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.

Post-translational modification of cysteine residues can regulate protein function and is essential for catalysis by cysteine-dependent enzymes. Covalent modifications neutralize charge on the reactive cysteine thiolate anion and thus alter the active site electrostatic environment. Although a vast number of enzymes rely on cysteine modification for function, precisely how altered structural and electrostatic states of cysteine affect protein dynamics remains poorly understood.Here we use X-ray crystallography, computer simulations, and enzyme kinetics to characterize how covalent modification of the active site cysteine residue in isocyanide hydratase (ICH) affects the protein conformational ensemble. ICH exhibits a concerted helical displacement upon cysteine modification that is gated by changes in hydrogen bond strength between the cysteine thiolate and the backbone amide of the highly strained residue Ile152. The mobile helix samples alternative conformations in crystals exposed to synchrotron X-ray radiation due to the X-rayinduced formation of a cysteine-sulfenic acid at the catalytic nucleophile (Cys101-SOH). This oxidized cysteine residue resembles the proposed thioimidate intermediate in ICH catalysis. Neither cysteine modification nor helical disorder were observed in X-ray free electron laser (XFEL) diffraction data. Computer simulations confirm cysteine modification-gated helical motion and show how structural changes allosterically propagate through the ICH dimer. Mutations at a Gly residue (Gly150) that modulate helical mobility reduce the ICH catalytic rate and alter its presteady state kinetic behavior, establishing that helical mobility is important for ICH catalytic efficiency. Our results suggest that cysteine modification may be a common and likely underreported means for regulating protein conformational dynamics. Figure 2: Cys101 oxidation leads to a weakening of the Ile152-Cys101 H-bond. Electrostatic Poisson-Boltzmann surfaces (red negative, blue positive charge) calculated from the Cys101-Ile152 (A) and Cys-SOH-Ile152 (B) environments. Cysteine photooxidation neutralizes the negative charge of the sulfur atom, weakening the N-H … S hydrogen bond. (C-E) Schematic showing covalent modification of Cys101 weakens the hydrogen bond (red dotted/dashed line) to Ile152 and allows relaxation of backbone strain (curved arrow). (F) The catalytic thioimidate covalent intermediate resembles photooxidized Cys101-SOH. Both modifications neutralize negative charge on Cys Sγ.

No abstract

No abstract

No abstract