Caldovic Ljubica, Haskins Nantaporn, Mumo Amy, Majumdar Himani, Pinter Mary, Tuchman Mendel, Krufka Alison
Center for Genetic Medicine Research, Children's National Medical Center, Washington D.C., United States of America ; Department of Integrative Systems Biology, The George Washington University, Washington D.C., United States of America.
Center for Genetic Medicine Research, Children's National Medical Center, Washington D.C., United States of America ; Molecular and Cellular Biology Program, University of Maryland, College Park, Maryland, United States of America.
PLoS One. 2014 Jan 22;9(1):e85597. doi: 10.1371/journal.pone.0085597. eCollection 2014.
The urea cycle converts ammonia, a waste product of protein catabolism, into urea. Because fish dispose ammonia directly into water, the role of the urea cycle in fish remains unknown. Six enzymes, N-acetylglutamate synthase (NAGS), carbamylphosphate synthetase III, ornithine transcarbamylase, argininosuccinate synthase, argininosuccinate lyase and arginase 1, and two membrane transporters, ornithine transporter and aralar, comprise the urea cycle. The genes for all six enzymes and both transporters are present in the zebrafish genome. NAGS (EC 2.3.1.1) catalyzes the formation of N-acetylglutamate from glutamate and acetyl coenzyme A and in zebrafish is partially inhibited by L-arginine. NAGS and other urea cycle genes are highly expressed during the first four days of zebrafish development. Sequence alignment of NAGS proteins from six fish species revealed three regions of sequence conservation: the mitochondrial targeting signal (MTS) at the N-terminus, followed by the variable and conserved segments. Removal of the MTS yields mature zebrafish NAGS (zfNAGS-M) while removal of the variable segment from zfNAGS-M results in conserved NAGS (zfNAGS-C). Both zfNAGS-M and zfNAGS-C are tetramers in the absence of L-arginine; addition of L-arginine decreased partition coefficients of both proteins. The zfNAGS-C unfolds over a broader temperature range and has higher specific activity than zfNAGS-M. In the presence of L-arginine the apparent Vmax of zfNAGS-M and zfNAGS-C decreased, their Km(app) for acetyl coenzyme A increased while the Km(app) for glutamate remained unchanged. The expression pattern of NAGS and other urea cycle genes in developing zebrafish suggests that they may have a role in citrulline and/or arginine biosynthesis during the first day of development and in ammonia detoxification thereafter. Biophysical and biochemical properties of zebrafish NAGS suggest that the variable segment may stabilize a tetrameric state of zfNAGS-M and that under physiological conditions zebrafish NAGS catalyzes formation of N-acetylglutamate at the maximal rate.
尿素循环将蛋白质分解代谢的废物氨转化为尿素。由于鱼类直接将氨排放到水中，尿素循环在鱼类中的作用尚不清楚。六种酶，即N-乙酰谷氨酸合酶（NAGS）、氨基甲酰磷酸合成酶III、鸟氨酸转氨甲酰酶、精氨琥珀酸合成酶、精氨琥珀酸裂解酶和精氨酸酶1，以及两种膜转运蛋白，即鸟氨酸转运蛋白和丙氨酸-天冬氨酸载体，构成了尿素循环。所有六种酶和两种转运蛋白的基因都存在于斑马鱼基因组中。NAGS（EC 2.3.1.1）催化由谷氨酸和乙酰辅酶A形成N-乙酰谷氨酸，在斑马鱼中部分受L-精氨酸抑制。NAGS和其他尿素循环基因在斑马鱼发育的前四天高度表达。六种鱼类的NAGS蛋白序列比对揭示了三个序列保守区域：N端的线粒体靶向信号（MTS），其后是可变段和保守段。去除MTS产生成熟的斑马鱼NAGS（zfNAGS-M），而从zfNAGS-M中去除可变段则产生保守的NAGS（zfNAGS-C）。在没有L-精氨酸的情况下，zfNAGS-M和zfNAGS-C都是四聚体；添加L-精氨酸会降低两种蛋白质的分配系数。zfNAGS-C在更宽的温度范围内展开，并且比zfNAGS-M具有更高的比活性。在L-精氨酸存在的情况下，zfNAGS-M和zfNAGS-C的表观Vmax降低，它们对乙酰辅酶A的Km（app）增加，而对谷氨酸的Km（app）保持不变。发育中的斑马鱼中NAGS和其他尿素循环基因的表达模式表明，它们可能在发育第一天的瓜氨酸和/或精氨酸生物合成中以及此后的氨解毒中发挥作用。斑马鱼NAGS的生物物理和生化特性表明，可变段可能稳定zfNAGS-M的四聚体状态，并且在生理条件下斑马鱼NAGS以最大速率催化N-乙酰谷氨酸的形成。
