate released/mg of protein. The native enzyme is a 58-kDa monomer. The purified aspartoacylase activity is enhanced by divalent cations, nonionic detergents, and dithiothreitol. Low levels of dithiothreitol or beta-mercaptoethanol are required for enzyme stability. Aspartoacylase has a Km of 8.5 x 10(-4) M and a Vmax of 43,000 nmol/min/mg of protein. Inhibition of aspartoacylase by glycyl-L-aspartate and amino derivatives of D-aspartic acid suggests that the carbon backbone of the substrate is primarily involved in its interaction with the active site and that a blocked amino group is essential for the catalytic activity of aspartoacylase. Biochemical and immunocytochemical studies revealed that aspartoacylase is localized to white matter, whereas the N-acetylaspartic acid concentration is threefold higher in gray matter than in white matter. Our studies so far indicate that aspartoacylase is conserved across species during evolution and suggest a significant role for aspartoacylase and N-acetylaspartic acid in normal brain biology. 相似文献
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