Lisi George P, East Kyle W, Batista Victor S, Loria J Patrick
Department of Chemistry, Yale University, New Haven, CT 06520.
Department of Chemistry, Yale University, New Haven, CT 06520;
Proc Natl Acad Sci U S A. 2017 Apr 25;114(17):E3414-E3423. doi: 10.1073/pnas.1700448114. Epub 2017 Apr 10.
Imidazole glycerol phosphate synthase (IGPS) is a V-type allosteric enzyme, meaning that its catalytic rate is critically dependent on activation by its allosteric ligand, -[(5'-phosphoribulosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (PRFAR). The allosteric mechanism of IGPS is reliant on millisecond conformational motions for efficient catalysis. We engineered four mutants of IGPS designed to disrupt millisecond motions and allosteric coupling to identify regions that are critical to IGPS function. Multiple-quantum Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments and NMR chemical shift titrations reveal diminished enzyme flexibility and a reshaping of the allosteric connectivity in each mutant construct, respectively. The functional relevance of the observed motional quenching is confirmed by significant reductions in glutaminase kinetic activity and allosteric ligand binding affinity. This work presents relevant conclusions toward the control of protein allostery and design of unique allosteric sites for potential enzyme inhibitors with regulatory or therapeutic benefit.
咪唑甘油磷酸合酶（IGPS）是一种V型别构酶，这意味着其催化速率严重依赖于其别构配体-[(5'-磷酸核糖基)甲脒基]-5-氨基咪唑-4-甲酰胺核糖核苷酸（PRFAR）的激活。IGPS的别构机制依赖于毫秒级的构象运动以实现高效催化。我们设计了IGPS的四个突变体，旨在破坏毫秒级运动和别构偶联，以确定对IGPS功能至关重要的区域。多量子Carr-Purcell-Meiboom-Gill（CPMG）弛豫色散实验和NMR化学位移滴定分别揭示了每个突变体构建体中酶灵活性的降低和别构连接性的重塑。谷氨酰胺酶动力学活性和别构配体结合亲和力的显著降低证实了所观察到的运动淬灭的功能相关性。这项工作为控制蛋白质别构和设计具有调节或治疗益处的潜在酶抑制剂的独特别构位点提供了相关结论。
