Abstract
OBJECTIVES: Argininosuccinate synthase 1 (ASS1) is a key rate-limiting enzyme in the urea cycle and is highly conserved at the protein level. Its catalytic activity directly determines the efficiency of ammonia clearance. Hyperammonemia is a clinical syndrome caused by abnormally elevated blood ammonia levels and is characterized mainly by central nervous system dysfunction, with neurotoxicity and a high mortality rate. When liver function is impaired or key enzymes of the urea cycle are deficient, ammonia clearance is reduced, leading to ammonia accumulation and passage across the blood-brain barrier, resulting in disturbances of consciousness, coma, or even death. Among currently used ammonia-lowering agents, L-ornithine-L-aspartate (LOLA) can promote the urea cycle to reduce blood ammonia levels and protect the liver; however, high doses are prone to gastrointestinal adverse effects, limiting its clinical application. This study obtained biologically active human ASS1 protein through prokaryotic expression and analyzed its enzymatic kinetic properties, aiming to explore the ammonia-lowering effect of ASS1 combined with LOLA and to provide new ideas and potential enzymatic intervention strategies for the clinical treatment of hyperammonemia. METHODS: The full-length open reading frame of human ASS1 was amplified by reverse transcription polymerase chain reaction (RT-PCR), and both the PCR product and the pET-28a vector were digested with restriction enzymes. After purification by agarose gel electrophoresis, the pET-28a-ASS1 prokaryotic expression vector was successfully constructed using homologous recombination technology. The recombinant plasmid was transformed into Escherichia coli, and expression of the 6×His-ASS1 recombinant protein was induced by isopropyl β-D-thiogalactopyranoside (IPTG). The protein was purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. In vitro enzyme activity assays were performed to verify the biological activity of the recombinant protein. An enzymatic reaction system using citrulline (Cit) and aspartate (Asp) as substrates was established to determine the optimal temperature and pH conditions for ASS1 activity and to further analyze its enzymatic kinetic parameters. A hyperammonemia mouse model was established in C57BL/6 mice by intraperitoneal injection of NH(4)Cl (400 mg/kg). The ammonia-lowering effects of ASS1 combined with LOLA were evaluated. Mice were randomly grouped, and 4 hours after modeling, ASS1, LOLA, or combined treatment was administered via the tail vein for 4 hours, followed by measurement of blood ammonia and urea levels. In addition, normal C57BL/6 mice were treated with combined ASS1 and LOLA for 24 hours, after which liver and renal function indices were assessed. RESULTS: The pET28a-ASS1 vector was successfully constructed using homologous recombination, and soluble recombinant ASS1 protein was obtained in a prokaryotic expression system. After purification and concentration by Ni-NTA affinity chromatography, the protein purity reached 95.4%±1.2%, with a concentration of (10.5± 0.8) mg/mL. In vitro enzyme activity assays systematically characterized the enzymatic properties of ASS1, showing an optimal reaction temperature of 37 ℃ and an optimal pH of 8.0. Using Asp and Cit as substrates, the enzymatic kinetic parameters were determined. The K(m) and k(cat)/K(m) values of ASS1 for Asp and Cit were (31.00±1.24) μmol/L and 2.07 L/(μmol·s), and (46.53±2.75) μmol/L and 1.35 L/(μmol·s), respectively. In vivo experiments showed that in an acute hyperammonemia mouse model induced by intraperitoneal injection of NH(4)Cl (400 mg/kg), ASS1 (50 mg/kg) reduced blood ammonia levels by 48.86% by accelerating the urea cycle. When ASS1 (50 mg/kg) was combined with low-dose LOLA (50 mg/kg), the ammonia-lowering efficiency was further enhanced, with a reduction of 76.31%, restoring blood ammonia levels to the normal range in hyperammonemic mice. CONCLUSIONS: Recombinant ASS1 protein obtained by prokaryotic expression exhibits stable and well-defined biological activity, with catalytic function significantly influenced by temperature and pH and displaying clear enzymatic kinetic characteristics. In an NH4Cl-induced mouse model of hyperammonemia, combined application of ASS1 and LOLA showed a more stable and effective ammonia-lowering effect than monotherapy, demonstrating good synergistic effects. These findings provide a new enzymatic intervention strategy for the treatment of hyperammonemia, lay a theoretical foundation for the development of combination ammonia-lowering therapies, and indicate potential application prospects.