Protein Dynamics DOI: 10.1002/ange.201204903 Conformer Selection and Intensified Dynamics During Catalytic Turnover in Chymotrypsin** Peter Liuni, Araby Jeganathan, and Derek J. Wilson* During catalytic turnover, enzymes undergo thermally driven conformational fluctuations (dynamics) that are directly linked to catalytic efficiency. [1–3] In broad terms, this link exists because enzymes must sample specific dynamic modes in order to access high-energy structures along the catalytic reaction coordinate. [4] Mass spectrometry-based approaches for probing enzyme dynamics are developing rapidly, but have been limited thus far by the application of H/D exchange (HDX) under steady-state conditions, which results in averaging of the data over multiple catalytic states. [5, 6] Models that attempt to define the specific nature of the enzyme dynamics/activity relationship are therefore drawn overwhelmingly from Carr–Purcell–Meiboom–Gill (CPMG) relaxation dispersion NMR experiments on “active” enzymes. [3, 7, 8] This approach is extremely powerful, but is confined to a small set of highly reversible reactions in order to circumvent the issue of substrate depletion during the experiment. [2, 3, 9–12] From the limited set of “CPMG accessible” reactions, two models for catalysis-linked dynamics have been formulated. The “induced fit” model is characterized by a substrate-free (resting) state that samples a different set of dynamic modes compared to the substrate-bound, catalytically active state. This would imply that the dynamics observed in the resting state should be substantially different from those observed during catalysis. A number of studies have reported evidence supporting this model. [13, 14] In the “conformer selection” model, the conformational space sampled by the enzyme is independent of catalysis (i.e., the resting and active-state conformational dynamics are identical). Productive enzyme– substrate interactions occur when incoming substrate “selects” the appropriate conformer for binding. Conformer selection is supported by a substantial number of studies showing no difference in dynamics between free and sub- strate-bound enzyme. [2, 3, 5, 9–11] “Hybrid” models have also been proposed in which substrate binding occurs through conformer selection followed by “induced fit-like” substrate- directed conformational sampling during catalysis. [15] These models provide crucial insights into virtually all aspects of enzyme function including substrate binding, specificity, allostery and rate-limiting catalytic processes. However, their formulation from a relatively small pool of similar enzyme systems suggests that they may describe only a fraction of possible catalysis-linked dynamic modes. In this work, we probe conformational dynamics in an active, “CPMG inaccessible” enzyme system using an alter- native approach that combines time-resolved electrospray mass spectrometry (TRESI-MS) [16] and sub-second H/D exchange (HDX) labeling [17] to monitor dynamics in the pre-steady state. By this approach, catalytic processes are detected as time-dependent intensity changes in mass-to- charge (m/z) peaks corresponding to the accumulation and/or depletion of enzyme intermediates. For each species that becomes populated during the measurement, dynamics are probed simultaneously, by the rate and magnitude of deute- rium uptake. In contrast to CPMG NMR spectroscopy, these measurements are not “site specific”, however, they represent a straightforward and broadly applicable method for charac- terizing dynamics in active enzyme systems. A schematic illustration of the experimental setup is provided in Figure 1. Chymotrypsin-catalyzed hydrolysis of para-nitrophenyl acetate (p-NPA) has long been a model for pre-steady state enzyme catalysis, likely due to the fortuitous release of a chromophoric product (para-nitrophenol) upon acylation of the enzyme. [18] Here we use this reaction to determine if current models are sufficient to define catalysis-linked dynamics in a system that is substantially different from the set of reactions upon which the models were formulated (i.e., different class of enzyme, covalent modification, equilibrium Figure 1. A schematic depiction of the TRESI/HDX experimental setup. [15] A solution containing chymotrypsin in water (red) is passed though the inner capillary, mixing 1:4 with pNPA in D 2 O (blue) in the mixing region (green). The reacting mixture (purple) is then passed through a delay volume before undergoing electrospray ionization. The reaction/HDX labeling time is adjusted by changing the position of the inner capillary within the outer capillary. Details are provided in the Supporting Information. [*] P. Liuni, A. Jeganathan, Dr. D. J. Wilson Chemistry, York University 4700 Keele St., Toronto, ON (Canada) E-mail: dkwilson@yorku.ca Homepage: http://www.yorku.ca/dkwilson [**] This work is funded by the Natural Science and Engineering Research Council of Canada (NSERC), the Canadian Foundation for Innovation (CFI), and an Ontario Ministry of Research and Innovation (MRI) Early Researcher Award. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/anie.201204903. A ngewandte Chemi e 1 Angew. Chem. 2012, 124,1–5  2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim These are not the final page numbers! Ü Ü