Activity-Related Microsecond Dynamics Revealed by Temperature-Jump Forster Resonance Energy Transfer Measurements on Thermophilic Alcohol Dehydrogenase

• Authors: Morgan Vaughn, Emory University; Jianyu Zhang, University of California Berkeley; Thomas G. Spiro, University of Washington; Richard Dyer, Emory University; Judith P. Klinman, University of California Berkeley
• Journal Title: Journal of the American Chemical Society
• Volume: Volume 140, Number 3
• Publisher: American Chemical Society | 2018-01-24, Pages 900-903
• Type of Work: Article | Post-print: After Peer Review
• Publisher DOI: http://doi.org/10.1021/jacs.7b12369
• Subjects: Chemistry, Pharmaceutical; Biology, Cell
• Keywords: Science & Technology; Physical Sciences; Chemistry, Multidisciplinary; Chemistry; LACTATE-DEHYDROGENASE; ENZYME CATALYSIS; PROTEIN DYNAMICS; FLUORESCENCE; MOTIONS

Abstract

Previous studies of a thermophilic alcohol dehydrogenase (ht-ADH) demonstrated a range of discontinuous transitions at 30 °C that include catalysis, kinetic isotope effects, protein hydrogen-deuterium exchange rates, and intrinsic fluorescence properties. Using the Förster resonance energy transfer response from a Trp-NADH donor-acceptor pair in T-jump studies of ht-ADH, we now report microsecond protein motions that can be directly related to active site chemistry. Two distinctive transients are observed: a slow, kinetic process lacking a temperature break, together with a faster transient that is only detectable above 30 °C. The latter establishes a link between enzyme activity and microsecond protein motions near the cofactor binding site, in a region distinct from a previously detected protein network that communicates with the substrate binding site. Though evidence of direct dynamical links between microsecond protein motions and active site bond cleavage events is extremely rare, these studies highlight the potential of T-jump measurements to uncover such properties.