气候变化领域集成服务门户(CCPortal): Assessment of enzyme active site positioning and tests of catalytic mechanisms through X-ray

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DOI	10.1073/PNAS.2011350117
	Assessment of enzyme active site positioning and tests of catalytic mechanisms through X-ray–derived conformational ensembles
	Yabukarski F.; Biel J.T.; Pinney M.M.; Doukov T.; Powers A.S.; Fraser J.S.; Herschlag D.
发表日期	2021
ISSN	00278424
起始页码	33204
结束页码	33215
卷号	117 期号:52
英文摘要	How enzymes achieve their enormous rate enhancements remains a central question in biology, and our understanding to date has impacted drug development, influenced enzyme design, and deepened our appreciation of evolutionary processes. While enzymes position catalytic and reactant groups in active sites, physics requires that atoms undergo constant motion. Numerous proposals have invoked positioning or motions as central for enzyme function, but a scarcity of experimental data has limited our understanding of positioning and motion, their relative importance, and their changes through the enzyme’s reaction cycle. To examine positioning and motions and test catalytic proposals, we collected “room temperature” X-ray crystallography data for Pseudomonas putida ketosteroid isomerase (KSI), and we obtained conformational ensembles for this and a homologous KSI from multiple PDB crystal structures. Ensemble analyses indicated limited change through KSI’s reaction cycle. Active site positioning was on the 1- to 1.5-Å scale, and was not exceptional compared to noncatalytic groups. The KSI ensembles provided evidence against catalytic proposals invoking oxyanion hole geometric discrimination between the ground state and transition state or highly precise general base positioning. Instead, increasing or decreasing positioning of KSI’s general base reduced catalysis, suggesting optimized Ångstrom-scale conformational heterogeneity that allows KSI to efficiently catalyze multiple reaction steps. Ensemble analyses of surrounding groups for WT and mutant KSIs provided insights into the forces and interactions that allow and limit active-site motions. Most generally, this ensemble perspective extends traditional structure–function relationships, providing the basis for a new era of “ensemble–function” interrogation of enzymes. © 2020 National Academy of Sciences. All rights reserved.
英文关键词	Catalytic proposals; Conformational ensembles; Enzyme catalysis; Ketosteroid isomerase; X-ray crystallography
语种	英语
scopus关键词	steroid delta isomerase; bacterial protein; steroid delta isomerase; Article; catalysis; conformation; enzyme active site; enzyme analysis; enzyme mechanism; enzyme structure; genetic discrimination; nonhuman; priority journal; protein interaction; Pseudomonas putida; room temperature; structure activity relation; wild type; X ray crystallography; chemistry; enzymology; kinetics; metabolism; molecular dynamics; X ray crystallography; Bacterial Proteins; Catalytic Domain; Crystallography, X-Ray; Kinetics; Molecular Dynamics Simulation; Pseudomonas putida; Steroid Isomerases
来源期刊	Proceedings of the National Academy of Sciences of the United States of America
文献类型	期刊论文
条目标识符	http://gcip.llas.ac.cn/handle/2XKMVOVA/179677
作者单位	Department of Biochemistry, Stanford University, Stanford, CA 94305, United States; Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, CA 94143, United States; Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Menlo Park, CA 94025, United States; Department of Chemistry, Stanford University, Stanford, CA 94305, United States; Department of Computer Science, Stanford University, Stanford, CA 94305, United States; Stanford ChEM-H, Stanford University, Stanford, CA 94305, United States; Department of Chemical Engineering, Stanford University, Stanford, CA 94305, United States
推荐引用方式 GB/T 7714	Yabukarski F.,Biel J.T.,Pinney M.M.,等. Assessment of enzyme active site positioning and tests of catalytic mechanisms through X-ray–derived conformational ensembles[J],2021,117(52).
APA	Yabukarski F..,Biel J.T..,Pinney M.M..,Doukov T..,Powers A.S..,...&Herschlag D..(2021).Assessment of enzyme active site positioning and tests of catalytic mechanisms through X-ray–derived conformational ensembles.Proceedings of the National Academy of Sciences of the United States of America,117(52).
MLA	Yabukarski F.,et al."Assessment of enzyme active site positioning and tests of catalytic mechanisms through X-ray–derived conformational ensembles".Proceedings of the National Academy of Sciences of the United States of America 117.52(2021).