Arginine: at the crossroads of nitrogen metabolism

Abstract

L-arginine is the most nitrogen-rich amino acid, acting as a key precursor for the synthesis of nitrogen-containing metabolites and an essential intermediate in the clearance of excess nitrogen. Arginine's side chain possesses a guanidino group which has unique biochemical properties, and plays a primary role in nitrogen excretion (urea), cellular signaling (nitric oxide) and energy buffering (phosphocreatine). The post-translational modification of protein-incorporated arginine by guanidino-group methylation also contributes to epigenetic gene control. Most human cells do not synthesize sufficient arginine to meet demand and are dependent on exogenous arginine. Thus, dietary arginine plays an important role in maintaining health, particularly upon physiologic stress. How cells adapt to changes in extracellular arginine availability is unclear, mostly because nearly all tissue culture media are supplemented with supraphysiologic levels of arginine. Evidence is emerging that arginine-deficiency can influence disease progression. Here, we review new insights into the importance of arginine as a metabolite, emphasizing the central role of mitochondria in arginine synthesis/catabolism and the recent discovery that arginine can act as a signaling molecule regulating gene expression and organelle dynamics.

Keywords: Arginine Deficiency; Arginine Metabolism; Metabolite Signaling; Mitochondria; Protein Synthesis.

Figure 1. Glutamine and arginine metabolic pathways.

Glutamine and arginine metabolism overlap in mitochondria. Once imported into mitochondria, glutamine is deaminated into glutamate by GLS1 which releases NH₄⁺. Two pathways are present inside mitochondria to sequester NH₄⁺: (1) α-KG accepts NH₄⁺ to produce more glutamate catalyzed by GDH or (2) production of CP from NH₄⁺ and HCO₃^- catalyzed by CPS1. Once formed, CP can enter the urea cycle. Glutamate supports arginine synthesis in three different ways. Glutamate is a substrate, alongside acetyl-CoA, in the production of NAG which in turn activates CPS1. Glutamate can also be reduced to ornithine through sequential P5CS and OAT activity. Finally, glutamate is an anaplerotic substrate for the TCA cycle to produce aspartate. Both ornithine and aspartate are key components of the urea cycle. Ornithine can be converted to citrulline by addition of CP catalyzed by mitochondrial OTC. After which, citrulline is exported out into the cytosol for further processing by ASS1 and ASL to produce arginine. The reaction catalyzed by ASL releases fumarate, which can return to mitochondria as a TCA cycle intermediate. Arginine can be further hydrolyzed into ornithine by cytosolic ARG1 or the mitochondrial isoform, ARG2, releasing ornithine and urea as products. Mitochondrial arginine can also transfer its guanidino group to glycine catalyzed by GATM in the first biosynthetic step of the phosphagen creatine, while cytosolic arginine is used to produce NO. Chemical formula of arginine (bottom) and glutamine (upper) are shown. Each molecule of glutamine contains two reduced nitrogen (in blue) while one molecule of arginine contains four reduced nitrogen. Black arrows: single biochemical reaction. Blue arrows: translocation of metabolites across cellular compartments. Yellow boxes: Products of arginine metabolism. Magenta arrow: NAG is an allosteric cofactor of CPS1 for activation and not a substrate. Double arrows: reversible biochemical reaction. Dotted arrows: multi-step reaction to synthesize final product. Enzymes in the urea/citrulline cycles are in magenta while enzymes in the catabolism of glutamine/glutamate are in green. Creatine production enzyme GATM is in grey. The cyclization of GSA into P5C is a spontaneous reaction. NAGS N-acetylglutamate synthase, CPS1 carbamoyl phosphate synthetase 1, OTC ornithine transcarbamylase, ASS1 argininosuccinate synthase 1, ASL argininosuccinate lyase, ARG1 arginase 1, ARG2 arginase 2, GATM L-arginine:glycine amidinotransferase, NOS nitric oxide synthases, TCA cycle tricarboxylic acid cycle, GLS1 glutaminase 1, GDH glutamate dehydrogenase; P5CS pyrroline-5-carboxylate synthase; OAT ornithine aminotransferase; PYCR1/2 Pyrroline-5-carboxylate reductase 1 and 2; CP carbamoyl phosphate, NO nitric oxide, GSA glutamate γ-semialdehyde, P5C pyrroline-5-carboxylate, NAG N-acetylglutamate, α-KG α-ketoglutarate, Acetyl-CoA acetyl coenzyme A, ATP adenosine triphosphate.

Figure 2. The multiple roles of arginine in human health.

In addition to protein synthesis, arginine is utilized as a signaling molecule, the precursor for a variety of important biomolecules and the protein residue for post-translational modifications.

Figure 3. Metabolites produced from arginine.

Arginine catabolism contributes to numerous metabolic pathways. Arginine is a substrate for NO synthesis, polyamine production and urea formation. It is also a building block for creatine and proline synthesis. Chemical elements derived from arginine are highlighted in blue. NO nitric oxide.

Figure 4. Phosphagen shuttles for energy buffering and distribution.

(A) ATP is produced in mitochondria through OXPHOS, which links the TCA cycle and the electron transport chain (ETC) together. Once generated, ATP can be used to phosphorylate creatine through mitochondrial creatine kinase (CKMT) in the mitochondrial intermembrane space to produce phosphocreatine (Creatine-P). Creatine-P, in turn, rapidly diffuses throughout the cytosol, where it donates its phosphate group back to ADP by cytosolic creatine kinase (CKM) to replenish the local ATP levels at sites of high ATP utilization. Creatine then returns to mitochondria where it can be re-phosphorylated to restart the cycle. Therefore, the creatine/creatine-P system serves in both energy transportation and as an energy reservoir to meet ATP demands of a cell. (B) Mammals utilize creatine/creatine-P in the homeostasis of cellular bioenergetics, while bacteria and invertebrates express arginine kinases that convert arginine into phosphoarginine (arginine-P) as their phosphagen shuttle pathway. Black arrows: single biochemical reaction. Blue arrows: translocation of metabolites across membranes. Dotted arrow: reduced substrates from the TCA cycle donate their electrons to the ETC for ATP production. Creatine pathway enzymes are in green and the ETC, comprising of five complexes is in magenta for (A). Chemical formula of creatine/creatine-P and arginine/arginine-P are shown in (B). ATP adenosine triphosphate, ADP adenosine diphosphate, CKMT mitochondrial creatine kinase, CKM creatine kinase M-type, e electron transfer, Arginine-P phosphoarginine, Creatine-P phosphocreatine, OXPHOS oxidative phosphorylation, TCA cycle tricarboxylic acid cycle, ETC electron transport chain.

Figure 5. Post-translational modifications on arginine residues.

The methylation of arginine residues is catalyzed by the protein arginine methyltransferases (PRMTs). PRMTs of Type I, II and III can all produce monomethylarginine (MMA). In addition, Type I add other methyl groups to the same nitrogen atom to yield asymmetric dimethylarginine (ADMA), while Type II PRMTs attach the second methyl group to the other N-terminal nitrogen of the arginine residue, forming symmetric dimethylarginine (SDMA). Type III PRMT is only capable of mono-methylation. In contrast, peptidylarginine deiminases (PADs) performs the hydrolysis of the guanidino group to form citrulline.

Figure 6. Arginine acquisition pathways.

As a semi-essential amino acid, intracellular arginine level can be maintained in three different ways (Apiz Saab et al, 2023). Arginine can be directly taken up from the extracellular environment through the SLC7 family of transporters. In addition, extracellular proteins and debris can be scavenged through macropinocytosis. Once internalized, macropinsomes are sorted and trafficked, and ultimately merged with lysosomes. Captured cargo are then degraded within the lysosomal lumen and the resultant amino acids (such as arginine) can be released into the cytosol or re-distributed into other cellular compartments such as mitochondria. Lastly, dietary supply of glutamine, ornithine and citrulline can all be used as substrates for de novo synthesis of arginine through the urea/citrulline cycles. Black arrows: single biochemical reaction. Blue arrows: translocation of metabolites across cellular compartments. Grey arrows: Capture and lysosomal degradation of extracellular proteins. OTC ornithine transcarbamylase, ASS1 argininosuccinate synthase 1, ASL argininosuccinate lyase, α-KG α-ketoglutarate, TCA cycle tricarboxylic acid cycle.

Figure 7. Cell–cell competition for arginine in the tumor microenvironment.

In the tumor microenvironment (TME), infiltrating cytotoxic T cells face competition for key nutrients such as arginine. Cancer cells which are often auxotrophic for arginine taking in large amounts of arginine to maintain their proliferation and cell viability, which creates an immunosuppressive environment due to reduced arginine (Mussai et al, 2015). To make matters worse, immunosuppressive macrophages also take up arginine (Raber et al, ; Tharp et al, 2024) and secrete ARG1 (Rodriguez et al, ; Sosnowska et al, 2021) into the extracellular environment, hydrolyzing free arginine which further reduces nutrient availability in the TME. Cytotoxic T cells are highly susceptible to arginine deprivation, leading to OXPHOS inhibition (Crump et al, ; Fletcher et al, 2015), hampered glycolysis (Crump et al, 2021) and a reduction in cytokine production (Choi et al, 2009), proliferation (Czystowska-Kuzmicz et al, 2019), differentiation (Werner et al, 2016) and CD3ξ expression (Rodriguez et al, 2003). Black arrow: single biochemical reaction. Blue arrows: translocation of metabolite/enzyme across cellular compartments. ARG1 arginase 1.