Superstoichiometric binding of L-Phe to phenylalanine hydroxylase from Caenorhabditis elegans: evolutionary implications
Superstoichiometric binding of L-Phe to phenylalanine hydroxylase from Caenorhabditis elegans: evolutionary implications
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DOI：
10.1007/s00726-010-0611-6
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发表时间：
2010-11-01
期刊：
影响因子：
3.5
通讯作者：
Martinez, Aurora
中科院分区：
文献类型：
--
作者：
Flydal, Marte I.;Mohn, Tonje C.;Martinez, Aurora
苯丙氨酸羟化酶 (PAH) 催化 L-Phe 羟基化为 L-Tyr。功能失调的 PAH 会导致苯丙酮尿症，因此哺乳动物 PAH 受到高度调节，并与 L-Phe 表现出正协同性（希尔系数 (h) = 2）。 L-Phe 不与全长四聚体人 PAH 中的调节 ACT 结构域结合，并且通过与催化位点的同向结合引发协同性（Thrlfsson 等人，《Biochemistry》41：7573-7585，2002）。来自秀丽隐杆线虫的 PAH (cePAH) 缺乏与 L-Phe 的协同作用 (h = 0.9)，并且如本研究所示，结构分析揭示了全长 cePAH 的调节域上有一个额外的 L-Phe 结合位点。该位点涉及 GA(S)L/ISRP 基序，这些基序也存在于其他 L-Phe 结合蛋白（例如预苯酸脱水酶）的 ACT 结构域中。等温滴定量热法进一步证明 cePAH 的每个亚基有 2 个结合位点，而 hPAH 的每个亚基只有 1 个结合位点。调节位点的空间封闭，特别是来自相邻催化结构域的残基 Lys215/Tyr216，似乎阻碍了全长 hPAH 中的调节结合。因此，cePAH 的人源化突变体 Q215K/N216Y 的结合类似于 1.4 L-Phe/亚基。该突变体还对 L-Phe 显示出高催化活性和一定的正协同性 (h = 1.4)。我们的结果支持哺乳动物形式的 PAH 中正协同性的获得伴随着调节性 L-Phe 结合位点的关闭。与此同时，调节性 ACT 结构域的功能似乎从氨基酸结合转变为催化亚基之间构象变化的通讯。
Phenylalanine hydroxylase (PAH) catalyzes the hydroxylation of L-Phe to L-Tyr. Dysfunctional PAH results in phenylketonuria and mammalian PAH is therefore highly regulated and displays positive cooperativity for L-Phe (Hill coefficient (h) = 2). L-Phe does not bind to the regulatory ACT domain in full-length tetrameric human PAH and cooperativity is elicited by homotropic binding to the catalytic site (Thrlfsson et al. in Biochemistry 41:7573-7585, 2002). PAH from Caenorhabditis elegans (cePAH) is devoid of cooperativity for L-Phe (h = 0.9), and, as shown in this work, structural analysis reveal an additional L-Phe binding site at the regulatory domain of full-length cePAH. This site involves the GA(S)L/ISRP motifs, which are also found in ACT domains of other L-Phe binding proteins, such as prephenate dehydratase. Isothermal titration calorimetry further demonstrated 2 binding sites per subunit for cePAH versus similar to 1 for hPAH. Steric occlusion of the regulatory site, notably by residues Lys215/Tyr216 from the adjacent catalytic domain, appears to hinder regulatory binding in full-length hPAH. Accordingly, the humanized mutant Q215K/N216Y of cePAH binds similar to 1.4 L-Phe/subunit. This mutant also displays high catalytic activity and certain positive cooperativity for L-Phe (h = 1.4). Our results support that the acquisition of positive cooperativity in mammalian forms of PAH is accompanied by a closure of the regulatory L-Phe binding site. Concomitantly, the function of the regulatory ACT domain appears to be adapted from amino acid binding to serving the communication of conformational changes among catalytic subunits.