Parniak M A, Davis M D, Kaufman S
Laboratory of Neurochemistry, National Institute of Mental Health, Bethesda, Maryland 20205.
J Biol Chem. 1988 Jan 25;263(3):1223-30.
The pH optimum of rat liver phenylalanine hydroxylase is dependent on the structure of the cofactor employed and on the state of activation of the enzyme. The tetrahydrobiopterin-dependent activity of native phenylalanine hydroxylase has a pH optimum of about 8.5. In contrast, the 6,7-dimethyltetrahydropterin-dependent activity is highest at pH 7.0. Activation of phenylalanine hydroxylase either by preincubation with phenylalanine or by limited proteolysis results in a shift of the pH optimum of the tetrahydrobiopterin-dependent activity to pH 7.0. Activation of the enzyme has no effect on the optimal pH of the 6,7-dimethyltetrahydropterin-dependent activity. The different pH optimum of the tetrahydrobiopterin-dependent activity of native phenylalanine hydroxylase is due to a change in the properties of the enzyme when the pH is increased from pH 7 to 9.5. Phenylalanine hydroxylase at alkaline pH appears to be in an altered conformation that is very similar to that of the enzyme which has been activated by preincubation with phenylalanine as determined by changes in the intrinsic protein fluorescence spectrum of the enzyme. Furthermore, phenylalanine hydroxylase which has been preincubated at an alkaline pH in the absence of phenylalanine and subsequently assayed at pH 7.0 in the presence of phenylalanine shows an increase in tetrahydrobiopterin-dependent activity similar to that exhibited by the enzyme which has been activated by preincubation with phenylalanine at neutral pH. Activation of the enzyme also occurs when m-tyrosine or tryptophan replace phenylalanine in the assay mixture. The predominant cause of the increase in activity of the enzyme immediately following preincubation at alkaline pH appears to be the increase in the rate of activation by the amino acid substrate. However, in the absence of substrate activation, phenylalanine hydroxylase preincubated at alkaline pH displays an approximately 2-fold greater intrinsic activity than the native enzyme.
大鼠肝脏苯丙氨酸羟化酶的最适pH值取决于所使用的辅因子的结构以及酶的激活状态。天然苯丙氨酸羟化酶依赖四氢生物蝶呤的活性的最适pH值约为8.5。相比之下，依赖6,7-二甲基四氢蝶呤的活性在pH 7.0时最高。通过与苯丙氨酸预孵育或有限的蛋白水解激活苯丙氨酸羟化酶，会导致依赖四氢生物蝶呤的活性的最适pH值向pH 7.0偏移。酶的激活对依赖6,7-二甲基四氢蝶呤的活性的最佳pH值没有影响。天然苯丙氨酸羟化酶依赖四氢生物蝶呤的活性的不同最适pH值是由于当pH从7增加到9.5时酶的性质发生了变化。碱性pH下的苯丙氨酸羟化酶似乎处于一种改变的构象，这与通过酶的内在蛋白质荧光光谱变化所确定的、经与苯丙氨酸预孵育激活的酶的构象非常相似。此外，在没有苯丙氨酸的情况下在碱性pH下预孵育、随后在有苯丙氨酸存在的pH 7.0下测定的苯丙氨酸羟化酶，其依赖四氢生物蝶呤的活性增加，类似于在中性pH下经与苯丙氨酸预孵育激活的酶所表现出的增加。当间酪氨酸或色氨酸在测定混合物中替代苯丙氨酸时，酶也会被激活。在碱性pH下预孵育后酶活性立即增加的主要原因似乎是氨基酸底物激活速率的增加。然而，在没有底物激活的情况下，在碱性pH下预孵育的苯丙氨酸羟化酶显示出比天然酶大约高2倍的内在活性。
