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Stimulatory effect of arginine on acetylglutamate synthesis in isolated mitochondria of mouse and rat liver.

ABSTRACT: N-Acetyl-L-glutamate synthetase (EC 2.3.1.1) catalyses the synthesis of N-acetyl-L-glutamate, an allosteric activator of carbamoyl-phosphate synthetase I in the liver of ureotelic animals, and the first enzyme is activated specifically by arginine. We have proposed that arginine can stimulate acetylglutamine synthetase in vivo and thereby increase the mitochondrial content of acetylglutamate. The effects of arginine on acetylglutamate synthesis in isolated mitochondria were investigated in detail in the present work. When rat liver mitochondria were isolated and incubated with [14C]glutamate and unlabelled acetate as substrates, acetyl[14C]glutamate synthesis in the mitochondria was more extensive in the presence than in the absence of L-arginine. There was no significant difference between the specific radioactivities of intramitochondrial [14C]glutamate in the presence and absence of arginine. When rat liver mitochondria were incubated with [14C]acetate and unlabelled glutamate as substrates, arginine also stimulated acetyl[14C]glutamate synthesis in the isolated mitochondria. L-Lysine or L-homoarginine, which does not activate acetylglutamate synthetase, had no effect on acetylglutamate synthesis, in the isolated mitochondria. The arginine concentration giving half-maximal synthesis of acetylglutamate in isolated mitochondria was about 50 microM, which is in the range of physiological concentrations of arginine in the liver. As we previously reported [Kawamoto, Ishida, Mori & Tatibana (1982) Eur. J. Biochem. 123, 637-641], the sensitivity of acetylglutamate synthetase to arginine activation undergoes marked changes after food ingestion. The extent of arginine activation of acetylglutamate synthesis in isolated mitochondria correlated well with the sensitivity of acetylglutamate synthetase extracted from the mitochondria to arginine activation. These data lend further support to the idea that arginine itself activates the mitochondrial synthesis of acetylglutamate.

SUBMITTER: Kawamoto S

PROVIDER: S-EPMC1152883 | biostudies-other | 1985 Dec

REPOSITORIES: biostudies-other

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Project description:N-acetyl-L-glutamate kinase (NAGK) catalyzes the second, generally controlling, step of arginine biosynthesis. In yeasts, NAGK exists either alone or forming a metabolon with N-acetyl-L-glutamate synthase (NAGS), which catalyzes the first step and exists only within the metabolon. Yeast NAGK (yNAGK) has, in addition to the amino acid kinase (AAK) domain found in other NAGKs, a ~150-residue C-terminal domain of unclear significance belonging to the DUF619 domain family. We deleted this domain, proving that it stabilizes yNAGK, slows catalysis and modulates feed-back inhibition by arginine. We determined the crystal structures of both the DUF619 domain-lacking yNAGK, ligand-free as well as complexed with acetylglutamate or acetylglutamate and arginine, and of complete mature yNAGK. While all other known arginine-inhibitable NAGKs are doughnut-like hexameric trimers of dimers of AAK domains, yNAGK has as central structure a flat tetramer formed by two dimers of AAK domains. These dimers differ from canonical AAK dimers in the -110° rotation of one subunit with respect to the other. In the hexameric enzymes, an N-terminal extension, found in all arginine-inhibitable NAGKs, forms a protruding helix that interlaces the dimers. In yNAGK, however, it conforms a two-helix platform that mediates interdimeric interactions. Arginine appears to freeze an open inactive AAK domain conformation. In the complete yNAGK structure, two pairs of DUF619 domains flank the AAK domain tetramer, providing a mechanism for the DUF619 domain modulatory functions. The DUF619 domain exhibits the histone acetyltransferase fold, resembling the catalytic domain of bacterial NAGS. However, the putative acetyl CoA site is blocked, explaining the lack of NAGS activity of yNAGK. We conclude that the tetrameric architecture is an adaptation to metabolon formation and propose an organization for this metabolon, suggesting that yNAGK may be a good model also for yeast and human NAGSs.

Project description:The FDA has approved 31 small-molecule kinase inhibitors (KIs) for human use as of November 2016, with six having black box warnings for hepatotoxicity (BBW-H) in product labeling. The precise mechanisms and risk factors for KI-induced hepatotoxicity are poorly understood. Here, the 31 KIs were tested in isolated rat liver mitochondria, an in vitro system recently proposed to be a useful tool to predict drug-induced hepatotoxicity in humans. The KIs were incubated with mitochondria or submitochondrial particles at concentrations ranging from therapeutic maximal blood concentrations (Cmax) levels to 100-fold Cmax levels. Ten endpoints were measured, including oxygen consumption rate, inner membrane potential, cytochrome c release, swelling, reactive oxygen species, and individual respiratory chain complex (I-V) activities. Of the 31 KIs examined only three including sorafenib, regorafenib and pazopanib, all of which are hepatotoxic, caused significant mitochondrial toxicity at concentrations equal to the Cmax, indicating that mitochondrial toxicity likely contributes to the pathogenesis of hepatotoxicity associated with these KIs. At concentrations equal to 100-fold Cmax, 18 KIs were found to be toxic to mitochondria, and among six KIs with BBW-H, mitochondrial injury was induced by regorafenib, lapatinib, idelalisib, and pazopanib, but not ponatinib, or sunitinib. Mitochondrial liability at 100-fold Cmax had a positive predictive power (PPV) of 72% and negative predictive power (NPV) of 33% in predicting human KI hepatotoxicity as defined by product labeling, with the sensitivity and specificity being 62% and 44%, respectively. Similar predictive power was obtained using the criterion of Cmax ≥1.1 µM or daily dose ≥100 mg. Mitochondrial liability at 1-2.5-fold Cmax showed a 100% PPV and specificity, though the NPV and sensitivity were 32% and 14%, respectively. These data provide novel mechanistic insights into KI hepatotoxicity and indicate that mitochondrial toxicity at therapeutic levels can help identify hepatotoxic KIs.

Project description:The efficient conversion of ammonia, a potent neurotoxin, into non-toxic metabolites was an essential adaptation that allowed animals to move from the aquatic to terrestrial biosphere. The urea cycle converts ammonia into urea in mammals, amphibians, turtles, snails, worms and many aquatic animals and requires N-acetylglutamate (NAG), an essential allosteric activator of carbamylphosphate synthetase I (CPSI) in mammals and amphibians, and carbamylphosphate synthetase III (CPSIII) in fish and invertebrates. NAG-dependent CPSI and CPSIII catalyze the formation of carbamylphosphate in the first and rate limiting step of ureagenesis. NAG is produced enzymatically by N-acetylglutamate synthase (NAGS), which is also found in bacteria and plants as the first enzyme of arginine biosynthesis. Arginine is an allosteric inhibitor of microbial and plant NAGS, and allosteric activator of mammalian NAGS.Information from mutagenesis studies of E. coli and P. aeruginosa NAGS was combined with structural information from the related bacterial N-acetylglutamate kinases to identify four residues in mammalian NAGS that interact with arginine. Substitutions of these four residues were engineered in mouse NAGS and into the vertebrate-like N-acetylglutamate synthase-kinase (NAGS-K) of Xanthomonas campestris, which is inhibited by arginine. All mutations resulted in arginine losing the ability to activate mouse NAGS, and inhibit X. campestris NAGS-K. To examine at what point in evolution inversion of arginine effect on NAGS occur, we cloned NAGS from fish and frogs and examined the arginine response of their corresponding proteins. Fish NAGS were partially inhibited by arginine and frog NAGS were activated by arginine.Difference in arginine effect on bacterial and mammalian NAGS most likely stems from the difference in the type of conformational change triggered by arginine binding to these proteins. The change from arginine inhibition of NAGS to activation was gradual, from complete inhibition of bacterial NAGS, to partial inhibition of fish NAGS, to activation of frog and mammalian NAGS. This change also coincided with the conquest of land by amphibians and mammals.

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Stimulatory effect of arginine on acetylglutamate synthesis in isolated mitochondria of mouse and rat liver.

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