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

ω-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 αketoglutaramate 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 ωamidase/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 [2-4] 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 errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author Manuscript Anal Biochem. Author manuscript; available in PMC 2010 August 15. Published in final edited form as: Anal Biochem. 2009 August 15; 391(2): 144–150. doi:10.1016/j.ab. 2009.05. 025. N IH PA Athor M anscript N IH PA Athor M anscript N IH PA Athor M anscript Intriguingly, neither asparagine nor glutamine is a substrate of rat liver ω-amidase [3]. On the other hand, both glutaramate and succinamate, where the α-keto group of αKGM and αKSM, respectively, is reduced to a -CH2group are also substrates of ω-amidase. The transamination and associated deamination reactions involving glutamine and asparagine are shown in more detail in Fig. 1.