Department of Biochemistry, Stanford University, Stanford, CA 94305, USA.
Stanford Synchrotron Radiation Light Source, SLAC National Accelerator Laboratory, Menlo Park, CA 94025, USA.
Sci Adv. 2022 Oct 14;8(41):eabn7738. doi: 10.1126/sciadv.abn7738.
Decades of structure-function studies have established our current extensive understanding of enzymes. However, traditional structural models are snapshots of broader conformational ensembles of interchanging states. We demonstrate the need for conformational ensembles to understand function, using the enzyme ketosteroid isomerase (KSI) as an example. Comparison of prior KSI cryogenic x-ray structures suggested deleterious mutational effects from a misaligned oxyanion hole catalytic residue. However, ensemble information from room-temperature x-ray crystallography, combined with functional studies, excluded this model. Ensemble-function analyses can deconvolute effects from altering the probability of occupying a state (-effects) and changing the reactivity of each state (-effects); our ensemble-function analyses revealed functional effects arising from weakened oxyanion hole hydrogen bonding and substrate repositioning within the active site. Ensemble-function studies will have an integral role in understanding enzymes and in meeting the future goals of a predictive understanding of enzyme catalysis and engineering new enzymes.
数十年的结构-功能研究已经确立了我们目前对酶的广泛理解。然而，传统的结构模型只是更广泛的构象变化状态的快照。我们以酶酮甾体异构酶 (KSI) 为例，证明了需要构象变化状态来理解功能。先前的 KSI 低温 X 射线结构的比较表明，由于氧阴离子孔催化残基未对准，会产生有害的突变效应。然而，来自室温 X 射线晶体学的组合功能研究排除了这种模型。构象-功能分析可以分解改变占据状态的概率（-效应）和改变每个状态的反应性（-效应）的影响；我们的构象-功能分析揭示了氧阴离子孔氢键变弱和底物在活性位点内重新定位引起的功能效应。构象-功能研究将在理解酶以及实现对酶催化的预测性理解和设计新酶的未来目标方面发挥重要作用。
