Adenosine triphosphate phosphoribosyltransferase (ATP-PRT) catalyzes the first step in histidine biosynthesis, a pathway essential to microorganisms and a validated target for antimicrobial drug design. The ATP-PRT enzyme catalyzes the reversible substitution reaction between phosphoribosyl pyrophosphate and ATP. The enzyme exists in two structurally distinct forms, a short- and a long-form enzyme. These forms share a catalytic core dimer but bear completely different allosteric domains and thus distinct quaternary assemblies. Understanding enzymatic transition states can provide essential information on the reaction mechanisms and insight into how differences in domain structure influence the reaction chemistry, as well as providing a template for inhibitor design. In this study, the transition state structures for ATP-PRT enzymes from Campylobacter jejuni and Mycobacterium tuberculosis (long-form enzymes) and from Lactococcus lactis (short-form) were determined and compared. Intrinsic kinetic isotope effects (KIEs) were obtained at reaction sensitive positions for the reverse reaction using phosphonoacetic acid, an alternative substrate to the natural substrate pyrophosphate. The experimental KIEs demonstrated mechanistic similarities between the three enzymes and provided experimental boundaries for quantum chemical calculations to characterize the transition states. Predicted transition state structures support a dissociative reaction mechanism with a D_N*A_N^‡ transition state. Weak interactions from the incoming nucleophile and a fully dissociated ATP adenine are predicted regardless of the difference in overall structure and quaternary assembly. These studies establish that despite significant differences in the quaternary assembly and regulatory machinery between ATP-PRT enzymes from different sources, the reaction chemistry and catalytic mechanism are conserved.

中文翻译：

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三磷酸腺苷磷酸核糖基转移酶的过渡态分析

三磷酸腺苷磷酸核糖基转移酶（ATP-PRT）催化组氨酸生物合成的第一步，这是微生物必不可少的途径，也是经过验证的抗菌药物设计目标。ATP-PRT酶催化磷酸核糖焦磷酸和ATP之间的可逆取代反应。该酶以两种结构上不同的形式存在，即一种短酶和一种长酶。这些形式共有一个催化核心二聚体，但具有完全不同的变构域，因此具有不同的四级组装。了解酶促过渡态可以提供有关反应机理的基本信息，并深入了解域结构的差异如何影响反应化学，以及提供抑制剂设计的模板。在这项研究中，ATP-PRT酶的过渡态结构对空肠弯曲菌和结核分枝杆菌（长型酶）和乳酸乳球菌（短型）进行了比较。使用膦酰乙酸（天然磷酸焦磷酸的另一种底物）在逆反应的反应敏感位置获得内在动力学同位素效应（KIEs）。实验性KIE证明了这三种酶之间的机理相似性，并为量子化学计算表征过渡态提供了实验边界。预测的过渡态结构支持D _N * A _N ^‡的解离反应机理过渡状态。预测来自传入的亲核试剂和完全解离的ATP腺嘌呤的弱相互作用，而不管整体结构和四级组装的差异如何。这些研究表明，尽管来自不同来源的ATP-PRT酶之间在四级组装和调控机制上存在显着差异，但反应化学和催化机理均得到了保留。