title: Identification of a long-range protein network that modulates active site dynamics in extremophilic alcohol dehydrogenases.
abstract: A tetrameric thermophilic alcohol dehydrogenase from Bacillus stearothermophilus (ht-ADH) has been mutated at an aromatic side chain in the active site (Trp-87). The ht-W87A mutation results in a loss of the Arrhenius break seen at 30 °C for the wild-type enzyme and an increase in cold lability that is attributed to destabilization of the active tetrameric form. Kinetic isotope effects (KIEs) are nearly temperature-independent over the experimental temperature range, and similar in magnitude to those measured above 30 °C for the wild-type enzyme. This suggests that the rigidification in the wild-type enzyme below 30 °C does not occur for ht-W87A. A mutation at the dimer-dimer interface in a thermolabile psychrophilic homologue of ht-ADH, ps-A25Y, leads to a more thermostable enzyme and a change in the rate-determining step at low temperature. The reciprocal mutation in ht-ADH, ht-Y25A, results in kinetic behavior similar to that of W87A. Collectively, the results indicate that flexibility at the active site is intimately connected to a subunit interaction 20 Å away. The convex Arrhenius curves previously reported for ht-ADH (Kohen, A., Cannio, R., Bartolucci, S., and Klinman, J. P. (1999) Nature 399, 496-499) are proposed to arise, at least in part, from a change in subunit interactions that rigidifies the substrate-binding domain below 30 °C, and impedes the ability of the enzyme to sample the catalytically relevant conformational landscape. These results implicate an evolutionarily conserved, long-range network of dynamical communication that controls C-H activation in the prokaryotic alcohol dehydrogenases.
authors.affiliation: Department of Chemistry, University of California, Berkeley, California 94720.
authors.forename: Zachary D
authors.initials: ZD
authors.lastname: Nagel
authors.affiliation: Department of Chemistry, University of California, Berkeley, California 94720; California Institute for Quantitative Biosciences (QB3), University of California, Berkeley, California 94720.
authors.forename: Shujian
authors.initials: S
authors.lastname: Cun
authors.affiliation: Department of Chemistry, University of California, Berkeley, California 94720; California Institute for Quantitative Biosciences (QB3), University of California, Berkeley, California 94720; Department of Molecular and Cell Biology, University of California, Berkeley, California 94720. Electronic address: klinman@berkeley.edu.
authors.forename: Judith P
authors.initials: JP
authors.lastname: Klinman
mesh_terms: D000426:Alcohol Dehydrogenase / Q000737:chemistry* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D002384:Catalysis; D020134:Catalytic Domain; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003080:Cold Temperature; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics; D006859:Hydrogen / Q000737:chemistry; D016296:Mutagenesis; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D013053:Spectrophotometry
chemical_list: D001426:Bacterial Proteins; D006859:Hydrogen; D000426:Alcohol Dehydrogenase
keywords: Alcohol Dehydrogenase; Conformational Sampling; Convex Arrhenius Curves; Corresponding State Hypothesis; Enzyme Catalysis; Extremophiles; Hydrogen Tunneling; Isotope Effects; Mutagenesis; Protein Dynamics