On the catalytic mechanism of tryptophan hydroxylase
On the catalytic mechanism of tryptophan hydroxylase
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DOI：
10.1021/ja994479a
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发表时间：
2000-05-17
影响因子：
15
通讯作者：
Fitzpatrick, PF
中科院分区：
文献类型：
--
作者：
Moran, GR;Derecskei-Kovacs, A;Fitzpatrick, PF
色氨酸羟化酶利用四氢生物蝶呤和分子氧催化色氨酸的羟基化。以酪氨酸为底物，形成的C4a-羟基蝶呤的量大大超过形成的二羟基苯丙氨酸的量，这与氨基酸羟基化之前的步骤中发生的氧-氧键断裂一致。使用L-吲哚-H-2(5)-色氨酸、L-4-H-2-或L-5-H-2-色氨酸作为底物，色氨酸的V/K值没有同位素效应。 L-吲哚-B-2(5)-色氨酸和L-5-H-2-色氨酸对V-max 值有反同位素效应，但对L-4-H-2-色氨酸没有影响。将测量的同位素效应与计算的色氨酸羟基化次级平衡同位素效应值进行比较表明，结果与阳离子物质的形成最为一致。产品中 L-5-H-2-色氨酸同位素标记的保留证实了色氨酸羟化酶中发生了 NIH 转变，并表明转变方向是从碳 5 到碳 4。当氘最初位于碳 4 上而不是碳 5 上时，氘的保留程度更高。
Tryptophan hydroxylase catalyzes the hydroxylation of tryptophan using tetrahydrobiopterin and molecular oxygen. With tyrosine as a substrate, the amount of C4a-hydroxypterin formed greatly exceeds the amount of dihydroxyphenylalanine formed, consistent with oxygen-oxygen bond cleavage occurring in a step prior to amino acid hydroxylation. With L-indole-H-2(5)-tryptophan, L-4-H-2- Or L-5-H-2-tryptophan as substrate there is no isotope effect on the V/K value for tryptophan. There is an inverse isotope effect on the V-max value with L-indole-B-2(5)-tryptophan and L-5-H-2-tryptophan, but no effect with L-4-H-2-tryptophan. Comparison of the measured isotope effects with values of calculated secondary equilibrium isotope effects for tryptophan hydroxylation indicate that the results are most consistent with the formation of a cationic species. Retention of the isotopic label from L-5-H-2-tryptophan in the product confirms that an NIH shift occurs in tryptophan hydroxylase and shows that the direction of shift is from carbon 5 to carbon 4. The degree of retention of the deuterium is higher when the deuterium is initially on carbon 4 rather than carbon 5.