Paper Citing NAMD - Abstract
Chien, Chin-Hsiang; Gao, Quan-Ze; Cooper, Arthur J. L.; Lyu, Jyun-Hong; Sheu, Sheh-Yi
Structural Insights into the Catalytic Active Site and Activity of Human Nit2/omega-Amidase KINETIC ASSAY AND MOLECULAR DYNAMICS SIMULATION
JOURNAL OF BIOLOGICAL CHEMISTRY, 287:25715-25726, JUL 27 2012
Human nitrilase-like protein 2 (hNit2) is a putative tumor suppressor, recently identified as omega-amidase. hNit2/omega-amidase plays a crucial metabolic role by catalyzing the hydrolysis of alpha-ketoglutaramate (the alpha-keto analog of glutamine) and alpha-ketosuccinamate (the alpha-keto analog of asparagine), yielding alpha-ketoglutarate and oxaloacetate, respectively. Transamination between glutamine and alpha-keto-gamma-methiolbutyrate closes the methionine salvage pathway. Thus, hNit2/omega-amidase links sulfur metabolism to the tricarboxylic acid cycle. To elucidate the catalytic specificity of hNit2/omega-amidase, we performed molecular dynamics simulations on the wild type enzyme and its mutants to investigate enzyme-substrate interactions. Binding free energies were computed to characterize factors contributing to the substrate specificity. The predictions resulting from these computations were verified by kinetic analyses and mutational studies. The activity of hNit2/omega-amidase was determined with alpha-ketoglutaramate and succinamate as substrates. We constructed three catalytic triad mutants (E43A, K112A, and C153A) and a mutant with a loop 116-128 deletion to validate the role of key residues and the 116-128 loop region in substrate binding and turnover. The molecular dynamics simulations successfully verified the experimental trends in the binding specificity of hNit2/omega-amidase toward various substrates. Our findings have revealed novel structural insights into the binding of substrates to hNit2/omega-amidase. A catalytic triad and the loop residues 116-128 of hNit2 play an essential role in supporting the stability of the enzyme-substrate complex, resulting in the generation of the catalytic products. These observations are predicted to be of benefit in the design of new inhibitors or activators for research involving cancer and hyperammonemic diseases.
