Mechanism of the aromatic aminotransferase encoded by the Aro8 gene from Saccharomyces cerevisiae.
Mechanism of the aromatic aminotransferase encoded by the Aro8 gene from Saccharomyces cerevisiae.
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DOI：
10.1016/j.abb.2011.09.008
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发表时间：
2011-12
影响因子：
3.9
通讯作者：
W. Karsten;Zoraya L Reyes;Kostyantyn D. Bobyk;P. Cook;L. Chooback
中科院分区：
文献类型：
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作者：
W. Karsten;Zoraya L Reyes;Kostyantyn D. Bobyk;P. Cook;L. Chooback
在酿酒酵母中通过α-氨基己二酸途径合成氨基酸L-赖氨酸。一种尚未鉴定的含PLP的氨基转移酶被认为在L-赖氨酸生物合成途径中催化α-酮己二酸形成α-氨基己二酸，可能是酵母Aro 8基因产物。对几种不同氨基酸和酮酸的筛选表明，该酶使用l-酪氨酸、l-苯丙氨酸、α-酮己二酸和l-α-氨基己二酸作为底物。在pH 7.5时，转氨酶的紫外-可见光谱在280和343 nm处显示最大值。随着pH降低，343 nm处的峰（未质子化的内部醛亚胺）消失，并在328和400 nm处观察到两个新峰，分别代表质子化醛亚胺的烯醇亚胺和酮烯胺互变异构体。在pH7.1时，向游离酶中加入α-酮己二酸，导致343 nm处的吸光度消失，并在328和424 nm处出现峰。V/Et和V/Kα-酮己二酸酯EtpH曲线在pH 6.5至9.6范围内不依赖于pH值，而V/Kl-酪氨酸EtpH-速率曲线降低至低于7.0±0.1的单个pKa。数据表明，活性酶的形式是与内部醛亚胺未质子化。我们的结论是该酶应归类为α-氨基己二酸氨基转移酶。
The amino acid l-lysine is synthesized in Saccharomyces cerevisiae via the α-aminoadipate pathway. An as yet unidentified PLP-containing aminotransferase is thought to catalyze the formation of α-aminoadipate from α-ketoadipate in the l-lysine biosynthetic pathway that could be the yeast Aro8 gene product. A screen of several different amino acids and keto-acids showed that the enzyme uses l-tyrosine, l-phenylalanine, α-ketoadipate, and l-α-aminoadipate as substrates. The UV–visible spectrum of the aminotransferase exhibits maxima at 280 and 343nm at pH 7.5. As the pH is decreased the peak at 343nm (the unprotonated internal aldimine) disappears and two new peaks at 328 and 400nm are observed representing the enolimine and ketoenamine tautomers of the protonated aldimine, respectively. Addition, at pH 7.1, of α-ketoadipate to free enzyme leads to disappearance of the absorbance at 343nm and appearance of peaks at 328 and 424nm. The V/Etand V/Kα-ketoadipateEtpH profiles are pH independent from pH 6.5 to 9.6, while the V/Kl-tyrosinepH-rate profile decreases below a single pKaof 7.0±0.1. Data suggest the active enzyme form is with the internal aldimine unprotonated. We conclude the enzyme should be categorized as a α-aminoadipate aminotransferase.