Impaired protein conformational landscapes as revealed in anomalous Arrhenius prefactors

Abstract

A growing body of data supports a role for protein motion in enzyme catalysis. In particular, the ability of enzymes to sample catalytically relevant conformational substates has been invoked to model kinetic and spectroscopic data. However, direct experimental links between rapidly interconverting conformations and the chemical steps of catalysis remain rare. We report here on the kinetic analysis and characterization of the hydride transfer step catalyzed by a series of mutant thermophilic alcohol dehydrogenases (ht-ADH), presenting evidence for Arrhenius prefactor values that become enormously elevated above an expected value of approximately 10 13 s −1 when the enzyme operates below its optimal temperature range. Restoration of normal Arrhenius behavior in the ht-ADH reaction occurs at elevated temperatures. A simple model, in which reduced temperature alters the ability of the ht-ADH variants to sample the catalytically relevant region of conformational space, can reproduce the available data. These findings indicate an impaired landscape that has been generated by the combined condition of reduced temperature and mutation at a single, active-site hydrophobic side chain. The broader implication is that optimal enzyme function requires the maintenance of a relatively smooth landscape that minimizes low energy traps.A complete understanding of the origins of the remarkable rate acceleration and specificity achieved by enzymes remains elusive. Increasingly, studies on the fundamental basis for enzymatic catalysis have focused on the requirement for a range of protein motions in order to achieve efficient enzyme catalysis. Among these, evidence for a class of motion termed conformational sampling (or preorganization) has begun to accumulate recently (cf. refs. 1-4). The impact of such motions can be seen either in the formation of an enzyme-substrate complex or in its subsequent conversion to product.For example, a recent study of adenylate kinase implicates a temperature-dependent preequilibrium among conformers that are either "competent" or "incompetent" toward ligand binding (5). In the context of catalysis, studies of single-enzyme molecules indicate multiple, slowly interconverting conformers that lead to product with different rate constants (6-8). Extensive work on dihydrofolate reductase has shown the role of slow loop closures that occur along the reaction coordinate with the same (millsecond) time constants as catalysis (9, 10).Although computational studies also suggest a role for rapid conformational sampling in enzyme reactions (11, 12), demonstrating the direct link of these motions to catalysis is quite challenging. In this study, we use the thermophilic alcohol dehydrogenase from Bacillus stearothermophilus (ht-ADH) as a system to examine the presence of rapidly interconverting protein substates during catalysis. The ht-ADH catalyzes the transfer of a hydride equivalent from an alcohol donor to an NAD þ cofactor, and previous studies have shown that ht-ADH carries out its reaction via quantum t...

Identification of a Long-range Protein Network That Modulates Active Site Dynamics in Extremophilic Alcohol Dehydrogenases

“…This indicates the much larger effect of an active site mutant on oligomeric protein stability than the subunit interface mutant Y25A. (31). A detailed characterization of these enzymes has led to the conclusion that the unusual Arrhenius curves observed for ht-ADH variants reflect a reversible distribution of the protein conformational landscape into inactive, low energy states at low temperature.…”

“…It is, thus, possible to identify an approximately 20-Å pathway of dynamical communication between the two targeted residues (Tyr-25 and Trp-87), comprised of the very same peptides that were previously demonstrated to rigidify below 30°C in ht-ADH (21). We conclude that intersubunit contacts in the family of tetrameric prokaryotic alcohol dehydrogenases can alter the protein conformational landscape (31), thereby controlling the active site configurations that are directly linked to Arrhenius behavior and the properties of hydride tunneling between substrate and cofactor.…”

“…It has been shown previously that both the Arrhenius behavior and KIEs for ht-ADH are sensitive to amino acid substitutions for conserved bulky hydrophobic residues in the NAD ϩ cofactor binding pocket (31,32). We have now analyzed a variant at a conserved residue in the substrate-binding pocket of ht-ADH (ht-W87A) that is shown to abrogate the Arrhenius break in the experimental temperature range.…”

Kinetics of low-temperature transitions and a reaction rate theory from non-equilibrium distributions

“…Results in the kinetics of catalytic reactions of the dehydrogenase and oxidase enzymes have shown an undoubted super-Arrhenius behaviour [25,53,54,68].…”

“…It often manifests because of collective phenomena, such as those amenable to treatment by the non-extensive thermodynamics of Tsallis, and covers an ample set of phenomena: rates of enzymatic catalysis-promoted processes [53][54][55], food preservation processes [56,57] and basic features of the dynamics of complex or glass-forming liquids and solids [58][59][60][61][62][63].…”