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Inhibition of N-Acetylglutamate Synthase by Various Monocarboxylic and Dicarboxylic Short-Chain Coenzyme a Esters and the Production of Alternative Glutamate Esters☆

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Inhibition of N-Acetylglutamate Synthase by Various Monocarboxylic and Dicarboxylic Short-Chain Coenzyme a Esters and the Production of Alternative Glutamate Esters☆ View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Biochimica et Biophysica Acta 1842 (2014) 2510–2516 Contents lists available at ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbadis Inhibition of N-acetylglutamate synthase by various monocarboxylic and dicarboxylic short-chain coenzyme A esters and the production of alternative glutamate esters☆ M. Dercksen a,b,⁎, L. IJlst a, M. Duran a, L.J. Mienie b, A. van Cruchten a, F.H. van der Westhuizen b, R.J.A. Wanders a a Laboratory Genetic Metabolic Diseases, Departments of Pediatrics and Clinical Chemistry, Academic Medical Centre, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands b Centre for Human Metabonomics, North-West University (Potchefstroom Campus), Hoffman street 11, Potchefstroom, South Africa, 2520 article info abstract Article history: Hyperammonemia is a frequent finding in various organic acidemias. One possible mechanism involves the Received 11 December 2012 inhibition of the enzyme N-acetylglutamate synthase (NAGS), by short-chain acyl-CoAs which accumulate due Received in revised form 9 April 2013 to defective catabolism of amino acids and/or fatty acids in the cell. The aim of this study was to investigate the Accepted 29 April 2013 effect of various acyl-CoAs on the activity of NAGS in conjunction with the formation of glutamate esters. NAGS Available online 2 May 2013 activity was measured in vitro using a sensitive enzyme assay with ultraperformance liquid chromatography– – Keywords: tandem mass spectrometry (UPLC MS/MS) product analysis. Propionyl-CoA and butyryl-CoA proved to be the N-acetylglutamate synthase most powerful inhibitors of N-acetylglutamate (NAG) formation. Branched-chain amino acid related CoAs Hyperammonemia (isovaleryl-CoA, 3-methylcrotonyl-CoA, isobutyryl-CoA) showed less pronounced inhibition of NAGS whereas Organic acidemia the dicarboxylic short-chain acyl-CoAs (methylmalonyl-CoA, succinyl-CoA, glutaryl-CoA) had the least inhibitory Branched-chain amino acids effect. Subsequent work showed that the most powerful inhibitors also proved to be the best substrates in the for- N-acylglutamates mation of N-acylglutamates. Furthermore, we identified N-isovalerylglutamate, N-3-methylcrotonylglutamate Acyl-CoAs and N-isobutyrylglutamate (the latter two in trace amounts), in the urines of patients with different organic acidemias. Collectively, these findings explain one of the contributing factors to secondary hyperammonemia, which lead to the reduced in vivo flux through the urea cycle in organic acidemias and result in the inadequate elimination of ammonia. © 2013 Elsevier B.V. All rights reserved. 1. Introduction transcarbamylase (OTC) are located in the mitochondrion whereas argininosuccinate synthase (ASS), argininosuccinate lyase (ASL), The degradation of protein and subsequently amino acids is an and arginase (ARG) reside in the cytosol [1]. The urea cycle plays a essential part of human metabolism. The catabolism of amino acids key role in the control of ammonia levels which need to be kept as and other nitrogen-containing molecules results in the production of low as possible in order to prevent the deleterious consequences of ammonia (NH3), which is converted into urea in the liver via the urea hyperammonemia. Ammonia is especially toxic to the central nervous cycle and excreted as such in the urine. The urea cycle consists of six system (CNS) since slight elevations in ammonia (>100 μM, normal: key enzymes, localized in different compartments within the cell, thus b45 μM) may cause metabolic encephalopathy and irreversible CNS requiring the obligatory participation of different mitochondrial damage [2]. membrane transporters (SLC25A13 and SLC25A15). N-acetylglutamate CPS catalyzes the initial and rate limiting step of the urea cycle and synthase (NAGS), carbamyl phosphate synthetase 1 (CPS) and ornithine is vital for the regulation of the pathway. Its allosteric activator N-acetylglutamate (NAG), is essential for the function of CPS in the urea cycle [3]. NAGS (E.C. 2.3.1.1), originally identified in E. coli by – Abbreviations: NAGS, N-acetylglutamate synthase; CoA, coenzyme A; UPLC MS/MS, Vogel et al. [4] and later on described in humans by Bachmann et al. Ultraperformance liquid chromatography–tandem mass spectrometry; UCDs, urea cycle defects; CPS, carbamyl phosphate synthetase; CNS, central nervous system; NAG, [5], is responsible for the synthesis of N-acetylglutamate (NAG) N-acetylglutamate; GC–MS, gas chromatography mass spectrometry through the conjugation of acetyl-CoA and L-glutamate. The activity ☆ Competing interest: All parties confirmed that they have no competing interest for of NAGS is stimulated by L-arginine, an allosteric activator [6]. Apart declaration. from the short term regulation by NAG, urea cycle activity is also con- ⁎ Corresponding author at: Centre for Human Metabonomics, North-West University trolled by several other mechanisms, including enzyme induction, the (Potchefstroom Campus), Hoffman street 11, Potchefstroom, South Africa, 2520. Tel.: +27 18 299 2302. concentration of intermediates and substrates, diet as well as hor- E-mail address: [email protected] (M. Dercksen). monal changes. Although the precise regulation of ureagenesis is 0925-4439/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbadis.2013.04.027 M. Dercksen et al. / Biochimica et Biophysica Acta 1842 (2014) 2510–2516 2511 still poorly understood, it proves to be crucial in the development of as succinyl-CoA ligase deficiency due to SUCLA2 gene mutations. primary and secondary hyperammonemia [7,8]. [19–24]. These reports and previous findings indicate a clear variation Urea cycle enzyme and transporter defects (UCDs), including NAGS in the occurrence of hyperammonemia associated with short-chain- deficiency, are a well defined group of inherited metabolic disorders and short-branched-chain organic acidemias [25]. The inhibition of characterized by episodes of mild to severe hyperammonemia [7,9]. NAGS is one of the factors which may explain the presence of Except for hyperammonemia caused by primary deficiencies in one of hyperammonemia, among other. Additional information of these disor- the enzymes or transporters involved in the urea cycle, this pathophys- ders is summarized in Supplementary Table 1. iological condition is also observed in numerous other conditions. These Since there is accumulation of a range of acyl-CoAs in the different include liver disease, anatomical changes such as the portocaval shunt, organic acidemias, we decided to study the effect of several acyl-CoAs hyperinsulinism and hyperammonemia due to glutamate dehydroge- on the activity of NAGS as well as their behavior as substrates for the nase hyperactivity, several organic acidemias, mitochondrial fatty acid enzyme, using an optimized UPLC–MS/MS assay. We argued that oxidation disorders and valproate toxicity [7,10–12]. Fig 1 illustrates these compounds may act as inhibitors and/or substitute substrates the localization of several CoA esters and part of the urea cycle within for NAGS which would lead to the aberrant in vivo regulation of the the mitochondrion. urea cycle and contribute (in addition to other factors) to episodes The elevation of blood ammonia encountered in the classical of hyperammonemia. organic acidemias, including isovaleric-, propionic- and methylmalonic acidemia can be life-threatening [7]. Isolated cases of hyperammonemia 2. Materials and Methods have been reported for some disorders e.g. 3-methylcrotonyl-CoA carboxylase- (MCC), multiple acyl-CoA dehydrogenase- (MAD) and All materials were supplied by Sigma-Aldrich (St. Louis, MO) unless succinyl-CoA ligase (associated with mutations in SUCLG1 gene) defi- otherwise specified. ciencies [13–16]. Hyperammonemia in human short-chain acyl-CoA dehydrogenase deficiency (SCADD) is virtually non-existent. Mice 2.1. Preparation and purification of human NAGS protein with SCADD however have shown elevated blood ammonia levels [17,18]. No cases associated with hyperammonemia have been docu- The construction of the NAGS wild type plasmid, as well as the ex- mented for glutaryl-CoA dehydrogenase- (GA1), short/branched-chain pression of human NAGS protein in E. coli was performed essentially acyl-CoA dehydrogenase- (SBCAD) isobutyryl-CoA dehydrogenase- as described by Schmidt et al. [26] with minor modifications. The (IBCAD), 3-ketothiolase- and multiple carboxylase deficiencies as well coding sequence for mature NAGS, without the mitochondrial targeting Tryptopan OH-Lysine Amino acid catabolism 2-Aminoadipate Lysine semialdehyde 2-Oxo-adipic acid OCD 2-Aminoadipate Valine Isoleucine Leucine semialdehyde mBCAT mBCAT mBCAT 2-Oxo-isovaleric acid 2-Oxo-3-methylvaleric acid 2-Oxo-isocaproic acid B-oxidation of short- chain acyl-CoA 2-Oxo-adipic acid Cytoplasm Mitochondrion Butyryl-CoA Glutaryl-CoA Isobutyryl-CoA 2-Methylbutyryl-CoA Isovaleryl-CoA SCAD GD IBCAD SBCAD IVD Urea Ornithine SLC25A15 Ornithine Methylacrylyl-CoA Tiglyl-CoA 3-Methylcrotonyl-CoA Arginase Acetyl-CoA +Glutamate Crotonyl-CoA 3-MCC + L-Arg Arginine NAGS 3-OH-Isobutyryl-CoA 2-Methyl-3-OH-butyryl-CoA 3-Methylglutaconyl-CoA N-Acetylglutamate ASL Fumarate + HCO - + NH +2ATP 3 4 OTC CPS + Argininosuccinate Carbamyl phosphate Methylmalonic semialdehyde TL 2 x Acetyl-CoA ASS Succinate Acetoacetate SL Propionyl-CoA Acetyl-CoA Aspartate Citrulline SLC25A13 Citrulline
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