Kowlessur D, Citron B A, Kaufman S
Laboratory of Neurochemistry, National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland 20892, USA.
Arch Biochem Biophys. 1996 Sep 1;333(1):85-95. doi: 10.1006/abbi.1996.0367.
Recombinant human liver phenylalanine hydroxylase (PAH) expressed in Escherichia coli has been purified to homogeneity. The recombinant enzyme exists in solution as a mixture of 80% tetramers and 20% dimers. A study of the kinetic properties of the enzyme indicates that compared to the recombinant and the native rat liver enzymes, the recombinant human enzyme is in an activated state. This conclusion is supported by the finding that its catalytic activity is only marginally stimulated by incubation with either phenylalanine or lysolecithin. In contrast, the native and the recombinant rat liver enzymes are activated 8- to 25-fold, respectively, when preincubated with phenylalanine or lysolecithin. In the absence of activators, the ratio of the hydroxylase activity in the presence of 6-methyl-5,6,7,8-tetrahydropterin compared to the activity in the presence of (6R)-5,6,7,8-tetrahydrobiopterin (BH4), which is an index of the state of activation of the enzyme, is 4 for the human recombinant PAH compared to a value of 12 for the recombinant rat liver enzyme. Furthermore, the Km for phenylalanine in the presence of BH4 is 0.050 mM, a value that is one-fifth that of the recombinant rat liver enzyme. Covalent modification of the human enzyme by phosphorylation with protein kinase A provides further evidence that the human enzyme is in a substantially activated state. Phosphorylation, which results in the incorporation of 0.6 mol of phosphate/mol of subunit, leads to only a modest activation of 1.5-fold compared to about a 3-fold activation seen after phosphorylation of the native and the recombinant rat liver enzymes. Moreover, the recombinant human liver enzyme is less sensitive than the rat liver enzyme to stimulation by lysolecithin when tryptophan is the substrate. Just as is true for the rat liver enzyme, the apparent Km values for tryptophan and pheylalanine vary with the pterin cofactor employed. The ability of 7-tetrahydrobiopterin (7-BH4) to substitute for the natural cofactor tetrahydrobiopterin has been studied in vitro. The apparent Km for 7-BH4 for the recombinant human enzyme is 0.2 mM and the Km for phenylalanine is 0.05 mM. The hydroxylase reaction is severely inhibited by 7-BH4 in the presence of physiological concentrations of BH4. This inhibition can be overcome by a decrease in the concentration of phenylalanine. The implications of these novel properties of human PAH for phenylalanine homoestasis in man are discussed.
在大肠杆菌中表达的重组人肝脏苯丙氨酸羟化酶（PAH）已被纯化至同质。重组酶在溶液中以80%的四聚体和20%的二聚体混合物形式存在。对该酶动力学性质的研究表明，与重组大鼠肝脏酶和天然大鼠肝脏酶相比，重组人酶处于活化状态。这一结论得到以下发现的支持：与苯丙氨酸或溶血卵磷脂一起孵育时，其催化活性仅受到轻微刺激。相比之下，天然大鼠肝脏酶和重组大鼠肝脏酶在与苯丙氨酸或溶血卵磷脂预孵育时，分别被激活8至25倍。在没有激活剂的情况下，6-甲基-5,6,7,8-四氢蝶呤存在时的羟化酶活性与（6R）-5,6,7,8-四氢生物蝶呤（BH4）存在时的活性之比（这是酶活化状态的一个指标），重组人PAH为4，而重组大鼠肝脏酶为12。此外，在BH4存在下，苯丙氨酸的Km为0.050 mM，该值是重组大鼠肝脏酶的五分之一。用蛋白激酶A对人酶进行磷酸化的共价修饰进一步证明人酶处于基本活化状态。磷酸化导致每摩尔亚基掺入0.6摩尔磷酸盐，与天然大鼠肝脏酶和重组大鼠肝脏酶磷酸化后约3倍的活化相比，仅导致1.5倍的适度活化。此外，当色氨酸作为底物时，重组人肝脏酶比大鼠肝脏酶对溶血卵磷脂的刺激更不敏感。正如大鼠肝脏酶一样，色氨酸和苯丙氨酸的表观Km值随所使用的蝶呤辅因子而变化。已在体外研究了7-四氢生物蝶呤（7-BH4）替代天然辅因子四氢生物蝶呤的能力。重组人酶对7-BH4的表观Km为0.2 mM，对苯丙氨酸的Km为0.05 mM。在生理浓度的BH4存在下，羟化酶反应受到7-BH4的严重抑制。这种抑制可以通过降低苯丙氨酸的浓度来克服。讨论了人PAH的这些新特性对人体苯丙氨酸稳态的影响。
