Substrate discrimination by the human GTP fucose pyrophosphorylase
Substrate discrimination by the human GTP fucose pyrophosphorylase
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DOI：
10.1021/bi0503605
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发表时间：
2005-08-16
期刊：
影响因子：
2.9
通讯作者：
Seley, KL
中科院分区：
文献类型：
--
作者：
Quirk, S;Seley, KL
鸟苷二磷酸 - L - 岩藻糖焦磷酸化酶（GFPP，EC 2.7.7.30）催化鸟苷三磷酸和β - L - 岩藻糖 - 1 - 磷酸的可逆缩合反应，形成核苷酸 - 糖鸟苷二磷酸 - β - L - 岩藻糖。该酶主要在哺乳动物的肝脏和肾脏中发挥作用，在糖脂和糖蛋白分解过程中回收游离的L - 岩藻糖。本研究首次阐明了该蛋白质区分底物分子和非底物分子的机制。GFPP从一系列碱基 - 、核糖 - 、磷酸 - 和己糖修饰的前体分子形成核苷酸 - 糖的能力表明，酶的活性位点能感知一系列驱动底物/非底物区分的底物取代基。这些取代基改变了前体分子与酶相互作用的能力，这可以通过米氏常数（Km）、结合亲和力（Ka）的变化或酶促转换数（kcat）的变化来衡量。在这项工作中，底物结合和酶分析相结合的结果表明，嘌呤碱基的性质是底物特异性的主要决定因素，其次是己糖 - 1 - 磷酸的性质，最后是核糖部分。结合是由焓驱动的，不涉及质子转移。对于大多数核苷酸 - 糖类似物，与GFPP的结合在熵上是不利的；然而，令人惊讶的是，少数测试的底物类似物与GFPP结合时具有有利的熵项。
GTP-L-fucose pyrophosphorylase (GFPP, E. C. 2.7.7.30) catalyzes the reversible condensation of guanosine triphosphate and beta-L-fucose-1-phosphate to form the nucleotide-sugar GDP-beta-L-fucose. The enzyme functions primarily in the mammalian liver and kidney to salvage free L-fucose during the breakdown of glycolipids and glycoproteins. The mechanism by which this protein discriminates between substrate and nonsubstrate molecules has been elucidated for the first time in this study. The ability of GFPP to form nucleotide-sugars from a series of base-, ribose-, phosphate-, and hexose-modified precursor molecules has revealed that the enzyme active site senses a series of substrate substituents that drive substrate/nonsubstrate discrimination. These substituents alter the ability of the precursor molecule to interact with the enzyme, as measured by either changes in the Michaelis constant, K-m, the binding affinity, K-a, or through changes in enzymatic turnover, k(cat). In this work, the combined substrate binding and enzyme analysis has revealed that the nature of the purine base is the major determinant in substrate specificity, followed by the nature of the hexose-1-P, and finally by the ribose moiety. Binding is enthalpy-driven and does not involve proton transfer. For the majority of nucleotide-sugar analogues, binding to GFPP is entropically unfavorable; however, surprisingly, a few of the substrate analogues tested bind to GFPP with a favorable entropic term.