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Daniel Mansuy

Publications and source records attributed to Daniel Mansuy.

At least 19 recordsLinked to original sources
PubMed2006

Analogies and surprising differences between recombinant nitric oxide synthase-like proteins from Staphylococcus aureus and Bacillus anthracis in their interactions with l-arginine analogs and iron ligands.

Genome sequencing has recently shown the presence of genes coding for NO-synthase (NOS)-like proteins in bacteria. The roles of these proteins remain unclear. The interactions of a series of l-arginine (l-arg) analogs and iron ligands with two recombinant NOS-like proteins from Staphylococcus aureus (saNOS) and Bacillus anthracis (baNOS) have been studied by UV-visible spectroscopy. SaNOS and baNOS in their ferric native state, as well as their complexes with l-arg analogs and with various ligands, exhibit spectral characteristics highly similar to the corresponding complexes of heme-thiolate proteins such as cytochromes P450 and NOSs. However, saNOS greatly differs from baNOS at the level of three main properties: (i) native saNOS mainly exists under an hexacoordinated low-spin ferric state whereas native baNOS is mainly high-spin, (ii) the addition of tetrahydrobiopterin (H4B) or H4B analogs leads to an increase of the affinity of l-arg for saNOS but not for baNOS, and (iii) saNOS Fe(II), contrary to baNOS, binds relatively bulky ligands such as nitrosoalkanes and tert-butylisocyanide. Thus, saNOS exhibits properties very similar to those of the oxygenase domain of inducible NOS (iNOS(oxy)) not containing H4B, as expected for a NOSoxy-like protein that does not contain H4B. By contrast, the properties of baNOS which look like those of H4B-containing iNOS(oxy) are unexpected for a NOS-like protein not containing H4B. The origin of these surprising properties of baNOS remains to be determined.

Arginine↗
PubMed2006

Differential effects of alkyl- and arylguanidines on the stability and reactivity of inducible NOS heme-dioxygen complexes.

NO-Synthases are heme proteins that catalyze the oxidation of L-arginine into NO and L-citrulline. Some non-amino acid alkylguanidines may serve as substrates of inducible NOS (iNOS), while no NO* production is obtained from arylguanidines. All studied guanidines induce uncoupling between electrons transferred from the reductase domain and those required for NO formation. This uncoupling becomes critical with arylguanidines, leading to the exclusive formation of superoxide anion O2*- as well as hydrogen peroxide H2O2. To understand these different behaviors, we have conducted rapid scanning stopped-flow experiments with dihydrobiopterin (BH2) and tetrahydrobiopterin (BH4) to study, respectively, the (i) autoxidation and (ii) activation processes of heme ferrous-O2 complexes (Fe(II)O2) in the presence of eight alkyl- and arylguanidines. The Fe(II)O2 complex is more easily autooxidized by alkylguanidines (10-fold) and arylguanidines (100-fold) compared to L-arginine. In the presence of alkylguanidines and BH4, the oxygen-activation kinetics are very similar to those observed with L-arginine. Conversely, in the presence of arylguanidines, no Fe(II)O2 intermediate is detected. To understand such variations in reactivity and stability of Fe(II)O2 complex, we have characterized the effects of alkyl- and arylguanidines on Fe(II)O2 structure using the Fe(II)CO complex as a mimic. Resonance Raman and FTIR spectroscopies show that the two classes of guanidine derivatives induce different polar effects on Fe(II)CO environment. Our data suggest that the structure of the substituted guanidine can modulate the stability and the reactivity of heme-dioxygen complexes. We thus propose differential mechanisms for the electron- and proton-transfer steps in the NOS-dependent, oxygen-activation process, contingent upon whether alkyl- or arylguanidines are bound.

Animals↗
PubMed2006

Design and synthesis of selective, high-affinity inhibitors of human cytochrome P450 2J2.

The active site topology, substrate specificity, and biological roles of the human cytochrome P450 CYP2J2, which is mainly expressed in the cardiovascular system, are poorly known even though recent data suggest that it could be a novel biomarker and potential target for therapy of human cancer. This paper reports a first series of high-affinity, selective CYP2J2 inhibitors that are related to terfenadine, with K(i) values as low as 160nM, that should be useful tools to determine the biological roles of CYP2J2.

Cytochrome P-450 CYP2J2↗
PubMed2006

Reactivity of the heme-dioxygen complex of the inducible nitric oxide synthase in the presence of alternative substrates.

Single turnover reactions of the inducible nitric oxide synthase oxygenase domain (iNOSoxy) in the presence of several non alpha-amino acid N-hydroxyguanidines and guanidines were studied by stopped-flow visible spectroscopy, and compared with reactions using the native substrates L-arginine (L-arg) or N(omega)-hydroxy-L-arginine (NOHA). In experiments containing dihydrobiopterin, a catalytically incompetent pterin, and each of the studied substrates, L-arg, butylguanidine (BuGua), para-fluorophenylguanidine (FPhGua), NOHA, N-butyl- and N-(para-fluorophenyl)-N'-hydroxyguanidines (BuNOHG and FPhNOHG), the formation of a iron(II) heme-dioxygen intermediate (Fe(II)O2) was always observed. The Fe(II)O2 species then decayed to iron(III) iNOSoxy at rates that were dependent on the nature of the substrate. Identical reactions containing the catalytically competent cofactor tetrahydrobiopterin (BH4), iNOSoxy and the three N-hydroxyguanidines, all exhibited an initial formation of an Fe(II)O2 species that was successively converted to an Fe(III)NO complex and eventually to high-spin iron(III) iNOSoxy. The formation and decay kinetics of the Fe(III)NO complex did not vary greatly as a function of the N-hydroxyguanidine structure, but the formation of Fe(III)NO was substoichiometric in the cases of BuNOHG and FPhNOHG. Reactions between BH4-containing iNOSoxy and BuGua exhibited kinetics similar to those of the corresponding reaction with L-arginine, with formation of an Fe(II)O2 intermediate that was directly converted to high-spin iron(III) iNOSoxy. In contrast, no Fe(II)O2 intermediate was observed in the reaction of BH4-containing iNOSoxy and FPhGua. Multi-turnover reaction of iNOS with FPhGua did not lead to formation of NO or to hydroxylation of the substrate, contrary to reactions with BuGua or L-arg. Our results reveal how different structural and chemical properties of NOS substrate analogues can impact on the kinetics and reactivity of the Fe(II)O2 intermediate, and support an important role for substrate pKa during NOS oxygen activation.

Animals↗
PubMed2005

Reduction of ferric haemoproteins by tetrahydropterins: a kinetic study.

We previously showed that one-electron transfer from tetrahydropterins to iron porphyrins is a very general reaction, with formation of an intermediate cation radical similar to the one detected in NO synthase. As a model reaction, the rates of reduction of eight haemoproteins by diMePH4 (6,7-dimethyltetrahydropterin) have been studied and correlated with their one-electron reduction potentials, E(m) (Fe(III)/Fe(II)). On the basis of kinetic data analyses, a bimolecular collisional mechanism is proposed for the electron transfer from diMePH4 to ferrihaemoproteins. Haemoproteins with reduction potentials below -160 mV were shown not to be reduced by diMePH4 to the corresponding ferrohaemoproteins. For haemoproteins with reduction potentials more positive than -160 mV, such as chloroperoxidase, cytochrome b5, methaemoglobin and cytochrome c, there was a good correlation between the second-order reduction rate constant and the redox potential, E(m) (Fe(III)/Fe(II)): [formula: see text]. The rate of reduction of cytochrome c by BH4 [(6R)-5,6,7,8-tetrahydrobiopterin] was determined to be similar to that of the reduction of cytochrome c by diMePH4. These results confirm the role of tetrahydropterins as one-electron donors to Fe(III) porphyrins.

Chloride Peroxidase↗
PubMed2005

First evidence that cytochrome P450 may catalyze both S-oxidation and epoxidation of thiophene derivatives.

Oxidation of 2-phenylthiophene (2PT) by rat liver microsomes, in the presence of NADPH and glutathione (GSH), led to three kinds of metabolites whose structures were established by 1H NMR and mass spectrometry. The first ones were 2PT-S-oxide dimers formed by Diels-Alder type dimerization of 2PT-S-oxide, while the second ones were GSH adducts derived from the 1,4-Michaël-type addition of GSH to 2PT-S-oxide. The third metabolites were GSH adducts resulting from a nucleophilic attack of GSH to the 4,5-epoxide of 2PT. Oxidation of 2PT by recombinant, human cytochrome P4501A1, in the presence of NADPH and GSH, also led to these three kinds of metabolites. These results provide the first evidence that cytochrome P450 may catalyze the oxidation of thiophene compounds with the simultaneous formation of two reactive intermediates, a thiophene-S-oxide and a thiophene epoxide.

Animals↗
PubMed2005

Nomega-hydroxy-L-arginine homologues and hydroxylamine as nitric oxide-dependent vasorelaxant agents.

Endothelium-independent relaxant activities of N(omega)-hydroxy-L-arginine (L-NOHA) homologues and hydroxylamine, a possible intermediate in nitric oxide (NO) formation, were examined in rat aortic rings. Addition of one -CH(2)- group to the -(CH(2))(x)- chain between the alpha-amino acid and the hydroxyguanidine group (x=4) almost abolished-while deletion of one or two -CH(2)- (x=1 or 2) enhanced-the relaxant activity of L-NOHA homologues. N(omega)-hydroxy-nor-L-arginine- (x=2) and hydroxylamine-induced relaxations were blunted by a NO scavenger and by inhibitors of the guanylyl cyclase pathway, but not by NO synthase or cytochrome P(450) inhibitors (except 7-ethoxyresorufin). However, aortic NO formation was detected (using electron paramagnetic resonance) in the presence of concentrations of these compounds higher than those producing relaxation. These findings support the view that endothelium-independent vasorelaxations induced by both L-NOHA homologues with a required chain length x</=3 and hydroxylamine are mediated by NO-dependent activation of guanylyl cyclase, through a 7-ethoxyresorufin-inhibited mechanism.

Animals↗
PubMed2005

Synthesis and evaluation of new omega-borono-alpha-amino acids as rat liver arginase inhibitors.

Recent studies have demonstrated that arginase plays important roles in pathologies such as asthma or erectile dysfunctions. We have synthesized new omega-borono-alpha-amino acids that are analogues of the previously known arginase inhibitors S-(2-boronoethyl)-l-cysteine (BEC) and 2-amino-6-boronohexanoic acid (ABH) and evaluated them as inhibitors of purified rat liver arginase (RLA). In addition to the distance between the B(OH)(2) and the alpha-amino acid functions, the position of the sulfur atom in the side chain also appears as a key determinant for the interaction with the active site of RLA. Furthermore, substitution of the alkyl side chain of BEC by methyl groups and conformational restriction of ABH by incorporation of its side chain in a phenyl ring led to inactive compounds. These results suggest that subtle interactions govern the affinity of inhibitors for the active site of RLA.

Amino Acids↗
PubMed2005

Relationship between the structure of guanidines and N-hydroxyguanidines, their binding to inducible nitric oxide synthase (iNOS) and their iNOS-catalysed oxidation to NO.

The binding of several alkyl- and aryl-guanidines and N-hydroxyguanidines to the oxygenase domain of inducible NO-synthase (iNOS(oxy)) was studied by UV/Vis difference spectroscopy. In a very general manner, monosubstituted guanidines exhibited affinities for iNOS(oxy) that were very close to those of the corresponding N-hydroxyguanidines. The highest affinities were observed for the natural substrates, L-arginine and N(omega)-hydroxy-L-arginine (K(d) at the microm level). The deletion of either the CO2H or the NH2 function of their amino acid moiety led to dramatic decreases in the affinity. However, alkylguanidines with a relatively small alkyl chain exhibited interesting affinities, the best being observed for a butyl chain (K(d) =20 microM). Arylguanidines also bound to iNOS(oxy), however, with lower affinities (K(d) > 250 microm). Many N-alkyl- and N-aryl-N'-hydroxyguanidines are oxidized by iNOS with formation of NO, whereas only few alkylguanidines led to significant production of NO under identical conditions, and all the arylguanidines tested to date were unable to lead to the production of NO. The k(cat) values of NO production from the oxidation by iNOS of the studied N-hydroxyguanidines were found to vary independently of their affinity for the protein. The k(cat) values determined for the two-step oxidation of alkylguanidines to NO were not clearly related to the K(d) of these substrates toward iNOS(oxy). However, there is a qualitative relationship between these k(cat) values and the apparent rate constants of dissociation of the complex between iNOS(oxy) and the corresponding N-alkyl-N'-hydroxyguanidine (k(off) (app)) that were determined by stopped-flow UV/Vis spectroscopy. These data indicate that a key factor for efficient oxidation of a guanidine by iNOS to NO is the ability of the corresponding N-hydroxyguanidine to bind to the active site without being too rapidly released before its further oxidation. This explains why 4,4,4-trifluorobutylguanidine is so far the best non-alpha-amino acid guanidine substrate of iNOS with formation of NO, because the k(off) (app) of the corresponding N-hydroxyguanidine is particularly low. This suggests that the rational design of guanidines as new NO donors upon in situ oxidation by NOSs should take into account both thermodynamic and kinetic characteristics of the interaction of the protein not only with the guanidine but also with the corresponding N-hydroxyguanidine.

Binding Sites↗
PubMed2004

Analysis of human cytochrome P450 2C8 substrate specificity using a substrate pharmacophore and site-directed mutants.

The structural determinants of substrate specificity of human liver cytochrome P450 2C8 (CYP2C8) were investigated using site-directed mutants chosen on the basis of a preliminary substrate pharmacophore and a three-dimensional (3D) model. Analysis of the structural features common to CYP2C8 substrates exhibiting a micromolar K(m) led to a substrate pharmacophore in which the site of oxidation by CYP2C8 is 12.9, 8.6, 4.4, and 3.9 A from features that could establish ionic or hydrogen bonds, and hydrophobic interactions with protein amino acid residues. Comparison of this pharmacophore with a 3D model of CYP2C8 constructed using the X-ray structure of CYP2C5 suggested potential CYP2C8 amino acid residues that could be involved in substrate recognition. Twenty CYP2C8 site-directed mutants were constructed and expressed in yeast to compare their catalytic activities using five CYP2C8 substrates that exhibit different structures and sizes [paclitaxel, fluvastatin, retinoic acid, a sulfaphenazole derivative (DMZ), and diclofenac]. Mutation of arginine 241 had marked effects on the hydroxylation of anionic substrates of CYP2C8 such as retinoic acid and fluvastatin. Serine 100 appears to be involved in hydrogen bonding interactions with a polar site of the CYP2C8 substrate pharmacophore, as shown by the 3-4-fold increase in the K(m) of paclitaxel and DMZ hydroxylation after the S100A mutation. Residues 114, 201, and 205 are predicted to be in close contact with substrates, and their mutations lead either to favorable hydrophobic interactions or to steric clashes with substrates. For instance, the S114F mutant was unable to catalyze the 6alpha-hydroxylation of paclitaxel. The S114F and F205A mutants were the best catalysts for retinoic acid and paclitaxel (or fluvastatin) hydroxylation, respectively, with k(cat)/K(m) values 5 and 2.1 (or 2.4) times higher, respectively, than those found for CYP2C8. Preliminary experiments of docking of the substrate into the experimentally determined X-ray structure of substrate-free CYP2C8, which became available quite recently [Schoch, G. A., et al. (2004) J. Biol. Chem. 279, 9497], were consistent with key roles for S100, S114, and F205 residues in substrate binding. The results suggest that the effects of mutation of arginine 241 on anionic substrate hydroxylation could be indirect and result from alterations of the packing of helix G with helix B'.

Amiodarone↗
PubMed2004

Alternative nitric oxide-producing substrates for NO synthases.

Nitric oxide (NO) is a key inter- and intracellular molecule involved in the maintenance of vascular tone, neuronal signaling, and host response to infection. The biosynthesis of NO in mammals involves a two-step oxidation of L-arginine (L-Arg) to citrulline and NO catalyzed by a particular class of heme-thiolate proteins, called NO-synthases (NOSs). The NOSs successively catalyze the Nomega-hydroxylation of the guanidine group of L-Arg with formation of Nomega-hydroxy-L-arginine (NOHA) and the oxidative cleavage of the CN(OH) bond of NOHA with formation of citrulline and NO. During the last decade, a great number of compounds bearing a CNH or CNOH function have been synthesized and studied as possible NO-producing substrates of recombinant NOSs. This includes derivatives of L-Arg and NOHA, N-alkyl (or aryl) guanidines, N,N'- or N,N-disubstituted guanidines, N-alkyl (or aryl) N'-hydroxyguanidines, N- (or O-) disubstituted N'-hydroxyguanidines, as well as amidoximes, ketoximes, and aldoximes. However, only those involving the NHC(NH2)=NH (or NOH) moiety have led to a significant formation of NO. All the N-monosubstituted N'-hydroxyguanidines that are well recognized by the NOS active site lead to NO with catalytic efficiences (kcat/Km) up to 50% of that of NOHA. This is the case of many N-aryl and N-alkyl N'-hydroxyguanidines, provided that the aryl or alkyl substituent is small enough to be accommodated by a NOS hydrophobic site located in close proximity of the NOS "guanidine binding site." As far as N-substituted guanidines are concerned, few compounds bearing a small alkyl group have been found to act as NO-producing substrates. The kcat value found for the best compound may reach 55% of the kcat of L-Arg oxidation. However, the best catalytic efficiency (kcat/Km) that was obtained with N-(4,4,4-trifluorobutyl) guanidine is only 100-fold lower than that of L-Arg. In a general manner, NOS II is a better catalyst that NOS I and III for the oxidation of exogenous guanidines and N-hydroxyguanidines to NO. This is particularly true for guanidines as the ones acting as substrates for NOS II have been found to be almost inactive for NOS I and NOS III. Thus, a good NO-producing guanidine substrate for the two latter isozymes remains to be found.

Alkylation↗
PubMed2004

JunD reduces tumor angiogenesis by protecting cells from oxidative stress.

Reactive oxygen species (ROS) are implicated in the pathophysiology of various diseases, including cancer. In this study, we show that JunD, a member of the AP-1 family of transcription factors, reduces tumor angiogenesis by limiting Ras-mediated production of ROS. Using junD-deficient cells, we demonstrate that JunD regulates genes involved in antioxidant defense, H2O2 production, and angiogenesis. The accumulation of H2O2 in junD-/- cells decreases the availability of FeII and reduces the activity of HIF prolyl hydroxylases (PHDs) that target hypoxia-inducible factors-alpha (HIFalpha) for degradation. Subsequently, HIF-alpha proteins accumulate and enhance the transcription of VEGF-A, a potent proangiogenic factor. Our study uncovers the mechanism by which JunD protects cells from oxidative stress and exerts an antiangiogenic effect. Furthermore, we provide new insights into the regulation of PHD activity, allowing immediate reactive adaptation to changes in O2 or iron levels in the cell.

Animals↗
PubMed2004

Inhibitor coordination interactions in the binuclear manganese cluster of arginase.

Arginase is a manganese metalloenzyme that catalyzes the hydrolysis of L-arginine to form L-ornithine and urea. The structure and stability of the binuclear manganese cluster are critical for catalytic activity as it activates the catalytic nucleophile, metal-bridging hydroxide ion, and stabilizes the tetrahedral intermediate and its flanking states. Here, we report X-ray structures of a series of inhibitors bound to the active site of arginase, and each inhibitor exploits a different mode of coordination with the Mn(2+)(2) cluster. Specifically, we have studied the binding of fluoride ion (F(-); an uncompetitive inhibitor) and L-arginine, L-valine, dinor-N(omega)-hydroxy-L-arginine, descarboxy-nor-N(omega)-hydroxy-L-arginine, and dehydro-2(S)-amino-6-boronohexanoic acid. Some inhibitors, such as fluoride ion, dinor-N(omega)-hydroxy-L-arginine, and dehydro-2(S)-amino-6-boronohexanoic acid, cause the net addition of one ligand to the Mn(2+)(2) cluster. Other inhibitors, such as descarboxy-nor-N(omega)-hydroxy-L-arginine, simply displace the metal-bridging hydroxide ion of the native enzyme and do not cause any net change in the metal coordination polyhedra. The highest affinity inhibitors displace the metal-bridging hydroxide ion (and sometimes occupy a Mn(2+)(A) site found vacant in the native enzyme) and maintain a conserved array of hydrogen bonds with their alpha-amino and -carboxylate groups.

Animals↗
PubMed2004

Importance of valine 567 in substrate recognition and oxidation by neuronal nitric oxide synthase.

Nitric oxide (NO) is synthesised by a two-step oxidation of -arginine (L-Arg) in the active site of nitric oxide synthase (NOS) with formation of an intermediate, N omega-hydroxy-L-Arg (NOHA). Crystal structures of NOSs have shown the importance of an active-site Val567 residue (numbered for rat neuronal NOS, nNOS) interacting with non-amino acid substrates. To investigate the role of this Val residue in substrate recognition and NO-formation activity by nNOS, we generated and purified four Val567 mutants of nNOS, Val567Leu, Val567Phe, Val567Arg and Val567Glu. We characterized these proteins and tested their ability to generate NO from the oxidation of natural substrates L-Arg and NOHA, and from N-hydroxyguanidines previously identified as alternative substrates for nNOS. The Val567Leu mutant displayed lower NO formation activities than the wild type (WT) in the presence of all tested compounds. Surprisingly, the Val567Phe mutant formed low amounts of NO only from NOHA. These two mutants displayed lower affinity for L-Arg and NOHA than the WT protein. Val576Glu and Val567Arg mutants were much less stable and did not lead to any formation of NO. These results suggest that Val567 is an important residue for preserving the integrity of the active site, for substrate binding, and subsequently for NO-formation in nNOS.

Amino Acid Substitution↗
PubMed2003

Very general formation of tetrahydropterin cation radicals during reaction of iron porphyrins with tetrahydropterins: model for the corresponding NO-synthase reaction.

Electron transfer from tetrahydropterins to iron porphyrins, with formation of intermediate tetrahydropterin cation radicals, is a very general reaction that was shown to occur not only with tetrahydrobiopterin, as originally found in NO-synthases, but also with another important biological cofactor, tetrahydrofolate, and various iron porphyrins, either in their ferric state, or in the Fe(II)O(2) state, as in the first model of the corresponding NO-synthase reaction described in this paper.

Journal Article↗
PubMed2003

Rabbit aorta converts 15-HPETE to trihydroxyeicosatrienoic acids: potential role of cytochrome P450.

Previous work showed that rabbit aorta metabolizes arachidonic acid via 15-lipoxygenase to 15-hydroperoxyeicosatetraenoic acid (15-HPETE), which undergoes an enzymatic rearrangement to 11-hydroxy-14,15-epoxyeicosatrienoic acid (11-H-14,15-EETA) and 15-hydroxy-11,12-epoxyeicosatrienoic acid (15-H-11,12-EETA). Hydrolysis of the epoxy group results in the formation of 11,14,15- and 11,12,15-trihydroxyeicosatrienoic acids (THETAs). Endothelial cells have several heme-containing enzymes including cytochromes P450 (CYP), nitric oxide synthase (eNOS), and prostacyclin (PGI(2)) synthase that catalyze the rearrangement of 15-HPETE to HEETAs. Incubation of arachidonic acid and 15-lipoxygenase, or 15-HPETE with rabbit aortic microsomes or rat liver microsomes, a rich source of CYP, resulted in the formation of a product that comigrated with THETAs and HEETAs on HPLC. Immunoblot analysis showed the presence of CYP2C8 and CYP2J2 in aortic tissue and when CYP2J2 or CYP2C8 was incubated with arachidonic acid and 15-lipoxygenase, the major products were 11,12,15- and 11,14,15-THETAs. Incubation of purified hematin, CYP2C11, eNOS or PGI(2) synthase enzymes with arachidonic acid and 15-lipoxygenase produced a different pattern of metabolites from rabbit aortic microsomes. Clotrimazole, a non-specific CYP inhibitor, and ebastine and terfenadone, specific CYP2J2 inhibitors, blocked the ability of aortic microsomes to produce THETAs while specific inhibitors of PGI(2) synthase, eNOS or CYP2C8/2C9 had no effect on THETA production. We suggest that a CYP, possibly CYP2J2, may function as the hydroperoxide isomerase converting 15-HPETE to HEETAs in rabbit vascular tissue. Further hydrolysis of the epoxy group of the HEETAs results in the formation of 11,12,15- and 11,14,15-THETAs. The HEETAs and THETAs are both vasodilators and may function as important regulators of vascular tone.

Animals↗
PubMed2003

Structure of mammalian cytochrome P450 2C5 complexed with diclofenac at 2.1 A resolution: evidence for an induced fit model of substrate binding.

The structure of the anti-inflammatory drug diclofenac bound in the active site of rabbit microsomal cytochrome P450 2C5/3LVdH was determined by X-ray crystallography to 2.1 A resolution. P450 2C5/3LVdH and the related enzyme 2C5dH catalyze the 4'-hydroxylation of diclofenac with apparent K(m) values of 80 and 57 microM and k(cat) values of 13 and 16 min(-1), respectively. Spectrally determined binding constants are similar to the K(m) values. The structure indicates that the pi-electron system of the dichlorophenyl moiety faces the heme Fe with the 3'- and 4'-carbons located 4.4 and 4.7 A, respectively, from the Fe. The carboxyl moiety of the substrate is hydrogen bonded to a cluster of waters that are also hydrogen bonded to the side chains of N204, K241, S289, and D290 as well as the backbone of the protein. The proximity of the diclofenac carboxylate to the side chain of D290 together with an increased binding affinity at lower pH suggests that diclofenac is protonated when bound to the enzyme. The structure exhibits conformational changes indicative of an adaptive fit to the substrate reflecting both the hydration and size of the substrate. These results indicate how structurally diverse substrates are recognized by drug-metabolizing P450 enzymes.

Animals↗