Carkaci-Salli Nurgul, Bewley Maria C, Tekin Izel, Flanagan John M, Vrana Kent E
Departments of Pharmacology, Pennsylvania State University College of Medicine, Hershey, PA, 17033, USA.
Biochemistry and Molecular Biology, Pennsylvania State University College of Medicine, Hershey, PA, 17033, USA.
Biochem Biophys Rep. 2023 Aug 15;35:101527. doi: 10.1016/j.bbrep.2023.101527. eCollection 2023 Sep.
Human tryptophan hydroxylase 2 (hTPH2) is the rate-limiting enzyme for serotonin biosynthesis in the brain. A number of naturally-occurring single nucleotide polymorphisms (SNPs) have been reported for hTPH2. We investigated the activity and kinetic characteristics of the most common missense polymorphism rs2887147 (A328 V/E; 0.92% allelic frequency for the two different reported SNPs at the same site) using bacterially expressed hTPH2. The recombinant full-length enzyme A328E had no measurable enzyme activity, but A328V displayed decreased enzyme activity (V). A328V also displayed substrate inhibition and decreased stability compared to the wild-type enzyme. By contrast, in constructs lacking the N-terminal 150 amino acid regulatory domain, the A328V substitution had no effect; that is, there was no substrate inhibition, enzyme stabilities (for wild-type and A328V) were dramatically increased, and V values were not different (while the A328E variant remained inactive). These findings, in combination with molecular modeling, suggest that substitutions at A328 affect catalytic activity by altering the conformational freedom of the regulatory domain. The reduced activity and substrate inhibition resulting from these polymorphisms may ultimately reduce serotonin synthesis and contribute to behavioral perturbations, emotional stress, and eating disorders.
人色氨酸羟化酶2（hTPH2）是大脑中5-羟色胺生物合成的限速酶。已有多项关于hTPH2自然发生的单核苷酸多态性（SNP）的报道。我们使用细菌表达的hTPH2研究了最常见的错义多态性rs2887147（A328V/E；同一位置两个不同报道的SNP的等位基因频率为0.92%）的活性和动力学特征。重组全长酶A328E没有可测量的酶活性，但A328V的酶活性（V）降低。与野生型酶相比，A328V还表现出底物抑制和稳定性降低。相比之下，在缺乏N端150个氨基酸调节域的构建体中，A328V替换没有影响；也就是说，没有底物抑制，酶稳定性（野生型和A328V）显著增加，V值没有差异（而A328E变体仍然无活性）。这些发现与分子建模相结合，表明A328处的替换通过改变调节域的构象自由度来影响催化活性。这些多态性导致的活性降低和底物抑制最终可能会减少5-羟色胺的合成，并导致行为紊乱、情绪压力和饮食失调。
