Ultrafast 2D IR Studies of Internal Hydrogen Bonding Dynamics in Enzymes: The Influence of Substrate and Inhibitor Binding*

In many enzymes, collections of individually weak interactions – including hydrogen bonds – play important roles in controlling conformations and lowering transition barriers. The most direct effects arise from interactions within an enzyme’s active site but more distal effects on the scaffold must also be considered. Our laboratory uses ultrafast two-dimensional infrared (2D IR) spectroscopy and vibrational labeling techniques to site-specifically probe conformational ensembles and dynamics in enzymes. I will discuss the use of a genetically-encoded label to examine how a competitive inhibitor perturbs dynamics at the interface of the Pyrococcus horikoshii ene-reductase homodimer, where a buried cluster of several crystallographically-defined water molecules contacts the label. I will also discuss how we use alkyl-azide labeled peptides to sense human SET7/9 methyltransferase dynamics as a function of ‘depth’ in the active site. Here, substrate and inhibitor binding induces changes in 2D IR signatures, including variations in unique dynamic cross-peaks observed at moderate ‘depths’ that we tentatively assign to rapid hydrogen bond exchange/fluctuation. Experimental challenges, ongoing work, and the expected utility of theoretical studies will be highlighted.