Phillips Robert S, Chen Hao Yuan, Faleev Nicolai G
Department of Chemistry, University of Georgia, Athens, Georgia 30602-2556, USA.
Biochemistry. 2006 Aug 8;45(31):9575-83. doi: 10.1021/bi060561o.
Tyrosine phenol-lyase (TPL) from Citrobacter freundii is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the reversible hydrolytic cleavage of l-Tyr to give phenol and ammonium pyruvate. The proposed reaction mechanism for TPL involves formation of an external aldimine of the substrate, followed by deprotonation of the alpha-carbon to give a quinonoid intermediate. Elimination of phenol then has been proposed to give an alpha-aminoacrylate Schiff base, which releases iminopyruvate that ultimately undergoes hydrolysis to yield ammonium pyruvate. Previous stopped-flow kinetic experiments have provided direct spectroscopic evidence for the formation of the external aldimine and quinonoid intermediates in the reactions of substrates and inhibitors; however, the predicted alpha-aminoacrylate intermediate has not been previously observed. We have found that 4-hydroxypyridine, a non-nucleophilic analogue of phenol, selectively binds and stabilizes aminoacrylate intermediates in reactions of TPL with S-alkyl-l-cysteines, l-tyrosine, and 3-fluoro-l-tyrosine. In the presence of 4-hydroxypyridine, a new absorption band at 338 nm, assigned to the alpha-aminoacrylate, is observed with these substrates. Formation of the 338 nm peaks is concomitant with the decay of the quinonoid intermediates, with good isosbestic points at approximately 365 nm. The value of the rate constant for aminoacrylate formation is similar to k(cat), suggesting that leaving group elimination is at least partially rate limiting in TPL reactions. In the reaction of S-ethyl-l-cysteine in the presence of 4-hydroxypyridine, a subsequent slow reaction of the alpha-aminoacrylate is observed, which may be due to iminopyruvate formation. Both l-tyrosine and 3-fluoro-l-tyrosine exhibit kinetic isotope effects of approximately 2-3 on alpha-aminoacrylate formation when the alpha-(2)H-labeled substrates are used, consistent with the previously reported internal return of the alpha-proton to the phenol product. These results are the first direct spectroscopic observation of alpha-aminoacrylate intermediates in the reactions of TPL.
弗氏柠檬酸杆菌的酪氨酸酚裂解酶（TPL）是一种依赖于磷酸吡哆醛（PLP）的酶，可催化L-酪氨酸的可逆水解裂解，生成苯酚和丙酮酸铵。TPL的反应机制是先形成底物的外部醛亚胺，然后α-碳去质子化生成醌型中间体。接着有人提出苯酚消除后会生成α-氨基丙烯酸席夫碱，该碱释放出亚氨基丙酮酸，最终水解生成丙酮酸铵。之前的停流动力学实验为底物和抑制剂反应中外部醛亚胺和醌型中间体的形成提供了直接的光谱证据；然而，此前尚未观察到预测的α-氨基丙烯酸中间体。我们发现，4-羟基吡啶是苯酚的非亲核类似物，在TPL与S-烷基-L-半胱氨酸、L-酪氨酸和3-氟-L-酪氨酸的反应中，它能选择性地结合并稳定氨基丙烯酸中间体。在4-羟基吡啶存在下，用这些底物可观察到在338nm处有一个新的吸收带，归属于α-氨基丙烯酸。338nm峰的形成与醌型中间体的衰减同时发生，在约365nm处有良好的等吸收点。氨基丙烯酸形成的速率常数的值与k(cat)相似，这表明离去基团的消除在TPL反应中至少部分是速率限制步骤。在4-羟基吡啶存在下S-乙基-L-半胱氨酸的反应中，观察到α-氨基丙烯酸随后有一个缓慢反应，这可能是由于亚氨基丙酮酸的形成。当使用α-(2)H标记的底物时，L-酪氨酸和3-氟-L-酪氨酸在α-氨基丙烯酸形成上都表现出约2-3的动力学同位素效应，这与之前报道的α-质子向苯酚产物的内部回返一致。这些结果是首次在TPL反应中对α-氨基丙烯酸中间体进行直接光谱观察。
