Gibbs B S, Wojchowski D, Benkovic S J
Department of Chemistry, Pennsylvania State University, University Park 16802.
J Biol Chem. 1993 Apr 15;268(11):8046-52.
Rat liver phenylalanine hydroxylase was expressed in both Escherichia coli and the Spodoptera frugiperda insect cell line, Sf9. Recombinant enzyme from E. coli was inactive and contained less than 0.1 iron atom/subunit. In contrast, recombinant enzyme expressed in Sf9 cells using a baculovirus vector was active and identical in several properties to phenylalanine hydroxylase from rat liver: the Km for 6-methyltetrahydropterin was 39 microM (compared with 35 microM for the rat liver enzyme), 1 atom of iron was "associated" per enzyme subunit, and electron paramagnetic resonance spectra showed that iron was distributed within two distinct environments. Putative iron-binding sites of phenylalanine hydroxylase were studied by mutating either histidine 284 or 289 to serine and expressing these mutant enzymes (PAH-H284S and PAH-H289S) in Sf9 cells. Mutants were expressed at levels similar to wild-type PAH, but contained < or = 0.1 iron/subunit and were inactive. Thus, both His284 and His289 apparently are required for iron binding and hydroxylation activity.
大鼠肝脏苯丙氨酸羟化酶在大肠杆菌和草地贪夜蛾昆虫细胞系Sf9中均有表达。来自大肠杆菌的重组酶无活性，每个亚基所含铁原子少于0.1个。相比之下，利用杆状病毒载体在Sf9细胞中表达的重组酶具有活性，且在多个特性上与大鼠肝脏苯丙氨酸羟化酶相同：6-甲基四氢蝶呤的Km为39微摩尔（大鼠肝脏酶为35微摩尔），每个酶亚基“结合”1个铁原子，电子顺磁共振光谱显示铁分布在两种不同的环境中。通过将组氨酸284或289突变为丝氨酸并在Sf9细胞中表达这些突变酶（PAH-H284S和PAH-H289S），对苯丙氨酸羟化酶的假定铁结合位点进行了研究。突变体的表达水平与野生型PAH相似，但每个亚基所含铁≤0.1个且无活性。因此，His284和His289显然都是铁结合和羟化活性所必需的。
