Parniak M A, Kaufman S
J Biol Chem. 1981 Jul 10;256(13):6876-82.
Upon reaction with N-ethylmaleimide, a single sulfhydryl residue/Mr = 50,000 subunit of phenylalanine hydroxylase is modified. This modification is accompanied by a 20-30-fold increase in hydroxylase activity when the activity is measured with tetrahydrobiopterin as cofactor. The N-ethylmaleimide-modified enzyme exhibits many of the characteristics of phenylalanine hydroxylase activated by partial proteolysis or by exposure to phospholipids. For example, a change from sigmoid to hyperbolic kinetics with varying phenylalanine concentration is observed, in addition to broadened substrate specificity and a dependence on phenylalanine hydroxylase stimulator protein at pH 6.8. The binding of phenylalanine to phenylalanine hydroxylase in the absence of pterin cofactor has also been studied. The native enzyme exhibits a sigmoidal phenylalanine binding curve. The N-ethylmaleimide-modified enzyme shows a hyperbolic response to phenylalanine binding in addition to an apparent decrease in total phenylalanine binding.
与N - 乙基马来酰亚胺反应时，苯丙氨酸羟化酶的单个巯基残基/分子量为50,000的亚基会被修饰。当以四氢生物蝶呤作为辅因子测定活性时，这种修饰伴随着羟化酶活性增加20 - 30倍。N - 乙基马来酰亚胺修饰的酶表现出许多通过部分蛋白酶解或暴露于磷脂而激活的苯丙氨酸羟化酶的特征。例如，除了底物特异性变宽以及在pH 6.8时对苯丙氨酸羟化酶刺激蛋白的依赖性外，还观察到随着苯丙氨酸浓度变化，动力学从S形变为双曲线形。在没有蝶呤辅因子的情况下，也研究了苯丙氨酸与苯丙氨酸羟化酶的结合。天然酶呈现出S形的苯丙氨酸结合曲线。N - 乙基马来酰亚胺修饰的酶除了总苯丙氨酸结合明显减少外，对苯丙氨酸结合呈现双曲线响应。
