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Ornithine Transcarbamylase Has a Spatially Extended Active Site as Predicted
Author(s) -
Ngu Lisa,
Ramos Kevin,
DeLateur Nicholas,
Beuning Penny,
Ondrechen Mary Jo
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.572.9
Subject(s) - active site , ornithine transcarbamylase , chemistry , carbamoyl phosphate synthetase , ornithine , catalysis , carbamyl phosphate , phosphate , substrate (aquarium) , kinetics , aspartate carbamoyltransferase , binding site , biochemistry , biophysics , enzyme , stereochemistry , allosteric regulation , biology , amino acid , physics , urea cycle , arginine , ecology , quantum mechanics
Partial Order Optimum Likelihood (POOL), developed at Northeastern University, is a machine learning technique to predict catalytically important residues based on the tertiary structure of a protein and on computed electrostatic properties. POOL has been shown to predict accurately the catalytic residues and discern between a compact and extended active site. Classic studies of enzymes typically identify catalytic residues that are in direct contact with the substrate. POOL predicts a spatially extended active site for ornithine transcarbamylase (OTC), for which dynamic processes are believed to play a role in its catalytic mechanism. OTC is reported to undergo induced‐fit conformational changes upon binding carbamoyl phosphate, followed by binding of ornithine. POOL predicts OTC to have an extended triple‐layer active site. Kinetics assay of Asp140, His272, Glu299 and Arg57 variants show these POOL‐predicted remote residues, located in the second and third layers, are important for catalysis. Specifically, variants D140N, H272L, E299Q and R57A (positive control), respectively have catalytic efficiency relative to wild type of 10%, 3%, 6% and 2% for ornithine and 21%, 45%, 66% and 54% for carbamoyl phosphate. Negative controls not predicted by POOL to be catalytically important were S61A and Q104L with relative catalytic efficiencies of 405% and 205% for ornithine and 88% and 64% for carbamoyl phosphate respectively. Our observations indicate the importance of remote residues for OTC activity and POOL's power to accurately predict catalytically important residues. Work supported by NSF‐MCB‐1158176.