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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 in Bacillus stearothermophilus Tryptophanyl-tRNA synthetase (TrpRS) using both multi-mutant and modular thermodynamic 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. A modular thermodynamic 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.

SUBMITTER: Carter CW

PROVIDER: S-EPMC5272822 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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Combining multi-mutant and modular thermodynamic cycles to measure energetic coupling networks in enzyme catalysis.

Carter Charles W CW Chandrasekaran Srinivas Niranj SN Weinreb Violetta V Li Li L Williams Tishan T

Structural dynamics (Melville, N.Y.) 20170126 3

We measured and cross-validated the energetics of networks in Bacillus stearothermophilus Tryptophanyl-tRNA synthetase (TrpRS) using both multi-mutant and modular thermodynamic 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. A modular thermodynamic cycle con ...[more]

PMID: 28191480

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