Protein tyrosine phosphatases (PTPs) play an important role in cellular signaling and have been implicated in human cancers, diabetes, and obesity. Despite shared catalytic mechanisms and transition states for the chemical steps of catalysis, catalytic rates within the PTP family vary over several orders of magnitude. These rate differences have been implied to arise from differing conformational dynamics of the closure of a protein loop, the WPD-loop, which carries a catalytically critical residue. The present work reports computational studies of the human protein tyrosine phosphatase 1B (PTP1B) and YopH from Yersinia pestis , for which NMR has demonstrated a link between their respective rates of WPD-loop motion and catalysis rates, which differ by an order of magnitude. We have performed detailed structural analysis, both conventional and enhanced sampling simulations of their loop dynamics, as well as empirical valence bond simulations of the chemical step of catalysis. These analyses revealed the key residues and structural features responsible for these differences, as well as the residues and pathways that facilitate allosteric communication in these enzymes. Curiously, our wild-type YopH simulations also identify a catalytically incompetent hyper-open conformation of its WPD-loop, sampled as a rare event, previously only experimentally observed in YopH-based chimeras. The effect of differences within the WPD-loop and its neighboring loops on the modulation of loop dynamics, as revealed in this work, may provide a facile means for the family of PTP enzymes to respond to environmental changes and regulate their catalytic activities.

Recent years have witnessed an explosion of interest in understanding the role of conformational dynamics both in the evolution of new enzymatic activities from existing enzymes and in facilitating the emergence of enzymatic activity de novo on scaffolds that were previously non-catalytic. There are also an increasing number of examples in the literature of targeted engineering of conformational dynamics being successfully used to alter enzyme selectivity and activity. Despite the obvious importance of conformational dynamics to both enzyme function and evolvability, many (although not all) computational design approaches still focus either on pure sequence-based approaches or on using structures with limited flexibility to guide the design. However, there exist a wide variety of computational approaches that can be (re)purposed to introduce conformational dynamics as a key consideration in the design process. Coupled with laboratory evolution and more conventional existing sequence- and structure-based approaches, these techniques provide powerful tools for greatly expanding the protein engineering toolkit. This Perspective provides an overview of evolutionary studies that have dissected the role of conformational dynamics in facilitating the emergence of novel enzymes, as well as advances in computational approaches that allow one to target conformational dynamics as part of enzyme design. Harnessing conformational dynamics in engineering studies is a powerful paradigm with which to engineer the next generation of designer biocatalysts.

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Understanding how enzyme catalysis varies with temperature is key to understanding catalysis itself, and ultimately, how to tune temperature optima. Temperature dependence studies inform on the change in heat capacity during the reaction, Δ ‡ , and we have recently demonstrated that this can expose links between the protein free energy landscape and enzyme turnover. By quantifying Δ ‡ , we capture information on the changes to the distribution of vibrational frequencies during enzyme turnover. The primary experimental tool to probe the role of vibrational modes in a chemical/biological process is isotope effect measurements, since isotopic substitution primarily affects the frequency of vibrational modes at/local to the position of isotopic substitution. We have monitored the temperature dependence of a range of isotope effects on the turnover of a hyper-thermophilic glucose dehydrogenase. We find a progressive effect on the magnitude of Δ ‡ with increasing isotopic substitution of D-glucose. Our experimental findings, combined with molecular dynamics simulations and quantum mechanical calculations, demonstrate that Δ ‡ is sensitive to isotopic substitution. The magnitude of the change in Δ ‡ due to substrate isotopic substitution indicates that small changes in substrate vibrational modes are 'translated' into relatively large changes in the (distribution and/or magnitude of) enzyme vibrational modes along the reaction. Therefore, the data suggest that relatively small substrate isotopic changes are causing a significant change in the temperature dependence of enzymatic rates.