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Catalytic Use of the Allosteric Constant by B. stearothermophilus TrpRS
Author(s) -
Carter Charles W.,
Kapustina Maryna,
Pham Yen,
Li Li,
Weinreb Violetta
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a646-b
Subject(s) - allosteric regulation , chemistry , conformational change , adenosine triphosphate , transition (genetics) , catalysis , adenylylation , stereochemistry , crystallography , biophysics , enzyme , biochemistry , biology , biosynthesis , gene
Do domain motions accelerate enzymatic rates? This question has engaged many, convinced few. During catalysis of tryptophan activation TrpRS relocates the PPi subsite, suggesting catalytic use of domain motion. MD simulations ± ATP and ± adenosine tetraphosphate (AQP), a transition state mimic, reveal a high‐energy conformational transition state coincident with the chemical transition state. ATP binding alone triggers induced‐fit ( JMB 2003, 325 :39). Resolving experimental induced‐fit free energy changes for ATP and AQP into dissociation and conformational free energies by regression methods, we have used a thermodynamic cycle relating conformational and binding equilibria to determine the allosteric constant (L = 2.3 × 10 −5 ) for the induced‐fit conformational change in unliganded TrpRS. MD simulations also suggest that most of the induced fit work arises from domain twisting ( JMB 2006, 362 :). The TrpRS:AQP complex untwists relative to the ATP complex, improving distances to the adenosine and PPi moieties, consistent with mutational analysis of TyrRS showing distributed transition‐state binding throughout these two binding subsites. An engineered mutant deleting the long‐range interactions necessary for the unfavorable twist angle indicates a decrease in transition state stabilization comparable to the free energy, ΔG = +6.4 Kcal/mole, stored during induced‐fit. Supported by NIGMS.