Assay and purification of ω-amidase/Nit2, a ubiquitously expressed putative tumor suppressor, that catalyzes the deamidation of the α-keto acid analogues of glutamine and asparagine

Abstract

Abstractω-Amidase [ω-amidodicarboxylate amidohydrolase, E.C. 3.5.1.3] isolated from rat liver cytosol is a versatile enzyme that catalyzes a large number of amidase, transamidase and ester hydrolysis reactions. ω-Amidase activity toward α-ketoglutaramate and α-ketosuccinamate (the α-keto acid analogues of glutamine and asparagine, respectively) is present in mammalian tissues, tumors, plants, bacteria and fungi. Despite its versatility, widespread occurrence and high specific activity, the enzyme has been little studied, possibly because the assay procedure previously required a substrate (α-ketoglutaramate) that is not commercially available. Here we report a simplified method for preparing α-ketoglutaramate and an assay procedure that measures α-ketoglutarate formation from α-ketoglutaramate in a 96-well-plate format. We also describe a 96-well plate assay procedure that measures ω-amidase-catalyzed hydroxaminolysis of commercially available succinamic acid. The product, succinyl hydroxamate, yields a stable brown color in the presence of acidic ferric chloride that can be quantitated spectrophometrically with negligible background interference. The two assay procedures (i.e. hydrolysis of α-ketoglutaramate and hydroxaminolysis of succinamate) were employed in purifying ω-amidase about ∼3,600-fold from rat liver cytosol. The ratio of α-ketoglutaramate hydrolysis to succinamate hydroxaminolysis remained constant during the purification. ω-Amidase has recently been shown to be identical to Nit2, a putative tumor suppressor protein. It is anticipated that these new assay procedures will help characterize the function of ω-amidase/Nit2 in tumor suppression, will provide the basis of high-throughput procedures to search for potent inhibitors and enhancers of ω-amidase, and will assist in identifying biological interactions between nitrogen metabolism and tumor biology.An apparent pyruvate-activated glutaminase and an apparent pyruvate-activated asparaginase were discovered more than 60 years ago [1]. Subsequently, Meister and co-workers [2][3][4] showed that both activities were actually due to a composite of two enzymes, namely a pyridoxal 5′-phosphate-dependent glutamine transaminase (Eq. 1) plus ω-amidase (Eq. 2), and a pyridoxal 5′-phosphate-dependent asparagine transaminase (Eq. 4) plus ω-amidase (Eq. 5). The net reactions are shown in Eqs 3 and 6. Transamination of glutamine and asparagine yield α-ketoglutaramate (αKGM) 1 and α-ketosuccinamate (αKSM), respectively, both of which are substrates of ω-amidase, yielding α-ketoglutarate and oxaloacetate, respectively [2]. *Corresponding author: Fax 1 914 594 4058, boris_krasnikov@nymc.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process er...

ω-Amidase: an underappreciated, but important enzyme in l-glutamine and l-asparagine metabolism; relevance to sulfur and nitrogen metabolism, tumor biology and hyperammonemic diseases

“…Commercially available succinamate [ − O 2 CCH 2 CH 2 C(O)NH 2 ] is the amide substrate, which in the presence of ω-amidase reacts with NH 2 OH to form the corresponding hydroxamate [ − O 2 CCH 2 CH 2 C(O)NHOH]. The hydroxamate in the presence of acidic ferric chloride generates a brown color that can be measured spectrophotometrically (Krasnikov et al 2009a). Hersh (1971Hersh ( , 1972 also showed that the rat liver enzyme has strong esterase activity.…”

“…The activity is present in all rat tissues investigated with highest specific activity in liver and kidney (Meister 1953;Cooper 1988) and, as noted above, the activity is present in both cytosolic and mitochondrial fractions. Purification of ω-amidase from rat liver cytosol to homogeneity required a 570-fold enrichment (Krasnikov et al 2009a), which indicates that 0.1-0.2 % of all the soluble protein in the rat liver cytosol is accounted for by this enzyme. We suggest that this high concentration attests to its metabolic importance as a housekeeping enzyme.…”

The Enzymology of 2-Hydroxyglutarate, 2-Hydroxyglutaramate and 2-Hydroxysuccinamate and Their Relationship to Oncometabolites

“…The resulting hydroxamate is readily quantitated as a brown complex in the presence of highly acidic ferric chloride [45]. In the present work, this procedure was adapted to measure the hydroxaminolysis of l -2-HGM (Equation (10)).…”

Structural Insights into the Catalytic Active Site and Activity of Human Nit2/ω-Amidase

“…Protein overexpression was induced with 2 mM isopropyl 1-thio-␤-D-galactopyranoside and maintained for 6 h; the overexpressed protein in the crude protein fraction obtained from the periplasmic fraction was further purified. We chose KGM, SM, and 3-PPN for assay of the enzymatic activity of hNit2/-amidase (11,23,31). The proteins were then subjected to kinetic analysis.…”

“…A link between cancer biology and the glutaminase II pathway has recently been strengthened by the finding that nitrilase-like protein 2 (Nit2), which was previously shown to be a putative tumor suppressor (22), is identical to -amidase (10,11,23). On the basis of sequence analysis, Pace et al reported that Nit2 belongs to the nitrilase (Nit) superfamily (24,25).…”