Probing remote residues important for catalysis in Escherichia coli ornithine transcarbamoylase
| Autor: | Kien Nguyen, Penny J. Beuning, Mary Jo Ondrechen, Lisa Ngu, Kevin Ramos, Nicholas A. DeLateur, Paul C. Whitford, Lee Makowski, Jenifer N. Winters |
|---|---|
| Rok vydání: | 2019 |
| Předmět: |
Ornithine
Luminescence Protein Conformation Protein Structure Prediction Biochemistry Substrate Specificity Small-Angle Scattering Scattering chemistry.chemical_compound Protein structure Animal Cells Carbamoyl phosphate Protein Interaction Mapping Macromolecular Structure Analysis Peptide sequence Neurons 0303 health sciences Multidisciplinary Crystallography Physics Electromagnetic Radiation 030302 biochemistry & molecular biology Condensed Matter Physics Enzymes Chemistry Physical Sciences Crystal Structure Medicine Cellular Types Protein Binding Research Article Protein Structure Carbamyl Phosphate Science Catalysis Olfactory Receptor Neurons Fluorescence Phosphates 03 medical and health sciences Escherichia coli Solid State Physics Protein Interaction Domains and Motifs Enzyme kinetics Amino Acid Sequence Binding site Molecular Biology Ornithine Carbamoyltransferase 030304 developmental biology Binding Sites Base Sequence Chemical Compounds Substrate (chemistry) Biology and Life Sciences Afferent Neurons Proteins Protein engineering Cell Biology Kinetics chemistry Amino Acid Substitution Cellular Neuroscience Mutagenesis Site-Directed Enzymology Neuroscience |
| Zdroj: | PLoS ONE PLoS ONE, Vol 15, Iss 2, p e0228487 (2020) |
| ISSN: | 1932-6203 |
| Popis: | Understanding how enzymes achieve their tremendous catalytic power is a major question in biochemistry. Greater understanding is also needed for enzyme engineering applications. In many cases, enzyme efficiency and specificity depend on residues not in direct contact with the substrate, termed remote residues. This work focuses on Escherichia coli ornithine transcarbamoylase (OTC), which plays a central role in amino acid metabolism. OTC has been reported to undergo an induced-fit conformational change upon binding its first substrate, carbamoyl phosphate (CP), and several residues important for activity have been identified. Using computational methods based on the computed chemical properties from theoretical titration curves, sequence-based scores derived from evolutionary history, and protein surface topology, residues important for catalytic activity were predicted. The roles of these residues in OTC activity were tested by constructing mutations at predicted positions, followed by steady-state kinetics assays and substrate binding studies with the variants. First-layer mutations R57A and D231A, second-layer mutation H272L, and third-layer mutation E299Q, result in 57- to 450-fold reductions in kcat/KM with respect to CP and 44- to 580-fold reductions with respect to ornithine. Second-layer mutations D140N and Y160S also reduce activity with respect to ornithine. Most variants had decreased stability relative to wild-type OTC, with variants H272L, H272N, and E299Q having the greatest decreases. Variants H272L, E299Q, and R57A also show compromised CP binding. In addition to direct effects on catalytic activity, effects on overall protein stability and substrate binding were observed that reveal the intricacies of how these residues contribute to catalysis. |
| Databáze: | OpenAIRE |
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