Marota J J, Shiman R
Biochemistry. 1984 Mar 13;23(6):1303-11. doi: 10.1021/bi00301a044.
We have found that rat liver phenylalanine hydroxylase oxidizes a stoichiometric amount of its cofactor, 6-methyl-5,6,7,8-tetrahydropterin (6MPH4), in a reaction that is independent of phenylalanine. The reaction requires oxygen, and one 6MPH4 is oxidized per subunit of enzyme. A quinonoid dihydropterin is directly produced in the reaction, and there is no evidence for the intermediate formation of a 4a-hydroxydihydropterin. Neither hydrogen peroxide nor superoxide anions were detected as products of the oxidation, and phenylalanine hydroxylase appears to be the sole electron acceptor from 6MPH4. Therefore, in a functional sense, phenylalanine hydroxylase is reduced by its cofactor. The reduced state of the enzyme is stable to activation by phenylalanine and during catalytic turnover, and the electrons on the reduced enzyme cannot be directly used to drive phenylalanine hydroxylation. Of greatest importance, enzyme reduction appears to be required for the formation of a catalytically active enzyme species. Phenylalanine hydroxylase is chemically and physically altered by reduction. Reduced enzyme exhibits (1) a greatly increased fluorescence, which is quantitatively related to the extent of reduction, (2) an altered UV-visible absorbance spectrum, (3) a greatly increased sensitivity to inactivation by hydrogen peroxide, and (4) a greatly decreased sensitivity to inhibition by Dopa which quantitatively correlates with the increase in enzyme fluorescence. Second-order rate constants, kr, for the reduction of the enzyme by 6MPH4 have been determined and found to vary with pH, temperature, buffer, and enzyme activation: at pH 6.8, 25 degrees C, and in phosphate buffer, for phenylalanine-activated enzyme kr = 15 X 10(6) min-1 M-1. Tris is a competitive inhibitor with respect to 6MPH4 of enzyme reduction and also of catalysis.
我们发现，大鼠肝脏苯丙氨酸羟化酶在一个与苯丙氨酸无关的反应中，能化学计量地氧化其辅因子6-甲基-5,6,7,8-四氢蝶呤（6MPH4）。该反应需要氧气，且每一个酶亚基氧化一个6MPH4。反应直接产生一种醌型二氢蝶呤，没有证据表明会中间形成4a-羟基二氢蝶呤。氧化产物中未检测到过氧化氢或超氧阴离子，苯丙氨酸羟化酶似乎是6MPH4的唯一电子受体。因此，从功能意义上讲，苯丙氨酸羟化酶被其辅因子还原。酶的还原状态在被苯丙氨酸激活以及催化周转过程中是稳定的，还原酶上的电子不能直接用于驱动苯丙氨酸羟化反应。最重要的是，酶的还原似乎是形成具有催化活性的酶物种所必需的。苯丙氨酸羟化酶在化学和物理性质上因还原而发生改变。还原酶表现出：（1）荧光大幅增强，其强度与还原程度定量相关；（2）紫外-可见吸收光谱改变；（3）对过氧化氢失活的敏感性大幅增加；（4）对多巴抑制的敏感性大幅降低，这与酶荧光的增加定量相关。已经测定了6MPH4还原该酶的二级速率常数kr，发现其随pH、温度、缓冲液和酶的激活状态而变化：在pH 6.8、25℃和磷酸盐缓冲液中，对于苯丙氨酸激活的酶，kr = 15×10⁶ min⁻¹ M⁻¹。Tris对于酶还原和催化反应而言，是6MPH4的竞争性抑制剂。
