Understanding how enzymes catalyze their reactions is an interesting and important problem. Many catalytic mechanisms have been studied to understand this problem. However, some mechanisms utilize residues that are not obviously important for catalysis because they are not in direct with the reacting substrate molecule(s). Through the use of Partial Order Optimum Likelihood (POOL), predictions have been made of residues that are important for catalytic activity. Ornithine transcarbamoylase (OTC), an enzyme involved in the urea cycle and in arginine biosynthesis pathways, has been predicted to have an extended three-layer active site. Using site-directed mutagenesis, OTC variants were generated based on these predictions. Kinetic assays show that the variants E299Q and R57A, located in the second and third layer of the active site, respectively, contribute to catalysis. This research uses small angle x-ray solution scattering (SAXS) to understand how these POOL-predicted distal residues, which may control dynamic structural change in the protein, actually influence catalysis from a distance. Three-dimensional reconstructions of SAXS profiles for OTC variants were constructed using x-ray solution scattering programs, including GNOM and GASBOR. Reconstructions of variants of third layer POOL-predicted residues suggest a structural rearrangement that may play a key role in understanding the catalytic mechanism of OTC. Supported by NSF MCB-1158176.

Small angle X-ray scattering based 3D reconstruction of ornithine transcarbamoylase suggests structural rearrangement

250th National Meeting (2015)

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