The Conversion of Phenylalanine to Tyrosine

苯丙氨酸羟化酶(PAH)中有一个抑制性调节结构域，位于大约1到100个氨基酸残基之间，当用自然产生的辅酶四氢生物蝶呤(BH4)检测该酶时，该酶的活性抑制10倍以上。当使用BH4的类似物6MPH4作为辅酶时，这种抑制作用消失。为了确定PAH中在抑制中起关键作用的区域，产生了缺失突变。我们产生了三个突变体，N-15，N-26和N-50。与野生型相比，N-15的Vmax增加了2倍，N-26的Vmax增加了12倍。进一步删除的N-50在Vmax上与N-26相比没有显著差异，但在Km上与BH4相比要小得多(1.8倍)。这些结果表明，第15-26位之间的区域在PAH的负调控中起着重要作用。目前，该区域正在通过定点突变进行进一步研究。此前有研究表明，实验性糖尿病会导致大鼠体内PAH水平升高。我们现在已经与爱尔兰的John Donlon博士合作表明，这种情况会导致肝脏BH4水平的增加。由于已知糖尿病会增加糖异生，我们研究了喂饲大鼠糖异生前体(例如甘油或果糖)的效果。这些方案导致肝脏PAH和BH4水平降低，这表明羟基酶及其蝶呤辅助因子在体内是协调调节的。

An inhibitory regulatory domain in phenylalanine hydroxylase (PAH), located in a position roughly between 1 and 100 amino acid residues, inhibits PAH activity more than 10-fold when the enzyme is assayed with the naturally-occurring coenzyme, tetrahydrobiopterin (BH4). The inhibitory effect disappears when 6MPH4, an analog of BH4, is used as the coenzyme. In order to define the region in PAH which plays a key role in the inhibition, deletion mutations were generated. We generated three mutants, N-15, N-26 and N-50. Compared to wild-type, Vmax for N-15 increased 2-fold, while for N-26, it increased 12-fold. Further deletion, N-50, does not show significant differences compared to N-26 in Vmax but much less in Km with BH4 (1.8-fold). These results show that the region between position 15 and 26 plays an important role in the negative regulation of PAH. Right now this region is being further studied by site directed mutagenesis. It was previously shown that experimental diabetes in rats leads to increased levels of PAH. We have now shown in collaboration with Dr. John Donlon in Ireland, that this condition leads to an increase in hepatic levels of BH4. Since diabetes is known to increase gluconeogenesis, we examined the effect of feeding rats gluconeogenic precursors (e.g., glycerol or fructose). These regimens led to a decrease in the hepatic levels of both PAH and BH4, an indication that the hydroxylase and its pterin cofactor are coordinately regulated in vivo.