A review of the effects of manipulation of the cysteine residues of rat aryl sulfotransferase IV.

Aryl sulfotransferase IV from rat liver has the broad substrate range that is characteristic of the enzymes of detoxication. With the standard assay substrates, 4-nitrophenol and 3'-phosphoadenosine 5'-phosphosulfate (PAPS), sulfation is optimum at pH 5.4 whereas the reaction is minimal in the physiological pH range. These properties preclude a physiological function for this cytosolic enzyme. Partial oxidation of the enzyme, however, results not only in an increase in the rate of sulfation but also in a shift of the pH optimum to the physiological pH range. The mechanism for this dependence on the redox environment involves oxidation at Cys66, the cysteine residue that is conserved throughout the phenol sulfotransferase family. As documented by mass spectroscopic methods, oxidation by GSSG leads to the formation of an internal disulfide between Cys66 and Cys232; for mutants at Cys232, the oxidation product is a mixed disulfide of Cys66 and glutathione. Both of these disulfide species activate the enzyme and allow it to function at a pH optimum in the physiological range. The activated enzyme differs from the reduced form by a more circumscribed substrate spectrum. All five mutants, in which each of the cysteines of the sulfotransferase subunit have been changed to serine, are catalytically active. Only Cys66 is required for the redox response.

Intramolecular isotope effects for benzylic hydroxylation of isomeric xylenes and 4,4'-dimethylbiphenyl by cytochrome P450: relationship between distance of methyl groups and masking of the intrinsic isotope effect.

Intramolecular isotope effects associated with the benzylic hydroxylation of a series of selectively deuterated isomeric xylenes and 4,4'-dimethylbiphenyl as catalyzed by various rat liver microsomal preparations and CYP2B1 were determined. Substrate analogs in which each methyl group contained either one (d2 substrates) or two (d4 substrates) deuterium atoms were used to determine the intrinsic isotope effect for the reaction. Specific values of the individual primary (P) and secondary isotope effects (S) were determined. P ranged from a low of 5.32 +/- 0.48 to a high of 7.57 +/- 0.42 depending upon the specific cytochrome P450 preparation used for catalysis. S had an average value of 1.03. The d3 substrates allowed exploration of the effect of distance on the magnitude of the observed isotope effect. The results indicate that the distance of 6.62 A that separates the carbon atoms of the para methyl groups of p-xylene is insufficient to suppress (mask) the intrinsic isotope effect for benzylic hydroxylation by all of the enzyme preparations examined. Conversely, a distance of 11.05 A, the minimal separation between the carbon atoms of the para methyl groups of p,p'-dimethylbiphenyl, is large enough to almost completely mask the intrinsic isotope effect for benzylic hydroxylation by the same set of enzymes.

Control of activity through oxidative modification at the conserved residue Cys66 of aryl sulfotransferase IV.

Oxidation at Cys66 of rat liver aryl suflotransferase IV alters the enzyme's catalytic activity, pH optima and substrate specificity. Although this is a cytosolic detoxification enzyme, the pH optimum for the standard assay substrate 4-nitrophenol is at pH 5.5; upon oxidation, the optimum changes to the physiological pH range. The principal effect of the change in pH optimum is activation, which is manifest by an increase in K'cat without any major influence on substrate binding. In contrast, with tyrosine methyl ester as a substrate, the enzyme's optimum activity occurs at pH 8.0; upon oxidation, it ceases to be a substrate at any pH. The presence of Cys66 was essential for activation to occur, thereby providing a putative reason underlying the conserved nature of this cysteine throughout the phenol sulfotransferase family. Mapping of disulfides by mass spectrometry showed the critical event to be the oxidation of Cys66 to form a disulfide with either Cys232 or glutathione, either one is effective. These results point to a mechanism for regulating the activity of a key enzyme in xenobiotic detoxication during cellular oxidative stress.

Substrate probe for the mechanism of aromatic hydroxylation catalyzed by cytochrome P450.

The effect of branch pathways on the observed intramolecular isotope effect and deuterium retention associated with 6- and 7-hydroxylation of selectively monodeuterated (R)- and (S)-warfarin with cytochrome P450 (CYP) 2C9 and CYP1A2 were studied. cDNA-expressed CYP2C9 was incubated with enantiomerically pure (S)-7d1- and (S)-6d1-warfarin, and expressed CYP1A2 was incubated with enantiomerically pure (R)-7d1- and (R)-6d1-warfarin. A high degree of deuterium retention was observed in all metabolites, independent of the stereochemistry of the substrate or CYP isoform. No deuterium kinetic isotope effect was observed for the formation of 6-hydroxy- or 7-hydroxywarfarin in the case of the (S)-6d1-warfarin metabolism by CYP2C9, or for the formation of 6-hydroxy-, 7-hydroxy-, and 8-hydroxywarfarin in the case of the (R)-6d1-warfarin metabolism by CYP1A2. Deuterium isotope effects of 1.17 and 1.23 accompanied formation of 7-hydroxywarfarin from (S)-7d1-warfarin by CYP2C9 and from (R)-7d1-warfarin by CYP1A2, respectively. These observations are consistent with the addition-rearrangement pathway for aromatic hydroxylation, in which a triplet-like active oxygen species initially adds to the pi system, resulting in a tetrahedral intermediate. The intermediate subsequently rearranges to generate the phenol, the final product of the reaction.

Inactivation of horseradish peroxidase by 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine.

In the presence of H2O2, horseradish peroxidase (HRP) catalyzes the one-electron oxidation of a porphyrinogenic agent, 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine (DDEP), leading to formation of an ethyl radical, inactivation of the enzyme, and formation of an altered heme product. The loss of heme during the inactivation of HRP was dependent on the duration of exposure to DDEP as well as the concentration of H2O2 and DDEP. The pseudo first order rate constant for the oxidation of DDEP by compound I of HRP at pH 7.4 was 0.07 min-1, and the maximal extent of heme loss was 35%. The altered heme product, which was isolated by reverse phase HPLC, was characterized by the use of mass and 1H NMR spectrometry as a substitution product of a C2H4OH moiety for a meso proton of the prosthetic heme [meso-(hydroxyethyl)heme]. The source of the oxygen in the C2H4OH moiety appeared not to be H2O2 or H2O as 18O was not incorporated in the heme adduct when H2(18)O2 or H2(18)O was used. The DDEP-mediated reaction did not form the expected delta-meso-ethylheme adduct analogous to the ethyl radical-mediated inactivation of HRP by ethylhydrazine (EH) [Ator et al. (1987) J. Biol. Chem. 262, 14954-14960]. However, we have found that meso-(hydroxyethyl)heme was formed in the EH-mediated reaction, albeit in apparently lower amounts than delta-meso-ethylheme. Perhaps the proximity of the heme to the ethyl radical may play a role in determining the nature of the heme products formed.

pH-dependent one- and two-electron oxidation of 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine catalyzed by horseradish peroxidase.

The porphyrinogenic agent 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine (DDEP) is known to inactivate hepatic cytochrome P450 (P450) enzymes 2C11, 2C6, and 3A1 [Correia et al. (1987) Arch. Biochem. Biophys. 258, 436-451] by different mechanisms. The inactivation of P450 2C11 and 2C6 appears to be due to the ethylation of the heme in the active sites of the enzymes [Augusto et al. (1982) J. Biol. Chem. 257, 11288-11295], whereas the inactivation of P450 3A1 appears to involve the covalent binding of the heme to the apoprotein [Correia et al. (1987)]. Moreover, we have found that DDEP inactivates horseradish peroxidase (HRP) pretreated with hydrogen peroxide. In this system, DDEP was oxidized predominately to 3,5-dicarbethoxy-2,6-dimethyl-4-ethylpyridine (EDP) under weakly acidic conditions and predominately to 3,5-dicarbethoxy-2,6-dimethylpyridine (DP) under basic conditions. The loss of heme and the formation of altered heme products were also pH-dependent and were correlated with the formation of DP and the inactivation of HRP. Thus the inactivation of HRP appears to depend on the formation of an ethyl radical, which presumably reacts with the heme in the active site of the enzyme. Similar product ratios were obtained for the oxidation of DDEP by K3Fe(CN)6, indicating that product ratios of DP over EDP are mainly determined by the pH of buffer. These results, in addition to semiemperical calculations (AM1) for the oxidation of DDEP in the gas phase, are consistent with the idea that the inhibitor undergoes a single-electron oxidation to form the DDEP radical cation, the fate of which depends on the environment of the active site of the enzyme. The proposed formation of a radical cation by the abstraction of an electron from nitrogen is consistent with the finding of low intramolecular isotope effects of the metabolism of 3,5-dicarbethoxy-2,6-dimethyl-[4-2H,4-1H]-1,4-dihydropyridine by P450 2C11 and 3A4. Under basic or aprotic conditions, the radical dissociates to form DP and the ethyl radical, which reacts with the heme, thereby inactivating the enzyme. Under acidic or polar conditions, the radical undergoes an additional one-electron oxidation to form EDP.(ABSTRACT TRUNCATED AT 400 WORDS)

Reaction kinetics for nitrosation of cysteine and glutathione in aerobic nitric oxide solutions at neutral pH. Insights into the fate and physiological effects of intermediates generated in the NO/O2 reaction.

The critical regulatory function of nitric oxide (NO) in many physiologic processes is well established. However, in an aerobic aqueous environment NO is known to generate one or more reactive and potentially toxic nitrogen oxide (NOx) metabolites. This has led to the speculation that mechanisms must exist in vivo by which these reactive intermediates are detoxified, although the nature of these mechanisms has yet to be elucidated. This report demonstrates that among the primary bioorganic products of the reaction of cellular constituents with the intermediates of the NO/O2 reaction are S-nitrosothiol (S-NO) adducts. Anaerobic solutions of NO are not capable of nitrosating cysteine or glutathione, while S-NO adducts of these amino acids are readily formed in the presence of O2 and NO. Investigation of the kinetics for the formation of these S-NO adducts has revealed a rate equation of d[RSNO]/dt = kSNO[NO]2[O2], where kSNO = (6 +/- 2) x 10(6) M-2S-1, a value identical to that for the formation of reactive intermediates in the autoxidation of NO. Competition studies performed with a variety of amino acids, glutathione, and azide have shown that cysteine residues have an affinity for the NOx species that is 3 orders of magnitude greater than that of the nonsulfhydryl amino acids, and > 10(6) times greater than that of the exocyclic amino groups of DNA bases. The dipeptide alanyltyrosine reacts with the intermediates of the NO/O2 reaction with an affinity 150 times less than that of the sulfhydryl-containing compounds. Furthermore, Chinese hamster V79 lung fibroblasts depleted of glutathione display enhanced cytotoxicity on exposure to NO.(ABSTRACT TRUNCATED AT 250 WORDS)

Theoretical kinetics of sequential metabolism in vitro. Study of the formation of 16 alpha-hydroxyandrostenedione from testosterone by purified rat P450 2C11.

P450 2C11 from rat liver is known to metabolize testosterone to 2 alpha-, 16 alpha-, and 6 beta-hydroxytestosterone, and to androstenedione and 16 alpha-hydroxyandrostenedione. Because Waxman (J. Biol. Chem. 259, 15481-15490) has reported that the enzyme converts androstenedione to 16 alpha-hydroxyandrostenedione, it seemed likely that the metabolite was formed from testosterone by way of androstenedione. Indeed, we have found that P450 2C11 does not convert 16 alpha-hydroxytestosterone to 16 alpha-hydroxyandrostenedione to any significant extent and, therefore, that the metabolite is formed from testosterone almost solely by way of androstenedione. To determine whether some of the 16 alpha-hydroxyandrostenedione might be formed directly from the androstenedione-enzyme complex, we developed an approach by which it is possible to calculate the amount of the androstenedione, released into the medium, relative to the amount of the androstenedione-enzyme complex that is converted directly to 16 alpha-hydroxyandrostenedione under initial conditions when the concentration of released androstenedione will be negligible. The approach uses two factors: factor A is the androstenedione/(androstenedione + 16 alpha-hydroxyandrostenedione) present at the end of the incubation, and factor B corrects for the amount of released androstenedione that recombines with the enzyme and is converted to 16 alpha-hydroxyandrostenedione. Although the values of both factors A and B will vary with the concentrations of testosterone and preformed androstenedione present in the incubation mixtures and with the duration of incubation, the value of A*B will be independent of these parameters.(ABSTRACT TRUNCATED AT 250 WORDS)

Theory for the observed isotope effects on the formation of multiple products by different kinetic mechanisms of cytochrome P450 enzymes.

Cytochrome P450 systems are unusual in that many of them can convert a substrate to a number of different metabolites. Several kinetic mechanisms may be envisioned by which the metabolites may be formed. In each of the mechanisms, the substrate combines with the enzyme in different orientations to form a set of (ES) complexes that then are activated to a set of (EOS) complexes. The fate of these (EOS) complexes determines the kinetic mechanism. In the "parallel pathway" mechanism, the (EOS) complexes are so stable and rigid they cannot be converted either directly or indirectly to complexes with different orientations; the orientation of the (ES) complexes thus determines which metabolite will be formed. In the "nondissociative" mechanisms, the complexes are not rigid; instead they undergo interconversion while the substrate remains in the active site of the enzyme. In the "dissociative" mechanisms, the (EOS) complexes dissociate to (EO) and (S), but recombine to form (EOS) complexes with either the same or different orientations. Steady-state equations describing the deuterium isotope effects for these kinetics mechanisms have been derived and solved for competitive experiments, in which equal concentrations of both deuterated and nondeuterated substrates are present in incubation mixtures, and for noncompetitive experiments, in which only one of the substrates is present. The equations reveal that comparisons of the isotope effects on the formation of a metabolite by a pathway that does not involve the abstraction of a deuterium from a deuterated substrate (the non-deuterium abstraction pathway) in both experiments can differentiate between the kinetic mechanisms. A value of 1.0 for (v)H/(v)D in the competitive experiment, but < 1.0 to > 1.0 for the value of (Vmax/Km)H/(Vmax/Km)D in the noncompetitive experiment, is diagnostic for the "dissociative" mechanisms. Values of 1.0 in both kinds of experiments are diagnostic for the "parallel pathway" mechanism. Values of < 1.0 in both types of experiments are diagnostic for the "nondissociative" mechanisms. The equations also predict possible unusual substrate-inhibitor interactions when the "dissociative" mechanism is operative.

Deuterium isotope effects on A-ring and D-ring metabolism of testosterone by CYP2C11: evidence for dissociation of activated enzyme-substrate complexes.

Cytochrome P450 systems are unusual in that many of them can convert a substrate to a number of different metabolites by several possible kinetic mechanisms. Steady-state equations describing the deuterium isotope effects for mechanisms in which different orientations of the substrate relative to the perferryl oxygen in the active site of the enzyme are achieved before a hydrogen (or possibly an electron) is abstracted have been derived and solved (Gillette et al., 1994). These equations have been used to elucidate the kinetic mechanisms by which CYP2C11 converts testosterone to 2 alpha-hydroxytestosterone on the one hand and 16 alpha-hydroxytestosterone and androstenedione on the other. We have synthesized testosterone-2,2,4,6,6-2H5 and compared its metabolism by CYP2C11 with that of nondeuterated testosterone. In this system, deuterated 2 alpha-hydroxytestosterone would be formed by a deuterium abstraction pathway via the active oxygen intermediate (EOSw) and the D-ring metabolites would be formed by non-deuterium abstraction pathways from active oxygen intermediates represented by (EOSx). The results revealed that testosterone in the activated enzyme-substrate complexes, (EOSw) and (EOSx), does not change orientations while it is in the active site of CYP2C11. Instead, two of the noncompetitive experiments indicated that testosterone is able to dissociate from the (EOS) complexes and reassociate in either the same or different orientations. A third noncompetitive experiment suggested that testosterone in the (EOS) complexes does not change orientations while it is in the active site of CYP2C11, nor does it dissociate from the (EOS) complexes; instead, the pattern of metabolite formation is governed almost solely by the orientation of testosterone in the (ESw) and ESx) complexes.

Differential susceptibilities of the prosthetic heme of hemoglobin-based red cell substitutes. Implications in the design of safer agents.

One approach to the development of an effective red cell substitute has been chemical modification of human hemoglobin to optimize oxygen transport and plasma half-life. Human hemoglobin A0 and two of these modified hemoglobins, one prepared from the cross-linking of the alpha-chains at lysine residue 99 by bis(3,5-dibromosalicyl)fumarate (Hb-DBBF) and the other by acylation of lysine residue 82 of the beta-chain by mono-(3,5-dibromosalicyl)fumarate (Hb-FMDA), were tested by HPLC for their susceptibility to oxidative damage caused by H2O2. Such oxidative insult may occur during ischemia and reperfusion of tissues after transfusion of red cell substitutes to patients with hypovolemic shock and trauma. Hb-DBBF was extremely susceptible to damage of its heme and protein moieties with stoichiometric amounts of H2O2, whereas Hb-FMDA was highly resistant, even at 10-fold molar excess and at an acidic pH of 4.7. Hemoglobin A0 was of intermediate susceptibility, exhibiting alteration of heme and protein moieties at acidic but not neutral pH. Since the degradation of heme can release the potentially toxic agent iron, Hb-FMDA may be a more promising candidate than Hb-DBBF for development as a red cell substitute. A similar approach may be used to assess the susceptibility of other hemoglobin-based red cell substitutes to oxidative damage in order to determine the molecular basis of heme and protein alteration.

Metabolism-based transformation of myoglobin to an oxidase by BrCCl3 and molecular modeling of the oxidase form.

The stoichiometric reductive debromination of BrCCl3 to a trichloromethyl radical by myoglobin caused the prosthetic heme to become covalently cross-linked to the protein moiety and transformed myoglobin from an oxygen storage protein to an oxidase. This was shown in experiments in which oxygen consumption was measured during redox cycling of the altered myoglobin in the presence of ascorbate or an enzymatic reducing system containing diaphorase and NADH. Redox cycling eventually led to loss of the protein-bound heme adduct and oxidase activity of myoglobin. We have used molecular modeling and the known structure of the protein-bound heme adduct to identify probable mechanisms for transformation of myoglobin to an oxidase. Based on these modeling studies, the most likely structure of the experimentally observed adduct involves ligation to the heme iron of the epsilon-nitrogen atom of histidine 97 and/or that of histidine 64. The model structures revealed access of solvent to the heme active site, which could facilitate oxygen reduction. The transformation of myoglobins and perhaps other hemoproteins to oxidases may have toxicological importance in causing the tissue damage resulting from exposure to various xenobiotics and endogenous chemicals as well as explaining how hemoproteins are inactivated during catalysis.

Reactions of the bioregulatory agent nitric oxide in oxygenated aqueous media: determination of the kinetics for oxidation and nitrosation by intermediates generated in the NO/O2 reaction.

The reaction kinetics of nitric oxide autoxidation in aerobic solutions were investigated by direct observation of the nitrite ion product and by trapping the strongly oxidizing and nitrosating intermediates formed in this reaction. The rate behavior observed for nitrite formation [rate = k3[O2][NO]2, k3 = (6 +/- 1.5) x 10(6) M-2 s-1 at 22 degrees C] was the same as found for oxidation of Fe(CN)6(4-) and of 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) and as for the nitrosation of sulfanilamide. There was a slight decrease in k3 to (3.5 +/- 0.7) x 10(6) M-2 s-1 at 37 degrees C. The second-order dependency for NO was observed at NO concentrations as low as 3 microM. The results of the competitive kinetics studies suggest that the key oxidizing intermediates, species which are both strong oxidants and nitrosating agents, are not one of those commonly proposed (NO2, N2O3, NO+, or O2NO-) but are one or more as yet uncharacterized NOx species.

Stereoselectivity of the aliphatic hydroxylation of 6-n-propylchromone-2-carboxylic acid in rat and guinea pig.

Following administration of 6-n-propylchromone-2-carboxylic acid (6-n-PCCA) (500 mumol/kg) to male rats, three metabolic products were detected and isolated from the 0-24 h urine. All were identified as resulting from oxidation exclusively along the 6-n-propyl moiety. Some 66% of the dose was excreted in the 0-24 h urine, 55% of which was 6-PCCA, with 15% as (6-1'-hydroxypropyl)chromone-2-carboxylic acid (6-1'-HPCCA), 22% as 6-(2'-hydroxypropyl)chromone-2-carboxylic acid (6-2'-HPCCA), and 4% as 6-3'-carboxypropyl)chromone-2-carboxylic acid (6-3'-CPCCA). Derivatization of the methyl esters of the hydroxylated metabolites with S-alpha-methoxy-alpha-(trifuloromethyl)-phenylacetyl chloride (Mosher's reagent) allowed the evaluation of urinary enantiomeric composition by HPLC and assignment of their absolute configurations by NMR. This was found to be 90:10 (R/S) for 6-2'-HPCCA, and 7:93 (R/S) for 6-1'-HPCCA. When rats were dosed with the racemic 1'- and 2-hydroxy metabolites; no stereoselective metabolism or excretion was observed. Administration of 6-n-PCCA to male guinea pigs revealed that this species was unable to metabolise this compound.

Inhibition of cytochromes P450 by nitric oxide and a nitric oxide-releasing agent.

The effect of nitric oxide (NO) on cytochrome P450-mediated benzyloxyresorufin and ethoxyresorufin O-dealkylase activity of rat hepatic postmitochondrial (S-9) or microsomal subfractions, or purified rat liver CYP2B1, was examined. Two distinct inhibitory phases were observed regardless of whether the NO was added prior to initiation of the reactions with NADPH or during the course of substrate turnover. The first was a reversible inhibition characterized by complete cessation of catalytic activity, the duration of which was NO concentration-dependent with an IC50 in the range of 8-60 microM. This phase was followed by a second, irreversible, inhibitory phase characterized by a varying extent of recovery of activity, with inhibition ranging from < 1 to approximately 100%. The extent of this diminution in substrate conversion rate was also NO concentration-dependent, with an IC50 in the range of 13-72 microM, and could be partially abrogated by the inclusion of bovine serum albumin in the reaction mixture. Lower IC50 values for both inhibitory phases were obtained in the case of benzyloxyresorufin O-dealkylase activity than in the case of ethoxyresorufin O-dealkylase activity, suggesting a differential susceptibility to inhibition by NO for the two O-dealkylase activities. A nitric oxide-releasing compound ([Et2NN(O)NO]Na,DEA/NO) caused qualitatively similar inhibitory effects on benzyloxyresorufin O-dealkylase activity when added prior to the initiation of the reactions with NADPH, or during the course of substrate turnover. Based upon these results, it is possible that NO may play a role in the regulation of P450 activity in vivo.

12 alpha-hydroxytestosterone. A hitherto unidentified testosterone metabolite produced by cytochrome P-450 2A2.

Several cytochrome P-450 enzymes are able to hydroxylate testosterone to many different metabolites, some of which remain to be identified. One hitherto unidentified metabolite represents a major metabolite of P-450 2A2, present in the liver of adult male rats, and a minor metabolite of several other cytochrome P-450 enzymes. Using the techniques of HPLC and GC/MS, we have obtained unequivocal evidence that this metabolite is 12 alpha-hydroxytestosterone.