Pressure and Temperature Effects on the Formation of Aminoacrylate Intermediates of Tyrosine Phenol-lyase Demonstrate Reaction Dynamics,ACS Catalysis

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Pressure and Temperature Effects on the Formation of Aminoacrylate Intermediates of Tyrosine Phenol-lyase Demonstrate Reaction Dynamics
ACS Catalysis ( IF 11.3 ) Pub Date : 2020-01-10 , DOI: 10.1021/acscatal.9b03967
Robert S. Phillips _{1,

2} , Steven Craig ₂ , Andrey Kovalevsky ₃ , Oksana Gerlits ₄ , Kevin Weiss ₃ , Andreea I. Iorgu ₅ , Derren J. Heyes ₅ , Sam Hay ₅

Affiliation

The structures of aminoacrylate intermediates of wild-type, F448A mutant, and perdeuterated tyrosine phenol-lyase (TPL) formed from l-tyrosine, 3-F-l-tyrosine, S-ethyl-l-cysteine, and l-serine, with bound 4-hydroxypyridine, were determined by X-ray crystallography. All the aminoacrylate Schiff’s base structures in chain A are identical regardless of the substrate used to form them. 4-Hydroxypyridine is also in an identical location, except for F448A TPL, where it is displaced about 1 Å due to the increased size of the active site. In chain B, we have found different complexes depending on the substrate. With wild-type TPL, l-tyrosine gave no density, 3-F-l-tyrosine gave a gem-diamine, and l-serine gave a gem-diamine in chain B. S-Ethyl-l-cysteine formed an aminoacrylate in chain B with both wild-type and F448A TPL, but perdeuterated TPL with S-ethyl-l-cysteine formed a gem-diamine of aminoacrylate. The kinetics of aminoacrylate intermediate formation from l-tyrosine and S-ethyl-l-cysteine were followed by stopped-flow spectrophotometry at temperatures from 281 to 320 K and hydrostatic pressures ranging from 1 bar to 1.5 kbar at 293 K. There are large negative values of ΔS^‡, ΔC_p^‡, ΔV^‡, and Δβ^‡ for aminoacrylate intermediate formation for l-tyrosine but not for S-ethyl-l-cysteine. Formation of the aminoacrylate intermediates from l-tyrosine and S-ethyl-l-cysteine shows heavy enzyme deuterium kinetic isotope effects with perdeuterated TPL that are strongly temperature- and pressure-dependent and may be normal or inverse depending on conditions. These results suggest that conformational dynamics as well as vibrational coupling play a key role in the mechanism of the elimination reaction of l-tyrosine catalyzed by TPL.

中文翻译：

压力和温度对酪氨酸酚裂解酶氨基丙烯酸酯中间体形成的影响表明反应动力学

从形成的野生型，突变体F448A，全氘化和酪氨酸酚裂合酶（TPL）的氨基丙烯酸酯的中间体的结构升-酪氨酸，3- F-升-酪氨酸，š乙基升-半胱氨酸，和升-丝氨酸，与通过X射线晶体学测定结合的4-羟基吡啶。链A中所有的氨基丙烯酸Schiff碱结构都是相同的，而与用于形成它们的底物无关。4-羟基吡啶也位于相同的位置，但F448A TPL除外，该位置由于活性位点的增加而位移了约1。在链B中，我们发现了不同的配合物，具体取决于底物。对于野生型TPL，1-酪氨酸不产生密度3-F- 1-酪氨酸给了一个宝石二胺，和升丝氨酸给予了宝石在链B.二胺小号-乙基-升形成在B链与野生型和F448A TPL氨基丙烯酸酯-半胱氨酸，但氘化TPL与小号-乙基-升-半胱氨酸形成的宝石氨基丙烯酸酯的二胺。氨基丙烯酸酯中间体的形成从动力学升-酪氨酸和小号乙基升-半胱氨酸之后进行停流光谱法在温度为281至320 K和静水压力范围为1巴至293 K. 1.5千巴有大的负ΔS ^‡的值，Δ Ç _p^‡，Δ V ^‡，和Δβ ^‡为氨基丙烯酸酯的中间形成为升-酪氨酸但不能用于小号乙基升-半胱氨酸。由1-酪氨酸和S-乙基-1-半胱氨酸形成的氨基丙烯酸酯中间体显示出重氘的TPL具有强烈的酶氘动力学同位素效应，其强烈地依赖于温度和压力，并且取决于条件可以是正态或逆态。这些结果表明构象动力学以及振动耦合在l消除反应机理中起着关键作用。TPL催化的酪氨酸。

更新日期：2020-01-10

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