Testing Geometrical Discrimination Within An Enzyme Active Site: Constrained Hydrogen Bonding in the Ketosteroid Isomerase Oxyanion Hole
Enzymes are classically proposed to accelerate reactions by binding substrates within active-site environments that are structurally preorganized to optimize binding interactions with reaction transition states rather than ground states. This is a remarkably formidable task considering the limited 0.1--1 {angstrom} scale of most substrate rearrangements. The flexibility of active-site functional groups along the coordinate of substrate rearrangement, the distance scale on which enzymes can distinguish structural rearrangement, and the energetic significance of discrimination on that scale remain open questions that are fundamental to a basic physical understanding of enzyme active sites and catalysis. We bring together 1.2--1.5 {angstrom} resolution X-ray crystallography, {sup 1}H and {sup 19}F NMR spectroscopy, quantum mechanical calculations, and transition-state analogue binding measurements to test the distance scale on which noncovalent forces can constrain the structural relaxation or translation of side chains and ligands along a specific coordinate and the energetic consequences of such geometric constraints within the active site of bacterial ketosteroid isomerase (KSI). Our results strongly suggest that packing and binding interactions within the KSI active site can constrain local side-chain reorientation and prevent hydrogen bond shortening by 0.1 {angstrom} or less. Further, this constraint has substantial energetic effects on ligand binding and stabilization of negative charge within the oxyanion hole. These results provide evidence that subtle geometric effects, indistinguishable in most X-ray crystallographic structures, can have significant energetic consequences and highlight the importance of using synergistic experimental approaches to dissect enzyme function.
- Research Organization:
- SLAC National Accelerator Lab., Menlo Park, CA (United States)
- Sponsoring Organization:
- USDOE
- DOE Contract Number:
- AC02-76SF00515
- OSTI ID:
- 953565
- Report Number(s):
- SLAC-REPRINT-2009-318; JACSAT; TRN: US201002%%1393
- Journal Information:
- J. Am. Chem. Soc 130:13696,2008, Vol. 130, Issue 41; ISSN 0002-7863
- Country of Publication:
- United States
- Language:
- English
Similar Records
A Preorganized Electric Field Leads to Minimal Geometrical Reorientation in the Catalytic Reaction of Ketosteroid Isomerase
Testing Electrostatic Complementarity in Enzyme Catalysis: Hydrogen Bonding in the Ketosteroid Isomerase Oxyanion Hole
Related Subjects
BASIC
BONDING
CATALYSIS
CHAINS
CHARGES
COORDINATES
CRYSTALLOGRAPHY
DISTANCE
ENZYMES
FLEXIBILITY
FUNCTIONALS
GROUND STATES
HOLES
HYDROGEN
INTERACTIONS
ISOMERASES
LIGANDS
NUCLEAR MAGNETIC RESONANCE
RELAXATION
RESOLUTION
SPECTROSCOPY
STABILIZATION
STOWING
SUBSTRATES
TESTING
Other
OTHER
CHEM