Premium
Remote Residues Affect Stability of Ornithine Transcarbamylase
Author(s) -
Ramos Kevin,
Ngu Lisa,
DeLateur Nicholas,
Ondrechen Mary,
Beuning Penny
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.572.29
Subject(s) - ornithine transcarbamylase , chemistry , kinetics , melting temperature , catalysis , mutation , carbamoyl phosphate synthetase , point mutation , biochemistry , enzyme , biophysics , biology , arginine , amino acid , urea cycle , gene , materials science , physics , quantum mechanics , composite material
Ornithine transcarbamylase (OTC) is known to undergo induced‐fit conformational changes upon the binding of carbamoyl phosphate (CP), which allows the sequential binding of L‐ornithine (ORN) to produce citrulline (CIT) and inorganic phosphate. Computational tools THEMATICS and POOL predict that catalytic residues of OTC extend into the second and third spheres around the active site of the protein. To test these predictions, mutations of POOL‐predicted residues were tested with a kinetics assay to determine relative catalytic efficiencies. The stabilities of these mutations are being characterized in this study by a ThermoFluor assay that measures the melting temperature of OTC variants. OTC variants C273A, D140N, Y229F, H272L, H272N, E299D, E299Q and R57A showed 5‐fold decrease or greater in catalytic efficiency with ORN and/or CP. Mutation of these residues also has been found to decrease the melting temperature of OTC up to 18 ᵒ C. Thus these variants show less stability compared to wild‐type OTC. It is also observed that when OTC is bound to CP, the stability is increased compared to the apoenzyme, or to OTC with ORN‐ or CIT‐bound. Supported by NSF‐MCB‐1158176.