372-75-8Relevant articles and documents
Dissecting structural and electronic effects in inducible nitric oxide synthase
Hannibal, Luciana,Page, Richard C.,Haque, Mohammad Mahfuzul,Bolisetty, Karthik,Yu, Zhihao,Misra, Saurav,Stuehr, Dennis J.
, p. 153 - 165 (2015)
Nitric oxide synthases (NOSs) are haem-thiolate enzymes that catalyse the conversion of L-arginine (L-Arg) into NO and citrulline. Inducible NOS (iNOS) is responsible for delivery of NO in response to stressors during inflammation. The catalytic performance of iNOS is proposed to rely mainly on the haem midpoint potential and the ability of the substrate L-Arg to provide a hydrogen bond for oxygen activation (O-O scission). We present a study of native iNOS compared with iNOS-mesohaem, and investigate the formation of a low-spin ferric haem-aquo or -hydroxo species (P) in iNOS mutant W188H substituted with mesohaem. iNOS-mesohaem and W188H-mesohaem were stable and dimeric, and presented substrate-binding affinities comparable to those of their native counterparts. Single turnover reactions catalysed by iNOSoxy with L-Arg (first reaction step) or N-hydroxy-L-arginine (second reaction step) showed that mesohaem substitution triggered higher rates of FeIIO2 conversion and altered other key kinetic parameters. We elucidated the first crystal structure of a NOS substituted with mesohaem and found essentially identical features compared with the structure of iNOS carrying native haem. This facilitated the dissection of structural and electronic effects. Mesohaem substitution substantially reduced the build-up of species P in W188H iNOS during catalysis, thus increasing its proficiency towards NO synthesis. The marked structural similarities of iNOSoxy containing native haem or mesohaem indicate that the kinetic behaviour observed in mesohaem-substituted iNOS is most heavily influenced by electronic effects rather than structural alterations.
Mesohaem substitution reveals how haem electronic properties can influence the kinetic and catalytic parameters of neuronal NO synthase
Tejero, Jesus,Biswas, Ashis,Haque, Mohammad Mahfuzul,Wang, Zhi-Qiang,Hemann, Craig,Varnado, Cornelius L.,Novince, Zachary,Hille, Russ,Goodwin, Douglas C.,Stuehr, Dennis J.
, p. 163 - 174 (2011)
NOSs (NO synthases, EC 1.14.13.39) are haem-thiolate enzymes that catalyse a two-step oxidation of L-arginine to generate NO. The structural and electronic features that regulate their NO synthesis activity are incompletely understood. To investigate how haem electronics govern the catalytic properties of NOS, we utilized a bacterial haem transporter protein to overexpress a mesohaem-containing nNOS (neuronal NOS) and characterized the enzyme using a variety of techniques. Mesohaem-nNOS catalysed NO synthesis and retained a coupled NADPH consumption much like the wild-type enzyme. However, mesohaem-nNOS had a decreased rate of Fe(III) haem reduction and had increased rates for haem-dioxy transformation, Fe(III) haem-NO dissociation and Fe(II) haem-NO reaction with O2. These changes are largely related to the 48 mV decrease in haem midpoint potential that we measured for the bound mesohaem cofactor. Mesohaem nNOS displayed a significantly lower Vmax and KmO2 value for its NO synthesis activity compared with wild-type nNOS. Computer simulation showed that these altered catalytic behaviours of mesohaem-nNOS are consistent with the changes in the kinetic parameters. Taken together, the results of the present study reveal that several key kinetic parameters are sensitive to changes in haem electronics in nNOS, and show how these changes combine to alter its catalytic behaviour. The Authors Journal compilation
Neuronal nitric oxide synthase isoforms α and μ are closely related calpain-sensitive proteins
Laine, Romuald,Ortiz De Montellano, Paul R.
, p. 305 - 312 (1998)
The neuronal nitric oxide synthase isoform nNOSμ, which is expressed in striated muscle, differs from nNOSα, the major brain isoform, by the insertion of 34 amino acid residues between the calmodulin- and flavin- binding domains [J Biol Chem 271:11204-11208 (1996)]. We show here that recombinant, purified nNOSμ, despite the peptide insertion, has the same spectroscopic properties, L-arginine k(cat) and K(m) values, optimal pH, and calmodulin binding affinity constant as nNOSα. However, nNOSμ consumes NADPH and reduces cytochrome c at approximately half the rate of nNOSα. The rates of degradation of the two proteins by rat brain and muscle homogenates show that nNOSμ is degraded more slowly than nNOSα. The in vitro half- lives of nNOSα and nNOSμ are 12 and 50 min, respectively, and calpain is important for this degradation. These short in vitro half-lives suggest that the nNOS isoforms are susceptible to rapid degradation in vivo. The elevated (20-fold) levels of calpain in diseased muscle tissue in Duchenne muscular dystrophy, and the hydrolytic sensitivity of both nNOSμ and nNOSα to this enzyme, may contribute to the deficiency of nNOS activity in the diseased tissue.
Arginine Deiminase Uses an Active-Site Cysteine in Nucleophilic Catalysis of L-Arginine Hydrolysis
Lu, Xuefeng,Galkin, Andrey,Herzberg, Osnat,Dunaway-Mariano, Debra
, p. 5374 - 5375 (2004)
Arginine deiminase (EC 3.5.3.6) catalyzes the hydrolysis of l-arginine to citrulline and ammonium ion, which is the first step of the microbial l-arginine degradation pathway. The deiminase conserves the active-site Cys-His-Asp motif found in several related enzymes that catalyze group-transfer reactions from the guanidinium center of arginine-containing substrates. For each of these enzymes, nucleophilic catalysis by the conserved Cys has been postulated but never tested. In this communication we report the results from rapid quench studies of single-turnover reactions carried out with recombinant Pseudomonas aeruginosa arginine deiminase and limiting [14C-1]l-arginine. The citrulline-formation and arginine-decay curves measured at 25 °C were fitted to yield apparent rate constants k = 3.6 ± 0.1 s-1 and k = 4.2 ± 0.1 s-1, respectively. The time course for the formation (k =13 s-1) and decay (k = 6.5 s-1) of 14C-labeled enzyme defined a kinetically competent intermediate. Under the same reaction conditions, the Cys406Ser mutant failed to form the 14C-labeled enzyme intermediate. These results, along with the recently reported enzyme X-ray structure (Galkin, A.; Kulakova, L.; Sarikaya, E.; Lim, K.; Howard, A.; Herzberg, O. J. Biol. Chem. 2004, 279, 14001-14008, evidence a reaction pathway in which l-arginine deimination proceeds via a covalent enzyme intermediate formed by ammonia displacement from the arginine guanidinum carbon by the active-site Cys406. Copyright
Directed evolution of an antitumor drug (Arginine Deiminase PpADI) for Increased Activity at Physiological pH
Zhu, Leilei,Tee, Kang Lan,Roccatano, Danilo,Sonmez, Burcu,Ni, Ye,Sun, Zhi-Hao,Schwaneberg, Ulrich
, p. 691 - 697 (2010)
Arginine deiminase (ADI; EC 3.5.3.6) has been studied as a potential antitumor drug for the treatment of arginine-auxotrophic tumors, such as hepatocellular carcinomas (HCCs) and melanomas. Studies with human lymphatic leukemia cell lines confirmed that ADI is an antiangiogenic agent for treating leukemia. The main limitation of ADI from Pseudomonas plecoglossicida (PpADI) lies in its pH-dependent activity profile, its pH optimum is at 6.5. A pH shift from 6.5 to 7.5 results in an approximately 80% drop in activity. (The pH of human plasma is 7.35 to 7.45.) In order to shift the PpADI pH optimum, a directed-evolution protocol based on an adapted citrulline-screening protocol in microtiter-plate format was developed and validated. A proof of concept for ADI engineering resulted in a pH optimum of pH 7.0 and increased resistance under physiological and slightly alkaline conditions. At pH 7.4, variant M2 (K5T/ D44E/H404R) is four times faster than the wild-type PpADI and retains -50% of its activity relative to its pH optimum, compared to -10% in the case of the wild-type PpADI.
L337H mutant of rat neuronal nitric oxide synthase resembles human neuronal nitric oxide synthase toward inhibitors
Fang, Jianguo,Ji, Haitao,Lawton, Graham R.,Xue, Fengtian,Roman, Linda J.,Silverman, Richard B.
, p. 4533 - 4537 (2009)
A common dichotomy exists in inhibitor design: should the compounds be designed to block the enzymes of animals in the preclinical studies or to inhibit the human enzyme? We report that a single mutation of Leu-337 in rat neuronal nitric oxide synthase (nNOS) to His makes the enzyme resemble human nNOS more than rat nNOS. We expect that the approach used in this study can unite the dichotomy and speed up the process of inhibitor design and development.
Mechanisms of catalysis and inhibition operative in the arginine deiminase from the human pathogen Giardia lamblia
Li, Zhimin,Kulakova, Liudmila,Li, Ling,Galkin, Andrey,Zhao, Zhiming,Nash, Theodore E.,Mariano, Patrick S.,Herzberg, Osnat,Dunaway-Mariano, Debra
, p. 149 - 161 (2009)
Giardia lamblia arginine deiminase (GlAD), the topic of this paper, belongs to the hydrolase branch of the guanidine-modifying enzyme superfamily, whose members employ Cys-mediated nucleophilic catalysis to promote deimination of l-arginine and its naturally occurring derivatives. G. lamblia is the causative agent in the human disease giardiasis. The results of RNAi/antisense RNA gene-silencing studies reported herein indicate that GlAD is essential for G. lamblia trophozoite survival and thus, a potential target for the development of therapeutic agents for the treatment of giardiasis. The homodimeric recombinant protein was prepared in Escherichia coli for in-depth biochemical characterization. The 2-domain GlAD monomer consists of a N-terminal domain that shares an active site structure (depicted by an in silico model) and kinetic properties (determined by steady-state and transient state kinetic analysis) with its bacterial AD counterparts, and a C-terminal domain of unknown fold and function. GlAD was found to be active over a wide pH range and to accept l-arginine, l-arginine ethyl ester, Nα-benzoyl-l-arginine, and Nω-amino-l-arginine as substrates but not agmatine, l-homoarginine, Nα-benzoyl-l-arginine ethyl ester or a variety of arginine-containing peptides. The intermediacy of a Cys424-alkylthiouronium ion covalent enzyme adduct was demonstrated and the rate constants for formation (k1 = 80 s-1) and hydrolysis (k2 = 35 s-1) of the intermediate were determined. The comparatively lower value of the steady-state rate constant (kcat = 2.6 s-1), suggests that a step following citrulline formation is rate-limiting. Inhibition of GlAD using Cys directed agents was briefly explored. S-Nitroso-l-homocysteine was shown to be an active site directed, irreversible inhibitor whereas Nω-cyano-l-arginine did not inhibit GlAD but instead proved to be an active site directed, irreversible inhibitor of the Bacillus cereus AD.
Ruthenium(III) readily abstracts NO from L-arginine, the physiological precursor to NO, in the presence of H2O2. A remarkably simple model system for NO synthases.
Marmion,Murphy,Nolan
, p. 1870 - 1871 (2001)
Reaction of [Ru(Hedta)Cl]- with H2O2 in the presence of arginine, produces NO, in the form of an Ru(II)-(NO+) complex and citrulline which is a remarkably simple model system for the physiological NO synthase reaction.
IMPROVED METHOD OF SYNTHESIS AND PURIFICATION OF CITRULLINE
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Paragraph 00033-00034; 00037-00038, (2020/12/29)
This invention provides for synthesis of citrulline from a transition metal complex of ornithine using cyanate to derivatize the terminal amino group of ornithine. The invention also provides improved methods for purification of citrulline produced by reaction of cyanate with ornithine via the steps of reprecipitation of copper complex of citrulline, removal of the complexing metal by sulfide precipitation, activated carbon adsorption and antisolvent crystallization.
l-Arginine and nitric oxide synthesis in the cells with inducible NO synthase
Kuropteva,Baider,Nagler,Bogatyrenko,Belaia
, p. 174 - 180 (2019/04/25)
The effect of citrulline and ammonium chloride on the nitric oxide formation by peritoneal macrophages and liver tissue cells was studied using ESR spectroscopy. In ex vivo models, the incubation of cells capable of expressing inducible NO synthase (iNOS) with interferon-γ resulted in a moderate increase in the amount of hemoglobin–nitric oxide nitrosyl complexes (Heme–NO NCs), whereas incubation with l-citrulline and ammonium chloride increased the amount of Heme–NO NCs by an order of magnitude. It was assumed that a separate cycle of L-arginine and nitric oxide synthesis exists in the peritoneal macrophages and liver cells, with the major participants of the cycle being the inducible NO synthase enzyme (iNOS) and enzymes that synthesize L-arginine from L-citrulline and a nitrogen source. Functioning of this cycle makes immunocompetent cells with iNOS able to produce NO for a long time and in large amounts.
