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Structural insights into the catalytic active site and activity of human Nit2/?-amidase: kinetic assay and molecular dynamics simulation.

ABSTRACT: Human nitrilase-like protein 2 (hNit2) is a putative tumor suppressor, recently identified as ?-amidase. hNit2/?-amidase plays a crucial metabolic role by catalyzing the hydrolysis of ?-ketoglutaramate (the ?-keto analog of glutamine) and ?-ketosuccinamate (the ?-keto analog of asparagine), yielding ?-ketoglutarate and oxaloacetate, respectively. Transamination between glutamine and ?-keto-?-methiolbutyrate closes the methionine salvage pathway. Thus, hNit2/?-amidase links sulfur metabolism to the tricarboxylic acid cycle. To elucidate the catalytic specificity of hNit2/?-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/?-amidase was determined with ?-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/?-amidase toward various substrates. Our findings have revealed novel structural insights into the binding of substrates to hNit2/?-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.

SUBMITTER: Chien CH

PROVIDER: S-EPMC3406660 | biostudies-literature | 2012 Jul

REPOSITORIES: biostudies-literature

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Structural insights into the catalytic active site and activity of human Nit2/ω-amidase: kinetic assay and molecular dynamics simulation.

Chien Chin-Hsiang CH Gao Quan-Ze QZ Cooper Arthur J L AJ Lyu Jyun-Hong JH Sheu Sheh-Yi SY

The Journal of biological chemistry 20120606 31

Human nitrilase-like protein 2 (hNit2) is a putative tumor suppressor, recently identified as ω-amidase. hNit2/ω-amidase plays a crucial metabolic role by catalyzing the hydrolysis of α-ketoglutaramate (the α-keto analog of glutamine) and α-ketosuccinamate (the α-keto analog of asparagine), yielding α-ketoglutarate and oxaloacetate, respectively. Transamination between glutamine and α-keto-γ-methiolbutyrate closes the methionine salvage pathway. Thus, hNit2/ω-amidase links sulfur metabolism to t ...[more]

PMID: 22674578

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Project description:The present report identifies the enzymatic substrates of a member of the mammalian nitrilase-like (Nit) family. Nit2, which is widely distributed in nature, has been suggested to be a tumor suppressor protein. The protein was assumed to be an amidase based on sequence homology to other amidases and on the presence of a putative amidase-like active site. This assumption was recently confirmed by the publication of the crystal structure of mouse Nit2. However, the in vivo substrates were not previously identified. Here we report that rat liver Nit2 is omega-amidodicarboxylate amidohydrolase (E.C. 3.5.1.3; abbreviated omega-amidase), a ubiquitously expressed enzyme that catalyzes a variety of amidase, transamidase, esterase and transesterification reactions. The in vivo amidase substrates are alpha-ketoglutaramate and alpha-ketosuccinamate, generated by transamination of glutamine and asparagine, respectively. Glutamine transaminases serve to salvage a number of alpha-keto acids generated through non-specific transamination reactions (particularly those of the essential amino acids). Asparagine transamination appears to be useful in mitochondrial metabolism and in photorespiration. Glutamine transaminases play a particularly important role in transaminating alpha-keto-gamma-methiolbutyrate, a key component of the methionine salvage pathway. Some evidence suggests that excess alpha-ketoglutaramate may be neurotoxic. Moreover, alpha-ketosuccinamate is unstable and is readily converted to a number of hetero-aromatic compounds that may be toxic. Thus, an important role of omega-amidase is to remove potentially toxic intermediates by converting alpha-ketoglutaramate and alpha-ketosuccinamate to biologically useful alpha-ketoglutarate and oxaloacetate, respectively. Despite its importance in nitrogen and sulfur metabolism, the biochemical significance of omega-amidase has been largely overlooked. Our report may provide clues regarding the nature of the biological amidase substrate(s) of Nit1 (another member of the Nit family), which is a well-established tumor suppressor protein), and emphasizes a) the crucial role of Nit2 in nitrogen and sulfur metabolism, and b) the possible link of Nit2 to cancer biology.

Dataset Information

Structural insights into the catalytic active site and activity of human Nit2/?-amidase: kinetic assay and molecular dynamics simulation.

Publications

Structural insights into the catalytic active site and activity of human Nit2/ω-amidase: kinetic assay and molecular dynamics simulation.

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