title: Combining multi-mutant and modular thermodynamic cycles to measure energetic coupling networks in enzyme catalysis.
abstract: We measured and cross-validated the energetics of networks inBacillus stearothermophilusTryptophanyl-tRNA synthetase (TrpRS) using bothmulti-mutantandmodularthermodynamic cycles. Multi-dimensional combinatorial mutagenesis showed that four side chains from this "molecular switch" move coordinately with the active-site Mg2+ion as the active site preorganizes to stabilize the transition state for amino acid activation. Amodularthermodynamic cycle consisting of full-length TrpRS, its Urzyme, and the Urzyme plus each of the two domains deleted in the Urzyme gives similar energetics. These dynamic linkages, although unlikely to stabilize the transition-state directly, consign the active-site preorganization to domain motion, assuring coupled vectorial behavior.
authors.affiliation: Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill , Chapel Hill, North Carolina 27599-7260, USA.
authors.forename: Charles W
authors.initials: CW
authors.lastname: Carter
authors.affiliation: Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill , Chapel Hill, North Carolina 27599-7260, USA.
authors.forename: Srinivas Niranj
authors.initials: SN
authors.lastname: Chandrasekaran
authors.affiliation: Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill , Chapel Hill, North Carolina 27599-7260, USA.
authors.forename: Violetta
authors.initials: V
authors.lastname: Weinreb
authors.affiliation: Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill , Chapel Hill, North Carolina 27599-7260, USA.
authors.forename: Li
authors.initials: L
authors.lastname: Li
authors.affiliation: Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill , Chapel Hill, North Carolina 27599-7260, USA.
authors.forename: Tishan
authors.initials: T
authors.lastname: Williams