Revisiting the Roles of Catalytic Residues in Human Ornithine Transcarbamylase.

Human ornithine transcarbamylase (hOTC) is a mitochondrial transferase protein involved in the urea cycle and is crucial for the conversion of toxic ammonia to urea. Structural analysis coupled with kinetic studies of Escherichia coli, rat, bovine, and other transferase proteins has identified residues that play key roles in substrate recognition and conformational changes but has not provided direct evidence for all of the active residues involved in OTC function. Here, computational methods were used to predict the likely active residues of hOTC; the function of these residues was then probed with site-directed mutagenesis and biochemical characterization. This process identified previously reported active residues, as well as distal residues that contribute to activity. Mutation of active site residue D263 resulted in a substantial loss of activity without a decrease in protein stability, suggesting a key catalytic role for this residue. Mutation of predicted second-layer residues H302, K307, and E310 resulted in significant decreases in enzymatic activity relative to that of wild-type (WT) hOTC with respect to l-ornithine. The mutation of fourth-layer residue H107 to produce the hOTC H107N variant resulted in a 66-fold decrease in catalytic efficiency relative to that of WT hOTC with respect to carbamoyl phosphate and a substantial loss of thermal stability. Further investigation identified H107 and to a lesser extent E98Q as key residues involved in maintaining the hOTC quaternary structure. This work biochemically demonstrates the importance of D263 in hOTC catalytic activity and shows that residues remote from the active site also play key roles in activity.

Site-Specific Reversible Protein and Peptide Modification: Transglutaminase-Catalyzed Glutamine Conjugation and Bioorthogonal Light-Mediated Removal.

Dynamic photoswitches in proteins that impart spatial and temporal control are important to manipulate and study biotic and abiotic processes. Nonetheless, approaches to install these switches into proteins site-specifically are limited. Herein we describe a novel site-specific method to generate photoremovable protein conjugates. Amine-containing chromophores (e.g., venerable  o-nitrobenzyl and less-explored o-nitrophenylethyl groups) were incorporated via transamidation into a glutamine side chain of α-gliadin, LCMV, and TAT peptides, as well as ß-casein and UmuD proteins by transglutaminase (TGase, EC 2.3.2.13). Subsequently, photolysis regenerated the native peptides and proteins. When this modification leads to the reduction or abolishment of certain activities, the process is referred to as caging, as in the case for E. coli polymerase manager protein UmuD. Importantly, this method is simple, robust, and easily adaptable, e.g., all components are commercially available.