The role of substrate strain in the mechanism of the carbon-carbon lyases.
The role of substrate strain in the mechanism of the carbon-carbon lyases.
复制标题
底物应变在碳-碳裂解酶机制中的作用。
DOI：
10.1016/j.bioorg.2014.06.002
复制
发表时间：
2014
影响因子：
5.1
通讯作者：
Faleev,NicolaiG
中科院分区：
文献类型：
--
作者：
Phillips,RobertS;Demidkina,TatyanaV;Faleev,NicolaiG
色氨酸吲哚裂解酶（TIL）和酪氨酸苯酚裂解酶（TPL）是细菌中的两种酶，分别催化吲哚和苯酚从L-色氨酸和L-酪氨酸中的可逆消除。这些PLP依赖性酶显示出高度的序列同源性（约40%的同一性），并且两者都形成同源四聚体结构。两种酶的稳态动力学研究表明，活性位点碱基对活性是必不可少的，α-氘代底物对kcat和kcat/Km表现出适度的初级同位素效应，表明底物去质子化是部分限速的。用TPL和TIL的预稳态动力学显示快速形成外部醛亚胺中间体，然后去质子化以得到在约500 nm处吸收的喹喔啉中间体。在苯酚和吲哚类似物、4-羟基吡啶和苯并咪唑存在下，TPL和TIL的喹喔啉中间体衰变为氨基丙烯酸酯中间体，λ max在340 nm左右。令人惊讶的是，当使用α-氘代底物时，存在显著的动力学同位素效应对喹喔啉中间体的形成和随后的衰变。TPL与结合的竞争性抑制剂，4-羟基苯基丙酸酯的晶体结构，确定了几个必要的催化残基：酪氨酸-71，苏氨酸-124，精氨酸-381，和苯丙氨酸-448。TIL和TPL的活性位点是高度保守的，除了以下残基：Arg-381（TPL）/Ile-396（TIL）; Thr-124（TPL）/Asp-137（TIL）和Phe-448（TPL）/His-463（TIL）。这些残基的突变导致催化活性急剧降低，而不改变底物特异性。保守的酪氨酸，Tyr-71（TPL）/Tyr-74（TIL）对于两种酶的消除活性是必需的，并且可能作为离去基团的质子供体发挥作用。TPL的Arg-381和Thr-124突变为丙氨酸导致非常低但可测量的催化活性。与3-氟-1-酪氨酸结合的Y 71 F和F448 H TPL的晶体学表明存在两种五键结构，松弛和紧张。在松弛结构中，底物芳环与Cβ-Cγ键共面，但在紧张结构中，底物芳环与Cβ-Cγ键共面约20°。在张力结构中，底物OH与Arg-381的胍盐和Thr-124的OH之间形成氢键，Phe-448和449的苯环提供空间张力。基于诱变效应，估计底物菌株对TPL催化的贡献约为108。因此，TPL和TIL的机制需要底物菌株和酸/碱催化，并且底物菌株可能负责TPL和TIL的非常高的底物特异性。
The carbon–carbon lyases, tryptophan indole lyase (TIL) and tyrosine phenol-lyase (TPL) are bacterial enzymes which catalyze the reversible elimination of indole and phenol froml-tryptophan andl-tyrosine, respectively. These PLP-dependent enzymes show high sequence homology (∼40% identity) and both form homotetrameric structures. Steady state kinetic studies with both enzymes show that an active site base is essential for activity, and α-deuterated substrates exhibit modest primary isotope effects onkcatandkcat/Km, suggesting that substrate deprotonation is partially rate-limiting. Pre-steady state kinetics with TPL and TIL show rapid formation of external aldimine intermediates, followed by deprotonation to give quinonoid intermediates absorbing at about 500 nm. In the presence of phenol and indole analogues, 4-hydroxypyridine and benzimidazole, the quinonoid intermediates of TPL and TIL decay to aminoacrylate intermediates, withλmaxat about 340 nm. Surprisingly, there are significant kinetic isotope effects on both formation and subsequent decay of the quinonoid intermediates when α-deuterated substrates are used. The crystal structure of TPL with a bound competitive inhibitor, 4-hydroxyphenylpropionate, identified several essential catalytic residues: Tyr-71, Thr-124, Arg-381, and Phe-448. The active sites of TIL and TPL are highly conserved with the exceptions of these residues: Arg-381(TPL)/Ile-396 (TIL); Thr-124 (TPL)/Asp-137 (TIL), and Phe-448 (TPL)/His-463 (TIL). Mutagenesis of these residues results in dramatic decreases in catalytic activity without changing substrate specificity. The conserved tyrosine, Tyr-71 (TPL)/Tyr-74 (TIL) is essential for elimination activity with both enzymes, and likely plays a role as a proton donor to the leaving group. Mutation of Arg-381 and Thr-124 of TPL to alanine results in very low but measurable catalytic activity. Crystallography of Y71F and F448H TPL with 3-fluoro-l-tyrosine bound demonstrated that there are two quinonoid structures, relaxed and tense. In the relaxed structure, the substrate aromatic ring is in plane with the Cβ–Cγbond, but in the tense structure, the substrate aromatic ring is about 20° out of plane with the Cβ–Cγbond. In the tense structure, hydrogen bonds are formed between the substrate OH and the guanidinium of Arg-381 and the OH of Thr-124, and the phenyl rings of Phe-448 and 449 provide steric strain. Based on the effects of mutagenesis, the substrate strain is estimated to contribute about 108to TPL catalysis. Thus, the mechanisms of TPL and TIL require both substrate strain and acid/base catalysis, and substrate strain is probably responsible for the very high substrate specificity of TPL and TIL.