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首页> 外文期刊>Biochemistry >Limited proteolysis and X-ray crystallography reveal the origin of substrate specificity and of the rate-limiting product release during oxidation of D-amino acids catalyzed by mammalian D-amino acid oxidase.
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Limited proteolysis and X-ray crystallography reveal the origin of substrate specificity and of the rate-limiting product release during oxidation of D-amino acids catalyzed by mammalian D-amino acid oxidase.

机译：有限的蛋白水解和X射线晶体学揭示了哺乳动物D-氨基酸氧化酶催化的D-氨基酸氧化过程中底物特异性和限速产物释放的起源。

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摘要

Limited proteolysis of D-amino acid oxidase holoenzyme with trypsin cleaves the protein at Arg 221 and near the C-terminus, producing stable 25, 13.4, and 2 kDa polypeptides [Torri-Tarelli, G., Vanoni, M. A., Negri, A., & Curti, B. (1990) J. Biol. Chem. 265, 21242-21246]. The 25 and 13.4 kDa polypeptides remain associated to form a nicked D-amino acid oxidase species. This nicked protein form maintains the ability to bind FAD, but exhibits altered catalytic efficiency toward the oxidation of various D-amino acids when compared to native DAAO. Changes in substrate specificity were first monitored by measuring the activity in the presence of different amino acid substrates at various times during proteolysis. Three amino acid substrates were then selected for further analysis of the properties of the nicked D-amino acid oxidase species produced by limited tryptic proteolysis: D-serine, D-arginine, and D-alanine. The three D-amino acids represented limiting cases of the observed changes of enzyme activityon nicking: loss of activity, increase of activity, and minor activity changes, respectively. D-serine was found to be no longer a substrate of D-amino acid oxidase. D-arginine exhibited a 2.5-fold increased apparent maximum velocity although its Km value increased 2-fold with the nicked enzyme in comparison to the native species. D-alanine was oxidized 1.5-fold faster by the nicked D-amino acid oxidase at infinite substrate concentration, and its Km value increased approximately 4-fold. The Kd for benzoate, which was determined kinetically with D-alanine as the enzyme substrate, increased 17-fold in the nicked species. Primary deuterium kinetic isotope effects on V and V/K during the oxidation of D-alanine were also measured. (D)V/K increased from 1.4 +/- 0.2 to 1.8 +/- 0.3 on nicking, while (D)V increased from 1.04 +/- 0.1 to 2.53 +/- 0.5. All the observed changes of the values of the kinetic parameters and of the observed isotope effects are consistent with the hypothesis that nicking of D-amino acid oxidase at position 221 decreases the strength of binding of both substrates and products to the enzyme active site. The information obtained by limited tryptic proteolysis nicely complements that gathered from the analysis of the three-dimensional structure of D-amino acid oxidase in complex with benzoate, which was recently determined [Mattevi, A., Vanoni, M. A., Todone, F., Rizzi, M., Teplyakov, A., Coda, A., Bolognesi, M., & Curti, B. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 7496-7501]. Arginine 221 is part of the 216-228 loop that covers the active site and contributes residues to substrate binding and catalysis. The limited proteolysis data support the hypothesis that this loop acts as a lid on the active site and controls both substrate specificity and the rate of turnover of D-amino acid oxidase.
机译：D-氨基酸氧化酶全酶用胰蛋白酶的有限蛋白水解作用可在Arg 221和C末端附近切割该蛋白质，从而产生稳定的25、13.4和2 kDa多肽[Torri-Tarelli，G.，Vanoni，MA，Negri，A. ，＆Curti，B.（1990）生物化学杂志。化学265，21242-21246]。 25和13.4kDa的多肽保持缔合以形成带切口的D-氨基酸氧化酶物质。与天然DAAO相比，这种带切口的蛋白质形式保持了结合FAD的能力，但对各种D-氨基酸的氧化表现出改变的催化效率。首先通过在蛋白水解过程中的不同时间测量在不同氨基酸底物存在下的活性来监测底物特异性的变化。然后选择三种氨基酸底物，以进一步分析有限的胰蛋白酶水解产生的带切口的D-氨基酸氧化酶种类的特性：D-丝氨酸，D-精氨酸和D-丙氨酸。这三个D-氨基酸代表了切刻时观察到的酶活性变化的极限情况：分别为活性损失，活性增加和较小的活性变化。发现D-丝氨酸不再是D-氨基酸氧化酶的底物。 D-精氨酸的表观最大速度增加了2.5倍，尽管与天然物种相比，有切口酶的Km值增加了2倍。在无限的底物浓度下，带切口的D-氨基酸氧化酶将D-丙氨酸的氧化速度提高了1.5倍，其Km值提高了约4倍。以D-丙氨酸为酶底物动力学测定的苯甲酸酯的Kd在有切口的物种中增加了17倍。还测量了D-丙氨酸氧化过程中氘对V和V / K的主要动力学同位素影响。 （D）V / K在切刻时从1.4 +/- 0.2增加到1.8 +/- 0.3，而（D）V从1.04 +/- 0.1增加到2.53 +/- 0.5。所有观察到的动力学参数值变化和观察到的同位素效应均与以下假设相一致：在221位的D-氨基酸氧化酶形成切口会降低底物和产物与酶活性位点的结合强度。通过有限的胰蛋白酶解获得的信息很好地补充了最近分析的D-氨基酸氧化酶与苯甲酸酯复合物的三维结构分析中收集的信息[Mattevi，A.，Vanoni，MA，Todone，F.， Rizzi，M.，Teplyakov，A.，Coda，A.，Bolognesi，M.，＆Curti，B.（1996年） Natl。学院科学美国专利93，7496-7501]。精氨酸221是216-228环的一部分，其覆盖活性位点并为残基结合和催化贡献残基。有限的蛋白水解数据支持以下假设：该环充当活性位点的盖子，并控制底物特异性和D-氨基酸氧化酶的周转率。

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