Mechanism of benzaldehyde lyase studied via thiamin diphosphate-bound intermediates and kinetic isotope effects
Mechanism of benzaldehyde lyase studied via thiamin diphosphate-bound intermediates and kinetic isotope effects
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DOI：
10.1021/bi702302u
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发表时间：
2008-03-25
期刊：
影响因子：
2.9
通讯作者：
Jordan, Frank
中科院分区：
文献类型：
--
作者：
Chakraborty, Surnit;Nemeria, Natalia;Jordan, Frank
在苯甲醛裂解酶上实现了硫胺素二磷酸结合的中间体的直接光谱观察，该酶进行（R）-安息香到苯甲醛的可逆和高度对映体特异性转化。关键的烯胺中间体可以观察到在λ（最大）393 nm的苯偶姻分解方向和脱羧酶反应开始与苯甲酰甲酸。以苯甲醛为底物，在314 nm处未检测到中间体，仅形成安息香。为了探测（R）-苯偶姻合成方向上的限速步骤，测定了在醛位置标记的苯甲醛的H-1/H-2动力学同位素效应，发现其很小（1.14 +/- 0.03），表明C2 α-羟基苄基硫胺素二磷酸的C2 α H的电离不是限速的。使用替代底物苯甲酰甲酸和苯丙氨酸（动机是观察到虽然是碳连接酶，但苯甲醛裂解酶也可以催化2-含氧酸的缓慢脱羧），能够通过1 '，4'-亚氨基嘧啶互变异构体观察到底物-硫胺素共价中间体，这是ThDP上具有四面体C2取代基的所有中间体的特征。苯甲醛裂解酶与发色底物类似物（E）-2-6-氧代-4（吡啶-3-基）-3-丁烯酸及其脱羧产物（E）-3-（吡啶-3-基）丙烯醛的反应使得能够检测与两者的共价加合物。加合物均未发生进一步反应。研究的一个重要发现是，所有的硫胺素相关的中间体是在苯甲醛裂解酶的手性环境，反映了他们的圆二色性签名。
Direct spectroscopic observation of thiamin diphosphate-bound intermediates was achieved on the enzyme benzaldehyde lyase, which carries out reversible and highly enantiospecific conversion of (R)-benzoin to benzaldehyde. The key enamine intermediate could be observed at lambda(max) 393 nm in the benzoin breakdown direction and in the decarboxylase reaction starting with benzoylformate. With benzaldehyde as substrate, no intermediates could be detected, only formation of benzoin at 314 nm. To probe the rate-limiting step in the direction of (R)-benzoin synthesis, the H-1/H-2 kinetic isotope effect was determined for benzaldehyde labeled at the aldehyde position and found to be small (1.14 +/- 0.03), indicating that ionization of the C2 alpha H from C2 alpha-hydroxybenzylthiamin diphosphate is not rate limiting. Use of the alternate substrates benzoylformic and phenylpyruvic acids (motivated by the observation that While a carboligase, benzaldehyde lyase could also catalyze the slow decarboxylation of 2-oxo acids) enabled the observation of the substrate-thiamin covalent intermediate via the 1',4'-iminopyrimidine tautomer, characteristic of all intermediates with a tetrahedral C2 substituent on ThDP. The reaction of benzaldehyde lyase with the chromophoric substrate analogue (E)-2-6xo-4(pyridin-3-yl)-3-butenoic acid and its decarboxylated product (E)-3-(pyridine-3-yl)acrylaldehyde enabled the detection of covalent adducts with both. Neither adduct underwent further reaction. An important finding of the studies is that all thiamin-related intermediates are in a chiral environment on benzaldehyde lyase as reflected by their circular dichroism signatures.