Detection and Time Course of Formation of Major Thiamin Diphosphate-Bound Covalent Intermediates Derived from a Chromophoric Substrate Analogue on Benzoylformate Decarboxylase
Detection and Time Course of Formation of Major Thiamin Diphosphate-Bound Covalent Intermediates Derived from a Chromophoric Substrate Analogue on Benzoylformate Decarboxylase
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DOI：
10.1021/bi801810h
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发表时间：
2009-02-10
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, Sumit;Nemeria, Natalia S.;Jordan, Frank
苯甲酰甲酸脱羧酶（BFDC）是一种典型的二磷酸硫胺素（ThDP）依赖性非氧化脱羧反应酶，本文以发色团交替底物（E）-2-氧-4-（吡啶-3-基）-3-丁烯酸（3-PKB）为底物，研究了BFDC脱羧酶的作用机理。添加3-PKB导致连续形成的两个瞬时中间体的出现，第一个是形成的脱羧ThDP结合的中间体，λ（max）在477 nm处，第二个对应于第一个脱羧后中间体烯胺，λ（max）在437 nm处。PKB-ThDP共价复合物和烯胺的形成/消耗的时间过程表明脱羧比PKB-ThDP共价加合物的形成慢。当脱羧产物3-（吡啶-3-基）丙烯醛（PAA）加入到BFDC中时。再次形成λ（max）为473 nm的吸光度，对应于PAA与ThDP的四面体加合物。将BFDC的良好形成的晶体添加到PAA的溶液中导致ThDP与PAA的BFDC结合的加合物的高分辨率（1.34埃）结构，证实了C2 α原子处的四面体性质，而不是烯胺的四面体性质，并且支持在473 nm处的λ（max）归属于PAA-ThDP加合物。PAA-ThDP共价复合物的结构是BFDC上产物-ThDP加合物的第一个实例。用3-PKB进行的类似研究表明发生了脱羧作用。还获得了当BFDC与苯甲醛孵育时烯胺中间体缓慢形成的证据，苯甲醛是脱羧反应的产物，因此证实其存在于反应途径上。
The mechanism of the enzyme benzoylformate decarboxylase (BFDC), which carries out a typical thiamin diphosphate (ThDP)-dependent nonoxidative decarboxylation reaction, was studied with the chromophoric alternate substrate (E)-2-oxo-4(pyridin-3-yl)-3-butenoic acid (3-PKB). Addition of 3-PKB resulted in the appearance of two transient intermediates formed consecutively, the first one to be formed a predecarboxylation ThDP-bound intermediate with lambda(max) at 477 nm, and the second one corresponding to the first postdecarboxylation intermediate the enamine with lambda(max) at 437 nm. The time course of formation/depletion of the PKB-ThDP covalent complex, and of the enamine showed that decarboxylation was slower than formation of the PKB-ThDP covalent adduct. When the product of decarboxylation 3-(pyridin-3-yl)acrylaldehyde (PAA) was added to BFDC. again an absorbance with lambda(max) at 473 mm was formed, corresponding to the tetrahedral adduct of PAA with ThDP. Addition of well-formed crystals of BFDC to a solution of PAA resulted in a high resolution (1.34 angstrom) structure of the BFDC-bound adduct of ThDP with PAA confirming the tetrahedral nature at the C2 alpha atom, rather than of the enamine, and supporting the assignment of the lambda(max) at 473 nm to the PAA-ThDP adduct. The structure of the PAA-ThDP covalent complex is the first example of a product-ThDP adduct on BFDC. Similar studies with 3-PKB indicated that decarboxylation had taken place. Evidence was also obtained for the slow formation of the enamine intermediate when BFDC was incubated with benzaldehyde, the product of the decarboxylation reaction thus confirming its presence on the reaction pathway.