Studies on the mechanism of action of D-amino acid oxidase. Evidence for removal of substrate -hydrogen as a proton.
Studies on the mechanism of action of D-amino acid oxidase. Evidence for removal of substrate -hydrogen as a proton.
复制标题
D-氨基酸氧化酶作用机制的研究。
DOI：
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发表时间：
1971
影响因子：
4.8
通讯作者：
R. Abeles
中科院分区：
文献类型：
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作者：
C. Walsh;A. Schonbrunn;R. Abeles
摘要d-氨基酸氧化酶催化β-氯丙氨酸氧化生成氯丙酮酸，并将其转化为丙酮酸，这是一个非氧化反应。产品组成取决于氧气：只有丙酮酸在100%的N2中产生，氯丙酮酸几乎完全在100%的O2中形成，混合物在中间水平产生。总酮酸生产（丙酮酸+氯丙酮酸）的速率是独立的氧，表明速率决定形成的两个酮酸的共同前体。β-氯丙氨酸的α-氢被氘和氚取代导致动力学同位素效应，其减慢氯丙氨酸转化，但不差异地影响产物分布。在2 H2O中进行孵育的氘溶剂同位素效应也存在类似的关系。底物动力学同位素效应表明，底物的α-C-H键的解离在共同中间体的形成过程中起着或至少部分起着速率决定作用。初步实验表明，l-氨基酸氧化酶也从l-氯丙氨酸产生丙酮酸和氯丙酮酸。这些结果表明，氯丙氨酸通过d-氨基酸氧化酶转化为丙酮酸和氯丙酮酸涉及一个共同的中间体，其中底物的α-氢已作为质子被除去。其他氨基酸底物的氧化很可能涉及类似的中间体。中间体可以是碳负离子或由底物碳负离子和黄素辅酶衍生的加合物。d-氨基酸氧化酶在厌氧条件下不催化氯丙氨酸和丝氨酸的α-氢与溶剂质子的交换。当[α-3H]脯氨酸、丙酮酸和NH 4+与酶厌氧孵育时，在此类孵育中形成的丙氨酸中未观察到3 H。这表明从底物释放的质子从E·FADII 2快速释放。
Abstract d-Amino acid oxidase catalyzes the oxidation of β-chloroalanine to chloropyruvate as well as its conversion to pyruvate, a nonoxidative reaction. The product composition depends on oxygen: only pyruvate is produced in 100% N2, chloropyruvate is formed almost exclusively in 100% O2, and mixtures result at intermediate levels. The rate of total keto acid production (pyruvate + chloropyruvate) is independent of oxygen, indicating the rate-determining formation of a common precursor of the two keto acids. Substitution of the α-hydrogen of β-chloroalanine by deuterium and tritium results in kinetic isotope effects which slow down chloroalanine conversion but do not differentially affect product distribution. A similar relationship holds for the deuterium solvent isotope effect with incubations conducted in 2H2O. The substrate kinetic isotope effects indicate that the dissociation of the α-C—H bond of the substrate is rate-determining or at least partially rate-determining in the formation of the common intermediate. Preliminary experiments indicate that l-amino acid oxidase also produces both pyruvate and chloropyruvate from l-chloroalanine. These results suggest that the conversion of chloroalanine to pyruvate and chloropyruvate by d-amino acid oxidase involves a common intermediate in which the α-hydrogen of the substrate has been removed as a proton. It is very probable that the oxidation of other amino acid substrates involves similar intermediates. The intermediate could be a carbanion or an adduct derived from a substrate carbanion and the flavin coenzyme. d-Amino acid oxidase does not catalyze the exchange of the α-hydrogen of chloroalanine and serine with solvent protons under anaerobic conditions. When [α-3H]proline, pyruvate, and NH4+ were incubated anaerobically with the enzyme, no 3H was observed in the alanine formed in such incubations. This indicates that the proton released from the substrate is rapidly released from E·FADII2.