Partial Order Optimum Likelihood (POOL) is a machine learning method that predicts catalytically important residues based on the tertiary structure of the protein, with computed chemical and electrostatic properties of the residues as input features. POOL has predicted spatially extended active sites, where residues that are not in direct contact with the substrate still contribute to catalysis for many enzymes, including for ornithine transcarbamoylase (OTC). OTC is part of the urea cycle and the arginine biosynthesis pathway; it catalyzes the reaction of carbamoyl phosphate (CP) with ornithine (ORN) to produce citrulline and inorganic phosphate. Enzyme variants were created through single-site directed mutagenesis and subsequently assayed for their kinetic activities, showing that predicted residues contribute to catalysis while non-POOL-predicted residues do not. Solution scattering techniques and molecular dynamics (MD) simulations were used to understand if these distal residues play a role in dynamical structural changes, thus affecting catalysis from a distance. Three-dimensional reconstructions of the solution scattering data for wild-type OTC and variants were generated using the programs GNOM and GASBOR from the ATSAS suite. Reconstructions of variants with mutations in distal positions show a structural rearrangement that in some cases reverses upon the addition of substrates.  Principal component analysis (PCA) of the MD simulations also suggests collective movements that are consistent with solution scattering data. Therefore, in addition to electrostatic effects, these distal residues may play a role in modulating dynamics and thus contribute to the catalytic mechanism of OTC. Supported by NSF MCB-1517290. <br/>

Distal residues of ornithine transcarbamoylase contribute to electrostatic and dynamics properties of the enzyme

254th National Meeting (2017)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.