Szeltner Zoltán, Juhász Tünde, Szamosi Ilona, Rea Dean, Fülöp Vilmos, Módos Károly, Juliano Luiz, Polgár László
Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, H-1025 Budapest, Pusztaszeri út 59-67, Hungary.
Biochim Biophys Acta. 2013 Jan;1834(1):98-111. doi: 10.1016/j.bbapap.2012.08.012. Epub 2012 Aug 19.
Prolyl oligopeptidase (POP) has emerged as a drug target for neurological diseases. A flexible loop structure comprising loop A (res. 189-209) and loop B (res. 577-608) at the domain interface is implicated in substrate entry to the active site. Here we determined kinetic and structural properties of POP with mutations in loop A, loop B, and in two additional flexible loops (the catalytic His loop, propeller Asp/Glu loop). POP lacking loop A proved to be an inefficient enzyme, as did POP with a mutation in loop B (T590C). Both variants displayed an altered substrate preference profile, with reduced ligand binding capacity. Conversely, the T202C mutation increased the flexibility of loop A, enhancing the catalytic efficiency beyond that of the native enzyme. The T590C mutation in loop B increased the preference for shorter peptides, indicating a role in substrate gating. Loop A and the His loop are disordered in the H680A mutant crystal structure, as seen in previous bacterial POP structures, implying coordinated structural dynamics of these loops. Unlike native POP, variants with a malfunctioning loop A were not inhibited by a 17-mer peptide that may bind non-productively to an exosite involving loop A. Biophysical studies suggest a predominantly closed resting state for POP with higher flexibility at the physiological temperature. The flexible loop A, loop B and His loop system at the active site is the main regulator of substrate gating and specificity and represents a new inhibitor target.
脯氨酰寡肽酶（POP）已成为神经疾病的药物靶点。在结构域界面处，由A环（第189 - 209位氨基酸残基）和B环（第577 - 608位氨基酸残基）组成的柔性环结构与底物进入活性位点有关。在此，我们测定了A环、B环以及另外两个柔性环（催化组氨酸环、螺旋桨天冬氨酸/谷氨酸环）发生突变的POP的动力学和结构特性。缺乏A环的POP被证明是一种低效酶，B环发生突变（T590C）的POP也是如此。这两种变体均表现出底物偏好谱改变，配体结合能力降低。相反，T202C突变增加了A环的灵活性，使催化效率超过天然酶。B环中的T590C突变增加了对较短肽段的偏好，表明其在底物门控中起作用。如之前细菌POP结构中所见，在H680A突变体晶体结构中A环和组氨酸环是无序的，这意味着这些环具有协同的结构动力学。与天然POP不同，A环功能异常的变体不受一种17肽的抑制，该17肽可能非生产性地结合到涉及A环的一个别构位点。生物物理研究表明，POP在生理温度下主要处于封闭的静息状态，具有较高的灵活性。活性位点处的柔性A环、B环和组氨酸环系统是底物门控和特异性的主要调节因子，代表了一个新的抑制剂靶点。
