Key points are not available for this paper at this time.
Human carbonyl reductase 1 (hCBR1) is an NADPH-dependent short chain dehydrogenase/reductase with broad substrate specificity and is thought to be responsible for the in vivo reduction of quinones, prostaglandins, and other carbonyl-containing compounds including xenobiotics. In addition, hCBR1 possesses a glutathione binding site that allows for increased affinity toward GSH-conjugated molecules. It has been suggested that the GSH-binding site is near the active site; however, no structures with GSH or GSH conjugates have been reported. We have solved the x-ray crystal structures of hCBR1 and a substrate mimic in complex with GSH and the catalytically inert GSH conjugate hydroxymethylglutathione (HMGSH). The structures reveal the GSH-binding site and provide insight into the affinity determinants for GSH-conjugated substrates. We further demonstrate that the structural isostere of HMGSH, S-nitrosoglutathione, is an ideal hCBR1 substrate (Km = 30 μm, kcat = 450 min-1) with kinetic constants comparable with the best known hCBR1 substrates. Furthermore, we demonstrate that hCBR1 dependent GSNO reduction occurs in A549 lung adenocarcinoma cell lysates and suggest that hCBR1 may be involved in regulation of tissue levels of GSNO. Human carbonyl reductase 1 (hCBR1) is an NADPH-dependent short chain dehydrogenase/reductase with broad substrate specificity and is thought to be responsible for the in vivo reduction of quinones, prostaglandins, and other carbonyl-containing compounds including xenobiotics. In addition, hCBR1 possesses a glutathione binding site that allows for increased affinity toward GSH-conjugated molecules. It has been suggested that the GSH-binding site is near the active site; however, no structures with GSH or GSH conjugates have been reported. We have solved the x-ray crystal structures of hCBR1 and a substrate mimic in complex with GSH and the catalytically inert GSH conjugate hydroxymethylglutathione (HMGSH). The structures reveal the GSH-binding site and provide insight into the affinity determinants for GSH-conjugated substrates. We further demonstrate that the structural isostere of HMGSH, S-nitrosoglutathione, is an ideal hCBR1 substrate (Km = 30 μm, kcat = 450 min-1) with kinetic constants comparable with the best known hCBR1 substrates. Furthermore, we demonstrate that hCBR1 dependent GSNO reduction occurs in A549 lung adenocarcinoma cell lysates and suggest that hCBR1 may be involved in regulation of tissue levels of GSNO. Human carbonyl reductase 1 (hCBR1), 3The abbreviations used are:hCBR1human carbonyl reductase 1GSNOS-nitrosoglutathioneHMGSHS-hydroxymethylglutathioneOH-PP3-(1-tert-butyl-4-amino-1H-indazol-3-yl)phenolhFDHhuman glutathione-dependent formaldehyde dehydrogenase 3The abbreviations used are:hCBR1human carbonyl reductase 1GSNOS-nitrosoglutathioneHMGSHS-hydroxymethylglutathioneOH-PP3-(1-tert-butyl-4-amino-1H-indazol-3-yl)phenolhFDHhuman glutathione-dependent formaldehyde dehydrogenase an NADPH-dependent enzyme belonging to the short chain dehydrogenase/reductase family, has been shown to be involved in the metabolism of structurally diverse carbonyl-containing substances. This is in contrast to other members of the short chain dehydrogenase/reductase family, such as 11β-hydroxysteroid dehydrogenases 1 and 2 that interconvert cortisone and cortisol, and the 17β-hydroxysteroid dehydrogenases, which have well defined androgen and estrogen substrates. Previously reported substrates of hCBR1 include prostaglandins and xenobiotics such as the anti-cancer anthracyclin doxorubicin and the vitamin K2 precursor menadione (1Forrest G.L. Gonzalez B. Chem. Biol. Interact. 2000; 129: 21-40Crossref PubMed Scopus (208) Google Scholar). More recently, hCBR1 has been linked to the detoxification of reactive aldehydes such as 4-oxonon-2-enal and its GSH conjugate that are believed to play a central role in oxidative stress-related neurodegenerative disorders including Alzheimer and Parkinson diseases (2Doorn J.A. Maser E. Blum A. Claffey D.J. Petersen D.R. Biochemistry. 2004; 43: 13106-13114Crossref PubMed Scopus (76) Google Scholar). Although extensive biological investigations of hCBR1, including RNA interference, pharmacology, and crystallography of human hCBR1 in complex with substrate mimics, have been carried out (3Tanaka M. Bateman R. Rauh D. Vaisberg E. Ramachandani S. Zhang C. Hansen K.C. Burlingame A.L. Trautman J.K. Shokat K.M. Adams C.L. PLoS Biol. 2005; 3: 764-776Crossref Scopus (203) Google Scholar), the endogenous physiological substrate(s) of this enzyme remain to be defined. One well accepted aspect of hCBR1 substrate recognition is the presence of a GSH-binding pocket predicted to be in close proximity to the catalytic site. GSH conjugates of otherwise poorly recognized substrates such as prostaglandin A1 are reduced by hCBR1 (4Tinguely J.N. Wermuth B. Eur. J. Biochem. 1999; 260: 9-14Crossref PubMed Scopus (26) Google Scholar), supporting this hypothesis. To identify additional physiological substrates of hCBR1, we initiated a structural biology effort to analyze the GSH-binding site of hCBR1, hypothesizing that cellular GSH adducts might serve as particularly good hCBR1 substrates. human carbonyl reductase 1 S-nitrosoglutathione S-hydroxymethylglutathione 3-(1-tert-butyl-4-amino-1H-indazol-3-yl)phenol human glutathione-dependent formaldehyde dehydrogenase human carbonyl reductase 1 S-nitrosoglutathione S-hydroxymethylglutathione 3-(1-tert-butyl-4-amino-1H-indazol-3-yl)phenol human glutathione-dependent formaldehyde dehydrogenase We solved the x-ray co-crystal structure of hCBR1 in complex with GSH, demonstrating that the GSH-binding pocket lies within the catalytic pocket of the enzyme. To next assess the viability of other candidate substrates, we attempted to obtain crystals of hCBR1 with GSH adducts of reported hCBR1 substrates (e.g. prostaglandin A1-GSH and Menadione-GSH) as well as other cellular GSH adducts not known to be hCBR1 substrates like formaldehyde-GSH. We were able to obtain diffraction quality crystals and solve the structure of the GSH-formaldehyde conjugate, hydroxymethylglutathione (HMGSH), in complex with hCBR1, but we were unable to co-crystallize other reported GSH adduct substrates. Although HMGSH is a thio-hemiacetal that cannot be reduced by hCBR1 because of the low reduction potential of NADPH, we considered the possibility that other physiological isosteres of HMGSH might be efficient substrates. We found that the nitrogen-containing GSH adduct S-nitrosoglutathione (GSNO) is an efficient substrate of hCBR1, which implicates this enzyme in physiological GSNO catabolism. Crystallization, Data Collection, and Refinement—Human carbonyl reductase 1 was overexpressed in Escherichia coli and purified as previously described (5Bateman R. Rauh D. Shokat K.M. Org. Biomol. Chem. 2007; 5: 3363-3367Crossref PubMed Scopus (21) Google Scholar). The crystals were obtained by the vapor diffusion method by growth in the presence of OH-PP (supplemental Fig. S1) as previously described (3Tanaka M. Bateman R. Rauh D. Vaisberg E. Ramachandani S. Zhang C. Hansen K.C. Burlingame A.L. Trautman J.K. Shokat K.M. Adams C.L. PLoS Biol. 2005; 3: 764-776Crossref Scopus (203) Google Scholar). The crystals were soaked successively (three times) in precipitant solution containing 5 mm of either freshly prepared HMGSH 4HMGSH was produced in situ by incubating stoichiometric amounts of GSH and formaldehyde and yielded the desired linear conjugate rather than the previously found bicyclic adduct ((2S,7R)-7-(carboxymethylcarbamoyl)-5-oxo-9-thia-1,6-diaza-,bicyclo4.4.1undecane-2-carboxylic acid) (5Bateman R. Rauh D. Shokat K.M. Org. Biomol. Chem. 2007; 5: 3363-3367Crossref PubMed Scopus (21) Google Scholar). or GSH. Crystals for hCBR1·NADP were grown from 20! polyethylene glycol 3350 and 0.2 m NaCl in space group P43212 with one molecule in the asymmetric unit. Single crystals were cryostabilized by rapid equilibration in precipitant solution containing 11.25! glycerol followed by flash freezing in a stream of nitrogen. The data set for hCBR1·NADP was measured in-house (Rigaku Raxis IV, UCSF). Data sets of hCBR1·NADP·OH-PP·GSH and hCBR1·NADP·OH-PP·HMGSH were measured at the 8.3.1 beamline of the Advance Light Source (Berkeley, CA). The data sets were integrated using DENZO and scaled with Scalepack (HKL2000 package (6Otwinowski Z. Minor W. Methods Enzymol. 1997; 276A: 307-326Crossref Scopus (38526) Google Scholar)). The structures were solved by molecular replacement with CNS (7Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16957) Google Scholar) or AMoRE (8Navaza J. Acta Crystallogr. Sect. D Biol. Crystallogr. 2001; 57: 1367-1372Crossref PubMed Scopus (658) Google Scholar). Starting coordinates were taken from hCBR1 in complex with OH-PP (Protein Data Bank code 1WMA) (3Tanaka M. Bateman R. Rauh D. Vaisberg E. Ramachandani S. Zhang C. Hansen K.C. Burlingame A.L. Trautman J.K. Shokat K.M. Adams C.L. PLoS Biol. 2005; 3: 764-776Crossref Scopus (203) Google Scholar). Crystallographic refinement and electron density map calculations were carried out using REFMAC5 (9Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13854) Google Scholar). The models of GSH and HMGSH were constructed and minimized using Moloc (10Gerber P.R. Muller K. J. Comput. Aided Mol. Des. 1995; 9: 251-268Crossref PubMed Scopus (506) Google Scholar). Topology files were generated using the Dundee PRODRG2 server (11Schuttelkopf A.W. van Aalten D.M. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 1355-1363Crossref PubMed Scopus (4253) Google Scholar). Model building was accomplished using COOT (12Emsley P. Cowtan K. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (23226) Google Scholar). Detailed data and refinement statistics are given in Table 1. Atomic coordinates for hCBR1·NADP, hCBR1·NADP·OH-PP·GSH, and hCBR1·NAPD·OH-PP·HMGSH have been deposited to the Protein Data Bank (Protein Data Bank codes 3BHI, 3BHJ, and 3BHM). Refined structures were validated with PROCHECK (13Laskowski E.R. Mayo Clin. Proc. 1993; 68: 1029-1030Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar). The figures were produced using PyMol 2002 (DeLano Scientific, San Carlos, CA).TABLE 1Data collection and refinement statisticshCBR1·NADPhCBR1·NADP·OH-PP·GSHhCBR1·NADP·OH-PP·HMGSHProtein Data Bank code3BHI3BHJ3BHMData collectionSpace groupP43 21 2P21 21 21P21 21 21Cell dimensionsa, b, c (Å)55.66, 55.66, 169.8854.64, 55.47, 95.7455.16, 55.94, 95.27α, β, γ (°)90, 90, 9090, 90, 9090, 90, 90Resolution (Å)2.27-50.00 (2.27-2.35)1.77-27.00 (1.77-1.83)1.66-50.00 (1.66-1.72)Rsym17.8 (40.2)2.9 (4.2)4.9 (32.1)Completeness (!)99.7 (100.0)93.3 (91.7)97.1 (81.8)Redundancy5.32.35.7I/σ9.29 (2.62)25.95 (16.13)27.19 (2.21)RefinementResolution (Å)2.27-50.00 (2.27-2.33)1.77-27.00 (1.77-1.82)1.8-48.22 (1.80-1.85)No. reflections13056/1241627133/2574927565/26182Rwork/Rfree20.2/28.217.3/22.319.7/26.9No. atomsProtein210221032035Ligand48142112Water165266200Ion13020B-factorsProtein17.813.816.8Ligand11.718.219.7Water21.127.128.9Ion21.535.8Root mean square deviationsBond lengths (Å)0.0250.0110.012Bond angles (°)2.0761.3341.346Ramachandran analysisResidues in most favored regions (!)92.893.292.8Residues in additional allowed regions (!)6.86.47.2Residues in generously allowed regions (!)0.40.40Residues in disallowed regions (!)000 Open table in a new tab Km Determination for the Substrate GSNO—Human carbonyl reductase 1 activity was determined spectrophotometrically as previously described (3Tanaka M. Bateman R. Rauh D. Vaisberg E. Ramachandani S. Zhang C. Hansen K.C. Burlingame A.L. Trautman J.K. Shokat K.M. Adams C.L. PLoS Biol. 2005; 3: 764-776Crossref Scopus (203) Google Scholar). The reactions contained 100 μm NADPH in 50 mm sodium phosphate (pH 6.8) and either 252 nm or 126 nm hCBR1. GSNO substrate was prepared by combining stoichiometric amounts (0.5 m each) of GSH, sodium nitrite, and HCl. Diethylenetriaminepentaacetic acid (1 mm) was added to prevent decomposition because of metal ion contamination. The yield of GSNO was verified spectrophotometrically by measuring the absorption of the created SNO group at 335 nm using an extinction coefficient of 0.92 mm-1 cm-1. The aqueous GSNO solution was added to samples to achieve concentrations of 151.4, 75.7, 37.85, 30.3, 22.7, and 15.1 μm. All of the reactions were performed in triplicate. The initial rates were calculated from the using a extinction coefficient of mm-1 for GSNO and NADPH Biochem. J. 1998; PubMed Scopus Google Scholar) and were to the for Km The kcat was determined at substrate μm NADPH and μm using hCBR1 that been purified by (5Bateman R. Rauh D. Shokat K.M. Org. Biomol. Chem. 2007; 5: 3363-3367Crossref PubMed Scopus (21) Google Scholar) and Determination of NADPH and GSNO were prepared in the described for Km substrate concentrations including 100 μm NADPH and 50 μm 50 μm NADPH and 100 μm or 50 μm NADPH and 50 μm GSNO. of GSNO contained acid (1 and the was verified spectrophotometrically The reactions were performed in and the in and GSNO was determined at the for was determined for human glutathione-dependent formaldehyde dehydrogenase A. M. J. J.S. 2001; PubMed Scopus Google Scholar) in an using Determination of and samples (1 were prepared containing mm sodium μm GSNO as and μm of either or The reactions were initiated by the of or hCBR1 and allowed to for 1 at was at nm to that the reactions were The samples were at of the the were prepared in the mm sodium phosphate (pH was determined by of the using the was determined in a to previously described Biochem. J. 1998; PubMed Scopus Google D.R. J.S. Biochem. 1995; PubMed Scopus Google Scholar). of 100 mm sodium phosphate (pH was added to of in aqueous was added with and followed by of 1 m sodium The samples were to for and for 1 and the was The was determined by the to a prepared with samples containing was determined in the The were prepared using in GSNO from A549 were using containing and of the were with and with mm 50 mm 1 mm at for The lysates were by and the was determined using the The were determined spectrophotometrically as by the of reduction in the of The reactions were performed in using containing 100 μm NADPH or with and the of 100 μm (supplemental Fig. S1) (3Tanaka M. Bateman R. Rauh D. Vaisberg E. Ramachandani S. Zhang C. Hansen K.C. Burlingame A.L. Trautman J.K. Shokat K.M. Adams C.L. PLoS Biol. 2005; 3: 764-776Crossref Scopus (203) Google Scholar). phosphate 6.8) and GSNO were in of GSH and HMGSH to carbonyl reductase 1 is an enzyme with broad substrate specificity of anthracyclin such as as well as such as prostaglandins (1Forrest G.L. Gonzalez B. Chem. Biol. Interact. 2000; 129: 21-40Crossref PubMed Scopus (208) Google Scholar). The by which hCBR1 is thought to carbonyl-containing substrates is a glutathione site that recognition of GSH adduct substrates. We to GSH substrates to the catalytic of hCBR1 using x-ray We solved the crystal structure of hCBR1 in complex with a of the enzyme (3Tanaka M. Bateman R. Rauh D. Vaisberg E. Ramachandani S. Zhang C. Hansen K.C. Burlingame A.L. Trautman J.K. Shokat K.M. Adams C.L. PLoS Biol. 2005; 3: 764-776Crossref Scopus (203) Google Scholar). Although x-ray structures of the and hCBR1 have been and are known to GSH-binding the of the GSH-binding site has not been defined to co-crystallize hCBR1 with GSH not yield we to GSH into hCBR1 crystals containing and the substrate mimic OH-PP (3Tanaka M. Bateman R. Rauh D. Vaisberg E. Ramachandani S. Zhang C. Hansen K.C. Burlingame A.L. Trautman J.K. Shokat K.M. Adams C.L. PLoS Biol. 2005; 3: 764-776Crossref Scopus (203) Google Scholar). The crystals to of hCBR1 with and GSH The binding site for GSH is in the of the enzyme active site with the toward the of the and the OH-PP substrate GSH adducts are from the that the of GSH in crystals is with the of substrate recognition of GSH adducts (supplemental Fig. GSH and hCBR1 are the GSH by chain including the of the to and and the to the carbonyl and the to a and in to the and chain the of in GSH is the of a and to the ion found in the structure (Protein Data Bank code The the of GSH and is the chain the structural aspect of the and in GSH is recognized in hCBR1. The structure of GSH to hCBR1 the pocket but not further reveal adducts of GSH structurally diverse carbonyl-containing compounds to the NADPH To this we GSH adducts with prostaglandin and the is not a substrate of the and not a carbonyl The complex that produced x-ray quality crystals was that of HMGSH, and which to The binding of HMGSH to hCBR1 was found to be to GSH c and are and the of GSH The is to and the to carbonyl and the to c and an the of HMGSH and the carbonyl is HMGSH is not a carbonyl-containing adduct and is not a substrate for reduction by hCBR1. HMGSH has been shown to be to by the enzyme M. R. Biol. 1995; PubMed Scopus Google M. Biol. 1997; PubMed Google Scholar). hCBR1 has not been shown to its substrates using we We no of this not The effort to GSH substrate recognition not to a structure for hCBR1, to a crystal of the enzyme that might be catalytically In this of crystals contained a carbonyl substrate mimic in with the catalytic of the active site and and to the of Although the presence of this crystal we a crystal this extensive crystal we were able to solve the structure of hCBR1 containing the crystals in the space group P43212 and to of the active site of the OH-PP structures of hCBR1 with hCBR1 no or other to the with the of a chain of the site. This is with the of to the the of is by a ion that is the chain of and of the The of and of the by a ion a previously reported of an hCBR1 The was reported to be catalytically but concentrations of mm) activity of the enzyme (4Tinguely J.N. Wermuth B. Eur. J. Biochem. 1999; 260: 9-14Crossref PubMed Scopus (26) Google Scholar). either a or in the of and the is for catalytic The site is at the of The of this is to the of the chain and to either the or Fig. the be by or which be crystallography not The of crystal structures of hCBR1 with substrates and substrate allowed an of of the binding pocket with to GSH of hCBR1 structures in the and in structures of hCBR1. This that a complex GSH the chain of this the by of GSH by The of the GSH in structures is in the to GSH substrate adducts of and is with the broad substrate specificity of hCBR1. We cannot be however, that binding of GSH in the GSH structure with OH-PP the binding of this GSH adducts a substrate mimic OH-PP is not GSNO by that the adduct of GSH we were able to structurally in the hCBR1 active site a from formaldehyde and that this adduct is not a substrate for the enzyme to isosteres of HMGSH as new substrates. physiological GSH involved in is a close structural of GSNO an for of Furthermore, the enzyme is able to that the NADPH-dependent hCBR1 be of the GSNO reduction by hCBR1 was spectrophotometrically by the in GSNO and NADPH at GSNO was prepared from of GSH, and and was reduced by hCBR1 in an NADPH-dependent (supplemental Fig. hCBR1 for the GSNO because reduction of is not by hCBR1 (supplemental Fig. The kinetic constants for GSNO reduction by hCBR1 are comparable with of its best known of kinetic constants for and GSNO reduction using NADPH and (4Tinguely J.N. Wermuth B. Eur. J. Biochem. 1999; 260: 9-14Crossref PubMed Scopus (26) Google were reported to be for menadione reduction by hCBR1 Eur. J. Biochem. PubMed Scopus Google The were reported to be for menadione reduction by hCBR1 Eur. J. Biochem. PubMed Scopus Google Scholar) Open table in a new tab of hCBR1 and GSNO has been reported to carbonyl to we the reduction of the of GSNO by hCBR1 of GSNO reduction by have been Biochem. J. 1998; PubMed Scopus Google Eur. J. Biochem. PubMed Scopus Google Scholar). In the GSH may in further reduction of to stoichiometric reduction from other we determined the of reduction of GSNO by We found the to be as determined by out reactions with amounts of either GSNO or the was GSNO reduction by (supplemental Fig. that the is the we that the of and reactions might be from the have been 1 and Biochem. J. 1998; PubMed Scopus Google Scholar). the is believed that occurs to the to an that to glutathione and further be to glutathione acid and Biochem. J. 1998; PubMed Scopus Google A. M. J. J.S. 2001; PubMed Scopus Google Scholar). and are We performed a by of and GSNO reduction with and NADPH, Table the concentrations of of reactions to be The most for and reactions was of yield for and More Biochem. J. 1998; PubMed Scopus Google Scholar) suggest that this of glutathione the the of and of and GSNO is that glutathione is the of GSNO of and for reactions prepared with μm μm or μm NADPH (hCBR1) in mm sodium phosphate were in the of The were in the of acid Open table in a new tab GSNO in assess hCBR1 to cellular GSNO we GSNO reduction in lung adenocarcinoma A549 which have previously been used to hCBR1 (3Tanaka M. Bateman R. Rauh D. Vaisberg E. Ramachandani S. Zhang C. Hansen K.C. Burlingame A.L. Trautman J.K. Shokat K.M. Adams C.L. PLoS Biol. 2005; 3: 764-776Crossref Scopus (203) Google Scholar). be responsible for GSNO we used the specificity of the reported GSNO reductase to the NADPH we to out reduction in the cell reduction by other like the that may NADPH for the of GSNO D. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), and reduction by GSNO To the reductase activity in cell lysates from other NADPH-dependent GSNO we an of hCBR1, (supplemental Fig. S1) (3Tanaka M. Bateman R. Rauh D. Vaisberg E. Ramachandani S. Zhang C. Hansen K.C. Burlingame A.L. Trautman J.K. Shokat K.M. Adams C.L. PLoS Biol. 2005; 3: 764-776Crossref Scopus (203) Google Scholar). GSNO reductase activity was measured in A549 cell lysates as measured by a in at nm This activity is to with this the hCBR1 not GSNO We NADPH-dependent GSNO reductase activity in the A549 cell The of of the NADPH-dependent that this of cellular GSNO reduction may be by hCBR1. in GSNO reduction was in the presence of Although GSNO reductase activity in is to A. M. J. J.S. 2001; PubMed Scopus Google Scholar), is NADPH-dependent activity A549 cell lysates are with the hCBR1 The of this NADPH-dependent activity may be with by the D. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) or a GSNO of the NADPH-dependent GSNO which is in at to mm has the to with in to the of which is and in the B. D. A. J.S. J. PubMed Scopus Google Scholar). The of GSNO is given its as a of which may include in and regulation K. R. T. M. B. J. 2000; PubMed Google J.S. 2005; PubMed Scopus Google A. T. J. J.S. Full Text Full Text PDF PubMed Scopus Google Scholar). Although have been reported to GSNO R.J. E. J. B. Biochem. J. 1997; PubMed Scopus Google Z. J. Biochem. PubMed Scopus Google M. R. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar), has been shown to endogenous levels of GSNO A. M. J. J.S. 2001; PubMed Scopus Google Scholar) by of a This enzyme is in that for reduction rather than the in vivo Although the Km of hCBR1 and for GSNO are a kcat This increased catalytic of may the enzyme to in the presence of low The physiological of the and in that physiological levels of NADPH in cell have been found to be with to levels are than This NADPH is the in vivo rather than and in the W. PubMed Scopus Google Scholar). Although concentrations have not been determined in A549 of has of = and = R. Biochem. J. PubMed Scopus Google D. P. Biochem. J. PubMed Scopus Google Scholar). The of and has been reported to be and the and NADPH is P.D. Biochem. PubMed Scopus Google Scholar). All of data are with a NADPH is the and is in to hCBR1 the that NADPH of GSNO GSH and hCBR1 the known NADPH-dependent reductase that in GSNO catabolism. We have hCBR1 as an NADPH-dependent GSNO enzyme (Km = 30 μm, kcat = 450 GSNO is reduced by hCBR1 with kinetic constants comparable with the best previously reported substrates of hCBR1, that GSNO may be as an hCBR1 substrate as the substrates of the enzyme. The that hCBR1 to be responsible for of the GSNO reductase activity in at one cell may suggest that this enzyme is at responsible for regulation of levels in We for the of human enzyme. with files
Bateman et al. (Wed,) studied this question.
Synapse has enriched 5 closely related papers on similar clinical questions. Consider them for comparative context: