Assessment of enzyme active site positioning and tests of catalytic mechanisms through X-ray-derived conformational ensembles

被引:49
作者
Yabukarski, Filip [1 ]
Biel, Justin T. [2 ]
Pinney, Margaux M. [1 ]
Doukov, Tzanko [3 ]
Powers, Alexander S. [4 ,5 ,6 ]
Fraser, James S. [2 ]
Herschlag, Daniel [1 ,6 ,7 ]
机构
[1] Stanford Univ, Dept Biochem, Stanford, CA 94305 USA
[2] Univ Calif San Francisco, Dept Bioengn & Therapeut Sci, San Francisco, CA 94143 USA
[3] SLAC Natl Accelerator Lab, Stanford Synchrotron Radiat Lightsource, Menlo Pk, CA 94025 USA
[4] Stanford Univ, Dept Chem, Stanford, CA 94305 USA
[5] Stanford Univ, Dept Comp Sci, Stanford, CA 94305 USA
[6] Stanford Univ, Stanford ChEM H, Stanford, CA 94305 USA
[7] Stanford Univ, Dept Chem Engn, Stanford, CA 94305 USA
基金
美国国家卫生研究院;
关键词
enzyme catalysis; catalytic proposals; conformational ensembles; X-ray crystallography; ketosteroid isomerase; NUCLEAR-MAGNETIC-RESONANCE; BARRIER HYDROGEN-BONDS; KETOSTEROID ISOMERASE; GENERAL BASE; DELTA(5)-3-KETOSTEROID ISOMERASE; CRYSTAL-STRUCTURES; PROTEIN DYNAMICS; BINDING-ENERGY; TRANSITION; TEMPERATURE;
D O I
10.1073/pnas.2011350117
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
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-angstrom 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 Angstrom-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.
引用
收藏
页码:33204 / 33215
页数:12
相关论文
共 89 条
[1]  
[Anonymous], 1963, The Feynman Lectures on Physics
[2]  
[Anonymous], 1999, The Weak Hydrogen Bond: in Structural Chemistry and Biology
[3]   DYNAMICS OF LIGAND-BINDING TO MYOGLOBIN [J].
AUSTIN, RH ;
BEESON, KW ;
EISENSTEIN, L ;
FRAUENFELDER, H ;
GUNSALUS, IC .
BIOCHEMISTRY, 1975, 14 (24) :5355-5373
[4]   A perspective on enzyme catalysis [J].
Benkovic, SJ ;
Hammes-Schiffer, S .
SCIENCE, 2003, 301 (5637) :1196-1202
[5]   The Protein Data Bank [J].
Berman, HM ;
Westbrook, J ;
Feng, Z ;
Gilliland, G ;
Bhat, TN ;
Weissig, H ;
Shindyalov, IN ;
Bourne, PE .
NUCLEIC ACIDS RESEARCH, 2000, 28 (01) :235-242
[6]   Relation between native ensembles and experimental structures of proteins [J].
Best, Robert B. ;
Lindorff-Larsen, Kresten ;
DePristo, Mark A. ;
Vendruscolo, Michele .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2006, 103 (29) :10901-10906
[7]   The dynamic energy landscape of dihydrofolate reductase catalysis [J].
Boehr, David D. ;
McElheny, Dan ;
Dyson, H. Jane ;
Wright, Peter E. .
SCIENCE, 2006, 313 (5793) :1638-1642
[8]   The role of dynamic conformational ensembles in biomolecular recognition [J].
Boehr, David D. ;
Nussinov, Ruth ;
Wright, Peter E. .
NATURE CHEMICAL BIOLOGY, 2009, 5 (11) :789-796
[9]  
Bruice T.C., 1970, ENZYMES, V2, P217
[10]   Validating Molecular Dynamics Simulations against Experimental Observables in Light of Underlying Conformational Ensembles [J].
Childers, Matthew Carter ;
Daggett, Valerie .
JOURNAL OF PHYSICAL CHEMISTRY B, 2018, 122 (26) :6673-6689