Arginine Binding Site Research Articles

Insights into human eNOS, nNOS and iNOS structures and medicinal indications from statistical analyses of their interactions with bound compounds.

83 Structures of human nNOS, 55 structures of human eNOS, 13 structures of iNOS, and about 126 reported NOS-bound compounds are summarized and analyzed. Structural and statistical analysis show that, at least one copy of each analyzed compound binds to the active site (the substrate arginine binding site) of human NOS. And binding features of the three isoforms show differences, but the binding preference of compounds is not in the way helpful for inhibitor design targeting nNOS and iNOS, or for activator design targeting eNOS. This research shows that there is a strong structural and functional similarity between oxygenase domains of human NOS isoforms, especially the architecture, residue composition, size, shape, and distribution profile of hydrophobicity, polarity and charge of the active site. The selectivity and efficacy of inhibitors over the rest of isoforms rely a lot on chance and randomness. Further increase of selectivity via rational improvement is uncertain, unpredictable and unreliable, therefore, to achieve high selectivity through targeting this site is complicated and requires combinative investigation. After analysis on the current two targeting sites in NOS, the highly conserved arginine binding pocket and H4B binding pocket, new potential drug-targeting sites are proposed based on structure and sequence profiling. This comprehensive analysis on the structure and interaction profiles of human NOS and bound compounds provides fresh insights for drug discovery and pharmacological research, and the new discovery here is practically applied to guide protein-structure based drug discovery.

Design and Synthesis of Novel PRMT1 Inhibitors and Investigation of Their Effects on the Migration of Cancer Cell.

Protein arginine methyltransferase 1 (PRMT1) can catalyze the protein arginine methylation by transferring the methyl group from S-adenosyl-L-methionine (SAM) to the guanidyl nitrogen atom of protein arginine, which influences a variety of biological processes including epithelial–mesenchymal transition (EMT) and EMT-mediated mobility of cancer cells. The upregulation of PRMT1 is involved in a diverse range of cancer, such as lung cancer, and there is an urgent need to develop novel and potent PRMT1 inhibitors. In this article, a series of 2,5-substituted furan derivatives and 2,4-substituted thiazole derivatives were designed and synthesized by targeting at the substrate arginine-binding site on PRMT1, and 10 compounds demonstrated significant inhibitory effects against PRMT1. Among them, the most potent inhibitor, compound 1r (WCJ-394), significantly affected the expression of PRMT1-related proteins in A549 cells and downregulated the expression of mesenchymal markers, by which WCJ-394 inhibited the TGF-β1-induced EMT in A549 cells and prevented the cancer cell migration. The current study demonstrated that WCJ-394 was a potent PRMT1 inhibitor, which could be used as the leading compound for further drug discovery.

Crystal Structure of H227A Mutant of Arginine Kinase in Daphnia magna Suggests the Importance of Its Stability.

Arginine kinase (AK) plays a crucial role in the survival of Daphnia magna, a water flea and a common planktonic invertebrate sensitive to water pollution, owing to the production of bioenergy. AK from D. magna (DmAK) has four highly conserved histidine residues, namely, H90, H227, H284, and H315 in the amino acid sequence. In contrast to DmAK WT (wild type), the enzyme activity of the H227A mutant decreases by 18%. To identify the structure-function relationship of this H227A mutant enzyme, the crystal 3D X-ray structure has been determined and an unfolding assay using anilino-1-naphthalenesulfonic acid (ANS) fluorescence has been undertaken. The results revealed that when compared to the DmAK WT, the hydrogen bonding between H227 and A135 was broken in the H227A crystal structure. This suggests that H227 residue, closed to the arginine binding site, plays an important role in maintaining the structural stability and maximizing the enzyme activity through hydrogen bonding with the backbone oxygen of A135.

Peptides Derived from Vascular Endothelial Growth Factor B Show Potent Binding to Neuropilin-1.

Vascular endothelial growth factors (VEGFs) regulate significant pathways in angiogenesis, myocardial and neuronal protection, metabolism, and cancer progression. The VEGF‐B growth factor is involved in cell survival, anti‐apoptotic and antioxidant mechanisms, through binding to VEGF receptor 1 and neuropilin‐1 (NRP1). We employed surface plasmon resonance technology and X‐ray crystallography to analyse the molecular basis of the interaction between VEGF‐B and the b1 domain of NRP1, and developed VEGF‐B C‐terminus derived peptides to be used as chemical tools for studying VEGF‐B ‐ NRP1 related pathways. Peptide lipidation was used as a means to stabilise the peptides. VEGF‐B‐derived peptides containing a C‐terminal arginine show potent binding to NRP1‐b1. Peptide lipidation increased binding residence time and improved plasma stability. A crystal structure of a peptide with NRP1 demonstrated that VEGF‐B peptides bind at the canonical C‐terminal arginine binding site. VEGF‐B C‐terminus imparts higher affinity for NRP1 than the corresponding VEGF‐A165 region. This tight binding may impact on the activity and selectivity of the full‐length protein. The VEGF‐B167 derived peptides were more effective than VEGF‐A165 peptides in blocking functional phosphorylation events. Blockers of VEGF‐B function have potential applications in diabetes and non‐alcoholic fatty liver disease.

The Development of Tetrazole Derivatives as Protein Arginine Methyltransferase I (PRMT I) Inhibitors.

Protein arginine methyltransferase 1 (PRMT1) can catalyze protein arginine methylation by transferring the methyl group from S-adenosyl-L-methionine (SAM) to the guanidyl nitrogen atom of protein arginine, which influences a variety of biological processes. The dysregulation of PRMT1 is involved in a diverse range of diseases, including cancer. Therefore, there is an urgent need to develop novel and potent PRMT1 inhibitors. In the current manuscript, a series of 1-substituted 1H-tetrazole derivatives were designed and synthesized by targeting at the substrate arginine-binding site on PRMT1, and five compounds demonstrated significant inhibitory effects against PRMT1. The most potent PRMT1 inhibitor, compound 9a, displayed non-competitive pattern with respect to either SAM or substrate arginine, and showed the strong selectivity to PRMT1 compared to PRMT5, which belongs to the type II PRMT family. It was observed that the compound 9a inhibited the functions of PRMT1 and relative factors within this pathway, and down-regulated the canonical Wnt/β-catenin signaling pathway. The binding of compound 9a to PRMT1 was carefully analyzed by using molecular dynamic simulations and binding free energy calculations. These studies demonstrate that 9a was a potent PRMT1 inhibitor, which could be used as lead compound for further drug discovery.

A novel mechanism of NO synthase uncoupling involving isolevuglandin adduction

We have previously shown that hypertension leads to modification of self‐proteins in antigen presenting cells by isolevuglandin (isoLG) lipid oxidation products. These modified proteins seem to be immunogenic. IsoLG‐modified peptides are presented in major histocompatibility complex (MHC) class 1 and drive CD8+ T cell proliferation. To understand alterations in the peptidome presented by MHC1 in hypertension, we created transgenic mice expressing a truncated form of MHC1 Kb lacking the transmembrane domain, driven by the CD11c promoter. The lack of a transmembrane domain causes MHC1 to be shed into the media when DCs are cultured. We treated these mice with either 2 weeks sham or angiotensin (ang) II infusion, isolated splenic DCs and placed these in culture for 72 hours. The shed soluble MHC1 Kb was harvested and associated peptides were analyzed by mass spectroscopy. One peptide identified in DCs from hypertensive mice was an identical match for a portion of the neuronal nitric oxide synthase (nNOS) containing an isoLG adduct at lysine 225. Because this lysine is positioned between the heme and arginine binding sites, we hypothesized that its modification would disrupt normal NO production and instead lead to superoxide production, i.e. NOS uncoupling. In subsequent studies, we found that nNOS expression is increased 5‐fold in DCs of ang II‐treated compared to sham‐treated mice (n = 6). Importantly, ang II infusion increased superoxide production of DCs from 37±3 to 111±5 pmol/mg protein (mean ± SE, p = 0.0001), and this was completely blocked by treatment of the cells with the selective nNOS inhibitor L‐vinyl NIO, suggesting that nNOS is a source of superoxide in DCs in hypertension. Next we expressed wild type nNOS and a mutant nNOS in which lysine 225 was mutated to an alanine (nNOS K225A) in human epithelial kidney (HEK) cells. Untransfected HEK cells failed to produce NO, as measured by electron spin resonance and the spin trap Fe[DETC]2. HEK cells transfected with either the WT or K225A nNOS produced 152 ± 5 vs 156 ± 6 arbitrary units of NO/mg protein (p = NS). However, exposure of the transfected HEK cells to tertbutyl hydroperoxide, which promotes isoLG formation, reduced NO production by cells expressing the wild‐type enzyme to 86 ± 6 units/mg protein, while having no effect on cells transfected with the mutant K225A (p = 0.003, n = 5 – 7), further implicating lysine 225 as a site of oxidative modification in nNOS. In summary, hypertension increases nNOS expression in DCs and this enzyme is altered by isoLG adduction at lysine 225. This leads to an increase in superoxide and reduced NO production in these immune cells. We conclude that isoLG adduction represents a novel mechanism of NOS uncoupling and a source of reactive oxygen species in dendritic cells.Support or Funding InformationAmerican Heart Association grants POST290900 and SFRN204200, and National Institutes of Health grants K01HL130497, R01HL125865, R01HL039006 and P01HL129941This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Design and synthesis of novel PRMT1 inhibitors and investigation of their binding preferences using molecular modelling

Protein arginine methyltransferase 1 (PRMT1) catalyses the methylation of substrate arginine by transferring the methyl group from SAM (S-adenosyl-l-methionine), which leads to the formation of S-adenosyl homocysteine (SAH) and methylated arginine. We have shown previously that the Asp84 on PRMT1 could be a potential inhibitor binding site. In the current study, 28 compounds were designed and synthesized that were predicted to bind the Asp84 and substrate arginine sites together. Among them, 6 compounds were identified as potential PRMT1 inhibitors, and showed strong inhibitory effects on cancer cell lines, especially HepG2. The most potent PRMT1 inhibitor, compound 13d, was selected for molecular dynamic simulations to investigate binding poses. Based on the free energy calculations and structural analysis, we predicted that the ethylenediamine group would tightly bind to Asp84, and the trifluoromethyl group should occupy part of substrate arginine binding site, which is consistent with our original goal. Our results show for the first time that PRMT1 inhibitors can target the Asp84 binding site, which will be helpful for future drug discovery studies.

Transition of yeast Can1 transporter to the inward-facing state unveils an α-arrestin target sequence promoting its ubiquitylation and endocytosis.

Substrate-transport-elicited endocytosis is a common control mechanism of membrane transporters avoiding excess uptake of external compounds, though poorly understood at the molecular level. In yeast, endocytosis of transporters is triggered by their ubiquitylation mediated by the Rsp5 ubiquitin-ligase, recruited by α-arrestin-family adaptors. We here report that transport-elicited ubiquitylation of the arginine transporter Can1 is promoted by transition to an inward-facing state. This conformational change unveils a region of the N-terminal cytosolic tail targeted by the Art1 α-arrestin, which is activated via the TORC1 kinase complex upon arginine uptake. Can1 mutants altered in the arginine-binding site or a cytosolic tripeptide sequence permanently expose the α-arrestin-targeted region so that Art1 activation via TORC1 is sufficient to trigger their endocytosis. We also provide evidence that substrate-transport elicited endocytosis of other amino acid permeases similarly involves unmasking of a cytosolic Art1-target region coupled to activation of Art1 via TORC1. Our results unravel a mechanism likely involved in regulation of many other transporters by their own substrates. They also support the emerging view that transporter ubiquitylation relies on combinatorial interaction rules such that α-arrestins, stimulated via signaling cascades or in their basal state, recognize transporter regions permanently facing the cytosol or unveiled during transport.

Structural mechanism for the arginine sensing and regulation of CASTOR1 in the mTORC1 signaling pathway.

The mTOR complex I (mTORC1) signaling pathway controls many metabolic processes and is regulated by amino acid signals, especially arginine. CASTOR1 has been identified as the cytosolic arginine sensor for the mTORC1 pathway, but the molecular mechanism of how it senses arginine is elusive. Here, by determining the crystal structure of human CASTOR1 in complex with arginine, we found that an exquisitely tailored pocket, carved between the NTD and the CTD domains of CASTOR1, is employed to recognize arginine. Mutation of critical residues in this pocket abolished or diminished arginine binding. By comparison with structurally similar aspartate kinases, a surface patch of CASTOR1-NTD on the opposite side of the arginine-binding site was identified to mediate direct physical interaction with its downstream effector GATOR2, via GATOR2 subunit Mios. Mutation of this surface patch disrupted CASTOR1’s recognition and inhibition of GATOR2, revealed by in vitro pull-down assay. Normal mode (NM) analysis revealed an ‘open’-to-‘closed’ conformational change for CASTOR1, which is correlated to the switching between the exposing and concealing of its GATOR2-binding residues, and is most likely related to arginine binding. Interestingly, the GATOR2-binding sites on the two protomers of CASTOR1 dimer face the same direction, which prompted us to propose a model for how dimerization of CASTOR1 relieves the inhibition of GATOR1 by GATOR2. Our study thus provides a thorough analysis on how CASTOR1 recognizes arginine, and describes a possible mechanism of how arginine binding induces the inter-domain movement of CASTOR1 to affect its association with GATOR2.

Tyrosine binding and promiscuity in the arginine repressor from the pathogenic bacterium Corynebacterium pseudotuberculosis

The arginine repressor (ArgR) regulates arginine biosynthesis in a number of microorganisms and consists of two domains interlinked by a short peptide; the N-terminal domain is involved in DNA binding and the C-terminal domain binds arginine and forms a hexamer made-up of a dimer of trimers. The crystal structure of the C-terminal domain of ArgR from the pathogenic Corynebacterium pseudotuberculosis determined at 1.9 Å resolution contains a tightly bound tyrosine at the arginine-binding site indicating hitherto unobserved promiscuity. Structural analysis of the binding pocket displays clear molecular adaptations to accommodate tyrosine binding suggesting the possible existence of an alternative regulatory process in this pathogenic bacterium.

Discovery of a Potent Class I Protein Arginine Methyltransferase Fragment Inhibitor.

Protein methyltransferases (PMTs) are a promising target class in oncology and other disease areas. They are composed of SET domain methyltransferases and structurally unrelated Rossman-fold enzymes that include protein arginine methyltransferases (PRMTs). In the absence of a well-defined medicinal chemistry tool-kit focused on PMTs, most current inhibitors were identified by screening large and diverse libraries of leadlike molecules. So far, no successful fragment-based approach was reported against this target class. Here, by deconstructing potent PRMT inhibitors, we find that chemical moieties occupying the substrate arginine-binding site can act as efficient fragment inhibitors. Screening a fragment library against PRMT6 produced numerous hits, including a 300 nM inhibitor (ligand efficiency of 0.56) that decreased global histone 3 arginine 2 methylation in cells, and can serve as a warhead for the development of PRMT chemical probes.

Computational development of selective nNOS inhibitors: binding modes and pharmacokinetic considerations.

Neuronal nitric oxide synthase (nNOS) produces the key signalling mediator nitric oxide, (NO). This gaseous, free radical molecule modulates a vast array of biological processes, from vascular pressure to immune responses and neurological signalling cascades. Overproduction of NO has been implicated in conditions including Alzheimer's disease, Parkinson's disease and schizophrenia. Inhibition of nNOS therefore offers a potential therapeutic approach for treatment of these conditions. This endeavour is made more complex by the fact that there are two other isoforms of nitric oxide synthase (NOS), endothelial NOS (eNOS) and inducible NOS (iNOS). The selectivity of nNOS inhibitors is therefore a key concern for therapeutic development. This review explores recent advances in the field of selective nNOS inhibition. A particular focus is placed on computational approaches towards the rational design of selective nNOS ligands with improved pharmacokinetic properties. These ligands have been targeted at four key binding sites of the nNOS enzyme - the tetrahydrobiopterin, calmodulin, nicotinamide adenine dinucleotide phosphate (NADPH) and arginine binding sites. The binding sites, and the compounds used to inhibit them, will be discussed in turn, along with the computational methods which have been employed in the field of nNOS inhibition.

Mutational analysis to identify the residues essential for the inhibition of N-acetyl glutamate kinase of Corynebacterium glutamicum.

N-acetyl glutamate kinase (NAGK) is a key enzyme in the synthesis of L-arginine that is inhibited by its end product L-arginine in Corynebacterium glutamicum (C. glutamicum). In this study, the potential binding sites of arginine and the residues essential for its inhibition were identified by homology modeling, inhibitor docking, and site-directed mutagenesis. The allosteric inhibition of NAGK was successfully alleviated by a mutation, as determined through analysis of mutant enzymes, which were overexpressed in vivo in C. glutamicum ATCC14067. Analysis of the mutant enzymes and docking analysis demonstrated that residue W23 positions an arginine molecule, and the interaction between arginine and residues L282, L283, and T284 may play an important role in the remote inhibitory process. Based on the results of the docking analysis of the effective mutants, we propose a linkage mechanism for the remote allosteric regulation of NAGK activity, in which residue R209 may play an essential role. In this study, the structure of the arginine-binding site of C. glutamicum NAGK (CgNAGK) was successfully predicted and the roles of the relevant residues were identified, providing new insight into the allosteric regulation of CgNAGK activity and a solid platform for the future construction of an optimized L-arginine producing strain.

Investigating the binding preferences of small molecule inhibitors of human protein arginine methyltransferase 1 using molecular modelling

Protein arginine methyltransferases (PRMTs) catalyse the methylation of arginine residues of target proteins. PRMTs utilise S-adenosyl methionine (SAM) as the methyl group donor, leading to S-adenosyl homocysteine (SAH) and monomethylarginine (mMA). A combination of homology modelling, molecular docking, Active Site Pressurisation, molecular dynamic simulations and MM-PBSA free energy calculations is used to investigate the binding poses of three PRMT1 inhibitors (ligands 1–3), which target both SAM and substrate arginine binding sites by containing a guanidine group joined by short linkers with the SAM derivative. It was assumed initially that the adenine moieties of the inhibitors would bind in sub-site 1 (PHE44, GLU137, VAL136 and GLU108), the guanidine side chain would occupy sub-site 2 (GLU 161, TYR160, TYR156 and TRP302), with the amino acid side chain occupying sub-site 3 (GLU152, ARG62, GLY86 and ASP84; pose 1). However, the SAH homocysteine moiety does not fully occupy sub-site 3, suggesting another binding pose may exist (pose 2), whereby the adenine moiety binds in sub-site 1, the guanidine side chain occupies sub-site 3, and the amino acid side chain occupies sub-site 2. Our results indicate that ligand 1 (pose 1 or 2), ligand 2 (pose 2) and ligand 3 (pose 1) are the predominant binding poses and we demonstrate for the first time that sub-site 3 contains a large space that could be exploited in the future to develop novel inhibitors with higher binding affinities.

Structural insights into regulation of vertebrate homolog N‐acetylglutamate synthase/kinase from Maricaulis maris

Novel bifunctional N‐acetylglutamate synthase/kinases (NAGS/K) that catalyze the first two steps of arginine biosynthesis and are homologous to vertebrate N‐acetylglutamate synthase (NAGS), an essential cofactor‐producing enzyme in the urea cycle, were identified in Maricaulis maris and several other bacteria. The crystal structures of M. maris NAGS/K (mmNAGS/K) with and without L‐arginine bound indicate that mmNAGS/K functions as a tetramer, in contrast to the hexameric structure of Neisseria gonorrhoeae NAGS. Each subunit has an amino acid kinase (AAK) domain, which is likely responsible for N‐acetylglutamate kinase (NAGK) activity and has a arginine binding site, and an N‐acetyltransferase (NAT) domain that contains the putative NAGS active site. The angle of rotation between AAK and NAT domains varies among crystal forms and subunits within the tetramer. A rotation of 26° is sufficient to close the predicted AcCoA binding site, thus reducing enzymatic activity. Since mmNAGS/K has the highest degree of sequence homology to vertebrate NAGS of NAGS and NAGK enzymes whose structures have been determined, the mmNAGS/K structure was used to develop a structural model of human NAGS that is fully consistent with the functional effects of the 14 missense mutations that were identified in NAGS‐deficient patients.

A novel N-acetylglutamate synthase architecture revealed by the crystal structure of the bifunctional enzyme from Maricaulis maris.

Novel bifunctional N-acetylglutamate synthase/kinases (NAGS/K) that catalyze the first two steps of arginine biosynthesis and are homologous to vertebrate N-acetylglutamate synthase (NAGS), an essential cofactor-producing enzyme in the urea cycle, were identified in Maricaulis maris and several other bacteria. Arginine is an allosteric inhibitor of NAGS but not NAGK activity. The crystal structure of M. maris NAGS/K (mmNAGS/K) at 2.7 Å resolution indicates that it is a tetramer, in contrast to the hexameric structure of Neisseria gonorrhoeae NAGS. The quaternary structure of crystalline NAGS/K from Xanthomonas campestris (xcNAGS/K) is similar, and cross-linking experiments indicate that both mmNAGS/K and xcNAGS are tetramers in solution. Each subunit has an amino acid kinase (AAK) domain, which is likely responsible for N-acetylglutamate kinase (NAGK) activity and has a putative arginine binding site, and an N-acetyltransferase (NAT) domain that contains the putative NAGS active site. These structures and sequence comparisons suggest that the linker residue 291 may determine whether arginine acts as an allosteric inhibitor or activator in homologous enzymes in microorganisms and vertebrates. In addition, the angle of rotation between AAK and NAT domains varies among crystal forms and subunits within the tetramer. A rotation of 26° is sufficient to close the predicted AcCoA binding site, thus reducing enzymatic activity. Since mmNAGS/K has the highest degree of sequence homology to vertebrate NAGS of NAGS and NAGK enzymes whose structures have been determined, the mmNAGS/K structure was used to develop a structural model of human NAGS that is fully consistent with the functional effects of the 14 missense mutations that were identified in NAGS-deficient patients.

Novel nanomolar imidazo[4,5-b]pyridines as selective nitric oxide synthase (iNOS) inhibitors: SAR and structural insights

Inducible arginine oxidation and subsequent NO production by correspondent synthase (iNOS) are important cellular answers to proinflammatory signals. Prolonged NO production has been proved in higher organisms to cause stroke or septic shock. Several classes of potent NOS inhibitors have been reported, most of them targeting the arginine binding site of the oxygenase domain. Here we disclose the SAR and the rational design of potent and selective iNOS inhibitors which may be useful as anti-inflammatory drugs.

Crystal Structure of the Intermediate Complex of the Arginine Repressor from Mycobacterium tuberculosis Bound with Its DNA Operator Reveals Detailed Mechanism of Arginine Repression

The concentration of l-arginine in Mycobacterium tuberculosis (Mtb) and in many other bacteria is controlled by a transcriptional factor called the arginine repressor (ArgR). It regulates the transcription of the biosynthetic genes of the arginine operon by interacting with the ∼16- to 20-bp ARG boxes in the promoter site of the operon. ArgRs in the arginine bound form are hexamers in which each protomer has two separately folded domains. The C-terminal domains form a hexameric core, whereas the N-terminal domains have the winged helix–turn–helix DNA-binding motif. Here, we present the crystal structure of the MtbArgR hexamer bound to three copies of the 16-bp DNA operator in the presence of trace amounts of l-arginine, determined to 2.15 Å resolution. In contrast to our previously published structure of the ternary MtbArgR–DNA complex in the presence of 10 mM l-arginine, the DNA operators do not form a double ARG box in the structure reported here. The present structure not only retains the noncrystallographic 32 symmetry of the core (as in the earlier structure), but it also has the 3-fold axis for the whole complex. The core trimers are rotated relative to one another as in the other holo hexamers of MtbArgR, although the l-arginine ligands have only partial density and do not fully occupy the arginine-binding sites. Refinement of the occupancies and B-factors of ligands resulted in a value of approximately 4.4 arginine ligands per hexamer. This has allowed the dissociation constant of arginine from the arginine-binding site to be estimated. The present structure also has two protomer conformations, folded and extended. However, they are different from the conformations in the complex determined at an l-arginine concentration of 10 mM and do not form an interlocking arrangement. The new complex is less stable than the earlier described complex bound with nine arginine residues. Thus, the former can be considered as an intermediate in a pathway to the latter. On the basis of the structure of this intermediate complex, a more detailed mechanism of the arginine biosynthesis regulation in Mtb is proposed.

Simple, recurring RNA binding sites for L-arginine

Seven new arginine binding motifs have been selected from a heterogeneous RNA pool containing 17, 25, and 50mer randomized tracts, yielding 131 independently derived binding sites that are multiply isolated. The shortest 17mer random region is sufficient to build varied arginine binding sites using five different conserved motifs (motifs 1a, 1b, 1c, 2, and 4). Dissociation constants are in the fractional millimolar to millimolar range. Binding sites are amino acid side-chain specific and discriminate moderately between L- and D-stereoisomers of arginine, suggesting a molecular focus on side-chain guanidinium. An arginine coding triplet (codon/anticodon) is highly conserved within the largest family of Arg sites (72% of all sequences), as has also been found in minimal, most prevalent RNA binding sites for Ile, His, and Trp.

Kinetic Consequences of Mutations at an Allosteric Site in Arginase from the Thermophile Bacillus Caldovelox

Arginase is a binuclear manganese metalloenzyme that catalyzes the hydrolysis of L-arginine to L-ornithine and urea. It is involved in ureagenesis and the control of arginine levels for production of proline, creatine, polyamines, and nitric oxide. The crystal structure of the enzyme from the extreme thermophile Bacillus caldovelox reveal a second arginine-binding site, located at the monomer-monomer interface. Binding of the guanidinum group of L-arginine by bidentate hydrogen bonds to Glu256 in one monomer and bifurcated hydrogen bond to Asp199 in the neighbor, was suggested to generate a catalytically competent conformation.