Determinants of substrate specificity in ω-aminotransferases
Determinants of substrate specificity in ω-aminotransferases
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DOI：
10.1074/jbc.m506977200
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发表时间：
2005-10-28
影响因子：
4.8
通讯作者：
John, RA
中科院分区：
文献类型：
--
作者：
Markova, M;Peneff, C;John, RA
鸟氨酸氨基转移酶和4-氨基丁酸氨基转移酶是具有不同底物特异性的磷酸吡哆醛依赖性酶。这些酶的原子结构已经表明：（i）活性位点的差异仅限于鸟氨酸氨基转移酶中两个酪氨酸残基占据的空间位置，以及（ii）在相关的、结构特征的氨基转移酶中，结合α-氨基酸的α-羧酸的保守精氨酸与谷氨酸残基紧密相互作用。为了确定这些残基的酶的特异性的贡献，我们分析了定点突变的鸟氨酸氨基转移酶的快速反应动力学，X-射线晶体学，和C-13 NMR光谱。如在氨基丁酸转氨酶中发现的，一个酪氨酸（Tyr-85）突变为异亮氨酸，使该酶与鸟氨酸的反应速率降低1000倍，与4-氨基丁酸的反应速率增加16倍，表明Tyr-85是对鸟氨酸特异性的主要决定因素。出乎意料的是，反应的后半部分，即酮戊二酸转化为谷氨酸的限制速率大大增加，尽管逆反应的动力学不受影响。一个突变体，其中的谷氨酸（Glu-235）与保守的精氨酸相互作用被丙氨酸取代保留其区域特异性的δ-氨基的鸟氨酸，但谷氨酸反应增强了650倍，而只有5倍的酮戊二酸反应速率增强。提出了一种模型，其中酶转化为磷酸吡哆胺形式破坏了内部谷氨酸-精氨酸相互作用，从而使酮戊二酸，但不是谷氨酸是一个很好的底物。
Ornithine aminotransferase and 4-aminobutyrate aminotransferase are related pyridoxal phosphate-dependent enzymes having different substrate specificities. The atomic structures of these enzymes have shown (i) that active site differences are limited to the steric positions occupied by two tyrosine residues in ornithine aminotransferase and (ii) that, uniquely among related, structurally characterized aminotransferases, the conserved arginine that binds the alpha-carboxylate of alpha-amino acids interacts tightly with a glutamate residue. To determine the contribution of these residues to the specificities of the enzymes, we analyzed site-directed mutants of ornithine aminotransferase by rapid reaction kinetics, x-ray crystallography, and C-13 NMR spectroscopy. Mutation of one tyrosine (Tyr-85) to isoleucine, as found in aminobutyrate aminotransferase, decreased the rate of the reaction of the enzyme with ornithine 1000-fold and increased that with 4-aminobutyrate 16-fold, indicating that Tyr-85 is amajor determinant of specificity toward ornithine. Unexpectedly, the limiting rate of the second half of the reaction, conversion of ketoglutarate to glutamate, was greatly increased, although the kinetics of the reverse reaction were unaffected. A mutant in which the glutamate (Glu-235) that interacts with the conserved arginine was replaced by alanine retained its regiospecificity for the delta-amino group of ornithine, but the glutamate reaction was enhanced 650-fold, whereas only a 5-fold enhancement of the ketoglutarate reaction rate resulted. A model is proposed in which conversion of the enzyme to its pyridoxamine phosphate form disrupts the internal glutamate-arginine interaction, thus enabling ketoglutarate but not glutamate to be a good substrate.