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首页> 外文期刊>Biochemistry >Glyoxylate carboligase: A unique thiamin diphosphate-dependent enzyme that can cycle between the 4′-aminopyrimidinium and 1′,4′- iminopyrimidine tautomeric forms in the absence of the conserved glutamate
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Glyoxylate carboligase: A unique thiamin diphosphate-dependent enzyme that can cycle between the 4′-aminopyrimidinium and 1′,4′- iminopyrimidine tautomeric forms in the absence of the conserved glutamate

机译：乙醛酸羧化酶：独特的硫胺素二磷酸依赖性酶，可在不存在保守的谷氨酸的情况下在4'-氨基嘧啶鎓和1'，4'-亚氨基嘧啶互变异构形式之间循环

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Glyoxylate carboligase (GCL) is a thiamin diphosphate (ThDP)-dependent enzyme, which catalyzes the decarboxylation of glyoxylate and ligation to a second molecule of glyoxylate to form tartronate semialdehyde (TSA). This enzyme is unique among ThDP enzymes in that it lacks a conserved glutamate near the N1′ atom of ThDP (replaced by Val51) or any other potential acid-base side chains near ThDP. The V51D substitution shifts the pH optimum to 6.0-6.2 (pK _a of 6.2) for TSA formation from pH 7.0-7.7 in wild-type GCL. This pK _a is similar to the pK _a of 6.1 for the 1′,4′-iminopyrimidine (IP)-4′-aminopyrimidinium (APH ~+) protonic equilibrium, suggesting that the same groups control both ThDP protonation and TSA formation. The key covalent ThDP-bound intermediates were identified on V51D GCL by a combination of steady-state and stopped-flow circular dichroism methods, yielding rate constants for their formation and decomposition. It was demonstrated that active center variants with substitution at I393 could synthesize (S)-acetolactate from pyruvate solely, and acetylglycolate derived from pyruvate as the acetyl donor and glyoxylate as the acceptor, implying that this substitutent favored pyruvate as the donor in carboligase reactions. Consistent with these observations, the I393A GLC variants could stabilize the predecarboxylation intermediate analogues derived from acetylphosphinate, propionylphosphinate, and methyl acetylphosphonate in their IP tautomeric forms notwithstanding the absence of the conserved glutamate. The role of the residue at the position occupied typically by the conserved Glu controls the pH dependence of kinetic parameters, while the entire reaction sequence could be catalyzed by ThDP itself, once the APH ~+ form is accessible.
机译：乙醛酸碳连接酶（GCL）是一种硫胺素二磷酸（ThDP）依赖性酶，它催化乙醛酸的脱羧反应并连接到乙醛酸的第二个分子上，形成酒石酸半醛（TSA）。该酶在ThDP酶中是独特的，因为它在ThDP的N1'原子附近（由Val51取代）或在ThDP附近的任何其他潜在的酸碱侧链上都没有保守的谷氨酸。对于野生型GCL，TSA形成的V51D取代将最适pH值从pH 7.0-7.7移至6.0-6.2（pK _a为6.2）。对于1'，4'-亚氨基嘧啶（IP）-4'-氨基嘧啶（APH〜+）质子平衡，该pK _a与6.1的pK _a相似，表明相同的基团同时控制ThDP质子化和TSA形成。在V51D GCL上，通过稳态和停止流圆二色性方法的组合，鉴定了关键的共价键与ThDP结合的中间体，生成了它们形成和分解的速率常数。已证明在I393处有取代的活性中心变异体可以仅合成丙酮酸中的（S）-乙酰乳酸，丙酮酸中的乙酰基乙醇酸酯为乙酰基供体，乙醛酸为受体，这表明该取代基在羧化酶反应中偏爱丙酮酸作为供体。与这些观察结果一致，尽管不存在保守的谷氨酸盐，但I393A GLC变体可以稳定其IP互变异构形式的衍生自乙酰基次膦酸酯，丙酰基次膦酸酯和乙酰基膦酸甲酯的预羧化中间体类似物。残基通常在保守的Glu占据的位置上的作用控制了动力学参数的pH依赖性，而一旦APH〜+形式可利用，则整个反应序列可由ThDP自身催化。

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