An official website of the United States government
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.
more »
« less
Low‐Barrier and Canonical Hydrogen Bonds Modulate Activity and Specificity of a Catalytic Triad
Abstract The position, bonding and dynamics of hydrogen atoms in the catalytic centers of proteins are essential for catalysis. The role of short hydrogen bonds in catalysis has remained highly debated and led to establishment of several distinctive geometrical arrangements of hydrogen atoms vis‐à‐vis the heavier donor and acceptor counterparts, that is, low‐barrier, single‐well or short canonical hydrogen bonds. Here we demonstrate how the position of a hydrogen atom in the catalytic triad of an aminoglycoside inactivating enzyme leads to a thirty‐fold increase in catalytic turnover. A low‐barrier hydrogen bond is present in the enzyme active site for the substrates that are turned over the best, whereas a canonical hydrogen bond is found with the least preferred substrate. This is the first comparison of these hydrogen bonds involving an identical catalytic network, while directly demonstrating how active site electrostatics adapt to the electronic nature of substrates to tune catalysis.
more »
« less
- Award ID(s):
- 1662080
- PAR ID:
- 10118058
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Angewandte Chemie International Edition
- Volume:
- 58
- Issue:
- 45
- ISSN:
- 1433-7851
- Page Range / eLocation ID:
- p. 16260-16266
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Two conserved second-sphere βArg (R) residues in nitrile hydratases (NHase), that form hydrogen bonds with the catalytically essential sulfenic and sulfinic acid ligands, were mutated to Lys and Ala residues in the Co-type NHase from Pseudonocardia thermophila JCM 3095 (PtNHase) and the Fe-type NHase from Rhodococcus equi TG328–2 (ReNHase). Only five of the eight mutants (PtNHase βR52A, βR52K, βR157A, βR157K and ReNHase βR61A) were successfully expressed and purified. Apart from the PtNHase βR52A mutant that exhibited no detectable activity, the kcat values obtained for the PtNHase and ReNHase βR mutant enzymes were between 1.8 and 12.4 s− 1 amounting to <1% of the kcat values observed for WT enzymes. The metal content of each mutant was also significantly decreased with occupancies ranging from ~10 to ~40%. UV–Vis spectra coupled with EPR data obtained on the ReNHase mutant enzyme, suggest a decrease in the Lewis acidity of the active site metal ion. X-ray crystal structures of the four PtNHase βR mutant enzymes confirmed the mutation and the low active site metal content, while also providing insight into the active site hydrogen bonding network. Finally, DFT calcu- lations suggest that the equatorial sulfenic acid ligand, which has been shown to be the catalytic nucleophile, is protonated in the mutant enzyme. Taken together, these data confirm the necessity of the conserved second- sphere βR residues in the proposed subunit swapping process and post-translational modification of the α-sub- unit in the α activator complex, along with stabilizing the catalytic sulfenic acid in its anionic form.more » « less
-
Abstract All threeN‐methylated andN‐protonated hydroxypyridinium BArF4–salt isomers were synthesized and their hydrogen bond donating abilities were investigated. DFT and G4 theory computations along with IR spectroscopic measurements were found to be effective methods for predicting the catalytic activities of these O–H and N–H Brønsted acids. A UV‐vis titration approach for rapidly quantifying hydrogen bond donating ability revealed that carbon‐hydrogen bonds also can participate in electrostatic interactions, but the presence of multiple equilibrium complexes results in a limitation of this method. In the methylated series of hydroxypyridines, the ortho and para isomers displayed modest rate enhancements relative to the meta derivative. Protonation introduces a new acidic site and the ortho hydroxypyridinium ion salt is a significantly more active catalyst than all of the other species examined. This is indicative of bidentate activation by the N–H and O–H acidic sites, and suggests a new design strategy for improving charge‐enhanced catalysts.more » « less
-
Transition metal carbides are attractive, low-cost alternatives to Pt group metals, exhibiting multifunctional acidic, basic, and metallic sites for catalysis. Their widespread applications are often impeded by a high surface affinity for oxygen, which blocks catalytic sites. However, recent reports indicate that the α-MoC phase is a stable and effective cocatalyst for reactions in oxidative or aqueous environments. In this work, we elucidate the factors affecting the stability and catalytic activity of α-MoC under mild electrooxidation conditions (0–0.8 V SHE) using density functional theory calculations, kinetics-informed surface Pourbaix diagram analysis, electronic structure analysis, and cyclic voltammetry. Both computational and experimental data indicate that α-MoC is significantly more resistant to electrooxidation by H2O than β-Mo2C. This higher stability is attributed to structural and kinetic factors, as the Mo-terminated α-MoC surface disfavors substitutional oxidation of partially exposed, less oxophilic C* atoms by hindering CO/CO2 removal. The α-MoC surface exposes H2O-protected [MoC2O2] and [MoC(CO)O2] oxycarbidic motifs available for catalysis in a wide potential window. At higher potentials, they convert to unstable [Mo(CO)2O2], resulting in material degradation. Using formic acid as a probe molecule, we obtain evidence for Pt-like O*-mediated O–H and C–H bond activation pathways. The largest kinetic barrier, observed for the C–H bond activation, correlates with the hydrogen affinity of the site in the order O*/Mofcc > O*/Ctop > O*/Motop. To mitigate the site-blocking effect of surface-bound H2O and bidentate formate, doping with Pt was investigated computationally to make the surface less oxophilic and more carbophilic, indicating a possible design strategy toward more active and selective carbide electrocatalysts.more » « less
-
The ability to site-selectively modify equivalent functional groups in a molecule has the potential to streamline syntheses and increase product yields by lowering step counts. Enzymes catalyze site-selective transformations throughout primary and secondary metabolism, but leveraging this capability for non-native substrates and reactions requires a detailed understanding of the potential and limitations of enzyme catalysis and how these bounds can be extended by protein engineering. In this review, we discuss representative examples of site-selective enzyme catalysis involving functional group manipulation and C–H bond functionalization. We include illustrative examples of native catalysis, but our focus is on cases involving non-native substrates and reactions often using engineered enzymes. We then discuss the use of these enzymes for chemoenzymatic transformations and target-oriented synthesis and conclude with a survey of tools and techniques that could expand the scope of non-native site-selective enzyme catalysis.more » « less
