Hill M A, Marota J J, Shiman R
Department of Biological Chemistry, Milton S. Hershey Medical Center, Pennsylvania State University, Hershey 17033.
J Biol Chem. 1988 Apr 25;263(12):5646-55.
Rat liver phenylalanine hydroxylase must be in a reduced form to be catalytically active (Marota, J.J. A., and Shiman, R. (1984) Biochemistry 23, 1303-1311). In this communication we show that a fatty acid hydroperoxide, 13-hydroperoxylinoleic acid (LOOH), can efficiently oxidize the reduced enzyme. In the process, the hydroperoxide is decomposed, oxygen consumed, and hydrogen peroxide formed. Enzyme reduction by the tetrahydropterin cofactor and reoxidation by LOOH can occur as two single steps or, when the enzyme concentration is low compared to that of the substrates, as part of a catalytic cycle. In this latter case, phenylalanine hydroxylase is a hydroperoxide-dependent tetrahydropterin oxidase. The reaction requires 1.0 mol of O2, 1.0 mol of tetrahydropterin, and 0.5 mol of LOOH to yield 1.0 mol of quinonoid dihydropterin, 0.4 mol of H2O2, and fatty acid products. Thus far, the catalytic and single-step reactions appear the same in all properties, consistent with the steady-state reaction following a ping-pong mechanism. Phenylalanine hydroxylase is an excellent catalyst for this reaction: the turnover number with LOOH is slightly greater than with phenylalanine; the Km(app) for LOOH is 11 +/- 4 microM; and the kcat/Km ratio for LOOH is about 25 times greater than for phenylalanine. LOOH and phenylalanine appear to react at different sites on phenylalanine appear to react at different sites on phenylalanine hydroxylase, and the reaction of LOOH is inhibited only slightly by phenylalanine and not at all by 5-deaza-6-methyltetrahydropterin, a competitive inhibitor of phenylalanine hydroxylation. The reaction of LOOH with phenylalanine hydroxylase strongly resembles the nonenzymatic reaction of LOOH with hematin, implying similar mechanisms for the two reactions and implicating the enzyme's non-heme iron as both the site of reaction of LOOH and of electron transfer during oxidation and reduction. The formation of hydrogen peroxide during a reaction of phenylalanine hydroxylase is unusual. Indirect evidence indicates a reduced oxygen species, formed on the enzyme during the reduction step, is (partially) released as H2O2 when the hydroperoxide reacts.
大鼠肝脏苯丙氨酸羟化酶必须处于还原状态才能具有催化活性（马罗塔，J.J.A.，和希曼，R.（1984年）《生物化学》23卷，1303 - 1311页）。在本通讯中，我们表明一种脂肪酸氢过氧化物，13 - 氢过氧化亚油酸（LOOH），能够有效地氧化还原态的酶。在此过程中，氢过氧化物分解，消耗氧气，并生成过氧化氢。四氢生物蝶呤辅因子使酶还原以及LOOH使其再氧化可作为两个单一步骤发生，或者当酶浓度相对于底物浓度较低时，作为催化循环的一部分发生。在后一种情况下，苯丙氨酸羟化酶是一种依赖氢过氧化物的四氢生物蝶呤氧化酶。该反应需要1.0摩尔的O₂、1.0摩尔的四氢生物蝶呤和0.5摩尔的LOOH，以产生1.0摩尔的醌型二氢生物蝶呤、0.4摩尔的H₂O₂和脂肪酸产物。到目前为止，催化反应和单步反应在所有性质上似乎都相同，这与遵循乒乓机制的稳态反应一致。苯丙氨酸羟化酶是该反应的优良催化剂：与LOOH反应的周转数略大于与苯丙氨酸反应的周转数；LOOH的表观Km为11±4微摩尔；LOOH的kcat/Km比值约为苯丙氨酸的25倍。LOOH和苯丙氨酸似乎在苯丙氨酸羟化酶的不同位点发生反应，并且LOOH的反应仅受到苯丙氨酸的轻微抑制，而完全不受5 - 脱氮 - 6 - 甲基四氢生物蝶呤（苯丙氨酸羟化反应的竞争性抑制剂）的抑制。LOOH与苯丙氨酸羟化酶的反应与LOOH与高铁血红素的非酶促反应非常相似，这意味着这两个反应具有相似的机制，并暗示酶的非血红素铁既是LOOH的反应位点，也是氧化还原过程中电子转移的位点。苯丙氨酸羟化酶反应过程中过氧化氢的形成是不寻常的。间接证据表明，在还原步骤中在酶上形成的一种还原态氧物种，在氢过氧化物反应时会（部分）以H₂O₂的形式释放出来。
