Mix-and-inject XFEL crystallography reveals gated conformational dynamics during enzyme catalysis

Significance Protein structures fluctuate owing to thermal motion and in response to functional changes such as ligand binding. As a consequence, it is challenging to determine which protein motions are functionally most important at equilibrium. Enzymes that are transiently covalently modified during catalysis offer a way to identify functional motions, as the modification can trigger catalytically important conformational changes. The covalent modification of the active-site cysteine in isocyanide hydratase weakens a critical hydrogen bond required for reactivity. Hydrogen bond disruption triggers a cascade of conformational changes whose modulation by mutation is detrimental to enzyme turnover. Most enzymes that form catalytic intermediates will experience similar transient changes in active-site electrostatics, suggesting that modification-gated conformational dynamics is common in enzymes. How changes in enzyme structure and dynamics facilitate passage along the reaction coordinate is a fundamental unanswered question. Here, we use time-resolved mix-and-inject serial crystallography (MISC) at an X-ray free electron laser (XFEL), ambient-temperature X-ray crystallography, computer simulations, and enzyme kinetics to characterize how covalent catalysis modulates isocyanide hydratase (ICH) conformational dynamics throughout its catalytic cycle. We visualize this previously hypothetical reaction mechanism, directly observing formation of a thioimidate covalent intermediate in ICH microcrystals during catalysis. ICH exhibits a concerted helical displacement upon active-site cysteine modification that is gated by changes in hydrogen bond strength between the cysteine thiolate and the backbone amide of the highly strained Ile152 residue. These catalysis-activated motions permit water entry into the ICH active site for intermediate hydrolysis. Mutations at a Gly residue (Gly150) that modulate helical mobility reduce ICH catalytic turnover and alter its pre-steady-state kinetic behavior, establishing that helical mobility is important for ICH catalytic efficiency. These results demonstrate that MISC can capture otherwise elusive aspects of enzyme mechanism and dynamics in microcrystalline samples, resolving long-standing questions about the connection between nonequilibrium protein motions and enzyme catalysis.