A convenient assay for mephenytoin 4-hydroxylase activity of human liver microsomal cytochrome P-450

A simple and rapid method for the determination of (S)-mephenytoin 4-hydroxylase activity by human liver microsomal cytochrome P-450 has been developed. [Methyl-14C] mephenytoin was synthesized by alkylation of S-nirvanol with 14CH3I and used as a substrate. After incubation of [methyl-14C]mephenytoin with human liver microsomes or a reconstituted monooxygenase system containing partially purified human liver cytochrome P-450, the 4-hydroxylated metabolite of mephenytoin was separated by thin-layer chromatography and quantified. The formation of the metabolite depended on the incubation time, substrate concentration, and cytochrome P-450 concentration and was found to be optimal at pH 7.4. The Km and Vmax rates obtained with a human liver microsomal preparation were 0.1 mM and 0.23 nmol 4-hydroxymephenytoin formed/min/nmol cytochrome P-450, respectively. The hydroxylation activity showed absolute requirements for cytochrome P-450, NADPH-cytochrome P-450 reductase, and NADPH in a reconstituted monooxygenase system. Activities varied from 5.6 to 156 pmol 4-hydroxymephenytoin formed/min/nmol cytochrome P-450 in 11 human liver microsomal preparations. The basic system utilized for the analysis of mephenytoin 4-hydroxylation can also be applied to the estimation of other enzyme activities in which phenol formation occurs.     

Hydroxylation of aniline and aminoantipyrine derivatives (1-phenyl-2,3-dimethylaminopyrazolon-5) in liver endoplasmic reticulum]

The absence of correlation between the effect of aniline and aminoantipyrine derivatives on cytochrome P-450 reduction rate and its oxidation rate draw to the conclusion that the reductase reaction is not a limiting step of hydroxylation for all substrates. Km is found to be directly proportional to Vmax of hydroxylated substrates. Hence, in these reactions the Km value is determined not by the value Ks but by the kappa+2/kappa+1 ratio. Km is not a characteristic of the affinity of cytochrome P-450 to substrates. The calculations were made to show that cytochrome P-450 formed two types of the enzyme-substrate complexes containing one or two substrate molecules. The complex in which one molecule of cytochrome P-450 binds one substrate molecule is considered to be active.     

Role of electrostatic interactions in the reaction of NADPH-cytochrome P-450 reductase with cytochromes P-450

Chemical modification of cytochrome P-450 reductase was used to determine the involvement of charged amino acids in the interaction between the reductase and two forms of cytochrome P-450. Acetylation of 11 lysine residues of the reductase with acetic anhydride yielded a 20-40% decrease in the apparent Km of the reductase for cytochrome P-450b or cytochrome P-450c using either 7-ethoxycoumarin or benzphetamine as substrates. A 20-45% decrease in the Vmax was observed except for cytochrome P-450b with 7-ethoxycoumarin as substrate, where there was a 27% increase. Modification of carboxyl groups on the reductase with 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC) and methylamine, glycine methyl ester, or taurine as nucleophiles inhibited the interaction with the cytochromes P-450. We were able to modify 4.0, 7.9, and 5.9 carboxyl groups using methylamine, glycine methyl ester, or taurine, respectively. The apparent Km for cytochrome P-450c or cytochrome P-450b was increased 1.3- to 5.2-fold in a reconstituted monooxygenase assay with 7-ethoxycoumarin or benzphetamine as substrate. There were varied effects on the Vmax. There was no significant change in the conformation of the reductase upon chemical modification with either acetic anhydride or EDC. These results strongly suggest that electrostatic interactions as well as steric constraints play a role in the binding and electron transfer step(s) between the reductase and cytochrome P-450.     

Interaction between NADPH-cytochrome P-450 reductase and cytochrome P-450 in the membrane of phosphatidylcholine vesicles.

Cytochrome P-450 and NADPH-cytochrome P-450 REDUctase, both purified from liver microsomes of phenobarbital-pretreated rabbits, have been incorporated into the membrane of phosphoaditylcholine vesicles by the cholate dialysis method. The reduction of cytochrome P-450 by NADPH in this system is biphasic, consisting of two first-order reactions. The rate constant of the fast phase, in which 80--90% of the total cytochrome is reduced, increases as the molar ratio of the reductase to the cytochrome is increased at a fixed ratio of the cytochrome to phosphatidylcholine, suggesting that the rate-limiting step of the fast phase is the interaction between the reductase and the cytochrome. The rate constant of the fast phase also increases when the amount of phosphatidylcholine, relative to those of the two proteins, is decreased. This latter observation suggests that the interaction between the two proteins is effected by their random collision caused by their lateral mobilities on the plane of the membrane of phosphatidylcholine vesicles. The rate constant of the slow phase as well as the fraction of cytochrome P-450 reducible in the slow phase, on the other hand, remains essentially constant even upon alteration in the ratio of the reductase to the cytochrome or in that of the two proteins to phosphatidylcholine. No satisfactory explanation is as yet available for the cause of the slow-phase reduction of cytochrome P-450. The overall activity of benzphetamine N-demethylation catalyzed by the reconstituted vesicles responds to changes in the composition of the sysTEM IN A SIMILAR WAY TO THE FAST-PHASE REDUCTION OF CYTOCHROME P-450, though the latter is not the rate-limiting step of the overall reaction.     

Single turnover studies with oxy-cytochrome P-450cam

The catalytic step of bacterial cytochrome P-450cam, i.e., the step of the reaction cycle in which the product 5-exo-hydroxycamphor is formed and released by the enzyme, has been studied by stopped-flow spectrophotometry. Our approach has been to observe a single-turnover reaction between reduced putidaredoxin and oxygenated camphor-bound cytochrome P-450cam. Multiple turnovers are prevented by using the inhibitor metyrapone to trap the cytochrome after product release, which prevents binding of another camphor molecule. The time course of the reaction has been measured at several wavelengths and has been found to be biphasic. The relatively slow second phase of the reaction is the reduction of ferric, metyrapone-bound cytochrome P-450cam. The first phase coincides with the formation of product stoichiometrically with cytochrome P-450cam, as measured by gas chromatography. A detailed kinetic study of the first phase reveals a hyperbolic dependence of initial rate upon putidaredoxin concentration at a fixed, limiting concentration of cytochrome P-450cam. The Vmax is 53 microM per second per microM cytochrome, and the Km for putidaredoxin is 33 microM. The hyperbolic relationship between initial rate and putidaredoxin concentration supports a model in which the cytochrome rapidly binds putidaredoxin, then undergoes one or more slower intracomplex steps.     

Interaction between cytochrome P-450 (P-450C21) and NADPH-cytochrome P-450 reductase from adrenocortical microsomes in a reconstituted system

The interaction between P-450C21 and NADPH-cytochrome P-450 reductase, both purified from bovine adrenocortical microsomes, has been investigated in a reconstituted system with a nonionic detergent, Emulgen 913, by kinetic analysis and gel filtrations. Steady state kinetic data in progesterone 21-hydroxylation showed formation of an equimolar complex between the two enzyme proteins at low Emulgen concentration. Steady state kinetic studies on the electron transfer from NADPH to P-450C21 via the reductase showed that a stable complex formation between the two enzyme proteins was not involved in the steady state electron transfer at high Emulgen concentration. In stopped flow experiments, a time course of the P-450C21 reduction showed biphasic kinetics composed of fast and slow phases. The dependence of kinetic parameters on Emulgen concentration indicates that the fast phase corresponds to the electron transfer within the complex and the slow phase to the electron transfer through a random collision between P-450C21 and the reductase. The stable complex formation between P-450C21 and the reductase has been clearly demonstrated by gel filtration. The stable complex was composed of several molecules of the two enzyme proteins at an equimolar ratio, which was active for progesterone 21-hydroxylation and had a tendency to dissociate at high Emulgen concentration.     

Effect of KCl on the interactions between NADPH:cytochrome P-450 reductase and either cytochrome c, cytochrome b5 or cytochrome P-450 in octyl glucoside micelles.

Significant dissociation of FMN from NADPH:cytochrome P-450 reductase resulted in loss of the activity for reduction of cytochrome b5 as well as cytochrome c and cytochrome P-450. However, the ability to reduce these electron acceptors was greatly restored upon incubation of FMN-depleted enzyme with added FMN. The reductions of cytochrome c and detergent-solubilized cytochrome b5 by NADPH:cytochrome P-450 reductase were greatly increased in the presence of high concentrations of KCl, although the stimulatory effect of the salt on cytochrome P-450 reduction was less significant. No apparent effect of superoxide dismutase could be seen on the rate or extent of cytochrome reduction in solutions containing high-salt concentrations. Complex formation of the flavoprotein with cytochrome c, which is known to be involved in the mechanism of non-physiological electron transfer, caused a perturbation in the absorption spectrum in the Soret-band region of cytochrome c, and its magnitude was enhanced by addition of KCl. Similarly, an appreciable increase in ellipticity in the Soret band of cytochrome c was observed upon binding with the flavoprotein. However, only small changes were found in absorption and circular dichroism spectra for the complex of NADPH:cytochrome P-450 reductase with either cytochrome b5 or cytochrome P-450. It is suggested that the high-salt concentration allows closer contact between the heme and flavin prosthetic groups through hydrophobic-hydrophobic interactions rather than electrostatic-charge pairing between the flavoprotein and the cytochrome which causes a faster rate of electron transfer. Neither alterations in the chemical shift nor in the line width of the bound FMN and FAD phosphate resonances were observed upon complex formation of NADPH:cytochrome P-450 reductase with the cytochrome.     

3-methoxybenzidine: A potent inhibitor of cholesterol side chain cleavage cytochrome P-450

The effect of 3-methoxybenzidine on the conversion of cholesterol to pregnenolone was investigated using a reconstituted enzyme system comprised of adrenodoxin, adrenodoxin reductase and cytochrome P-450scc purified from bovine adrenal cortex. Under conditions where the cytochrome P-450scc concentration was rate-limiting, 3-methoxybenzidine was found to be a potent inhibitor, causing 50% inhibition at 7 μM when using a cholesterol concentration of 70 μM. The parent compound, benzidine, was much less effective, exhibiting an Icn value of approximately 40 μM. No effect of 3-methoxybenzidine was observed on the adrenodoxin reductase and adrenodoxin-catalyzed reduction of cytochrome c by NADPH, and it is concluded that 3-methoxybenzidine acts on cytochrome P-450scc in inhibiting cholesterol side chain cleavage.     

Mechanism of inhibition by carbonyl cyanide m-chlorophenylhydrazone and sodium deoxycholate of cytochrome P-450-catalysed hepatic microsomal drug metabolism.

1. Treatment of liver microsomal fraction with 0.03-0.12% sodium deoxycholate and 0.005-0.06 mM carbonyl cyanide m-chlorophenylhydrazone decreases phospholipid-dependent hydrophobicity of the microsomal membrane, assayed by the kinetics of 8-anilinonaphthalene-1-sulphonate binding and ethyl isocyanide difference spectra. 2. Sodium deoxycholate at a concentration of 0.01% lacks its detergent properties, but competitively inhibits aminopyrine binding and activates the initial rate of NADPH-cytochrome P-450 reductase. In the presence of 0.03-0.09% sodium deoxycholate the rate-limiting factor in p-hydroxylation of aniline is the content of cytochrome P-450. and that for N-demethylation of aminopyrine is the activity of NADPH-cytochrome P-450 reductase. 3. Carbonyl cyanide m-chlorophenylhydrazone has no effect on the binding and metabolism of aniline; investigation of its inhibiting effect on aminopyrine N-demethylase established that the rate-limiting reaction is the dissociation of the enzyme-substrate complex in the microsomal preparations. 4. In the mechanism of action of carbonyl cyanide m-chlorophenylhydrazone the key step may be the electrostatic interaction of its protonated form and one of the forms of activated oxygen at the catalytic centre of cytochrome P-450. 5. at least two different phospholipid-dependent hydrophobic zones are assumed to exist in the microsomal membrane, both coupled with cytochrome P-450. One of them reveals selective sensitivity to the protonation action of carbonyl cyanide m-chlorophenylhydrazone and contains the 'binding protein' for type I substrates and NADPH-cytochrome P-450 reductase; the other contains the cytochrome P-450 haem group and binding sites for type II substrates.     

Kinetics of elementary steps in the cytochrome P-450 reaction sequence. IV. Mechanism of the NADPH reduction reaction of cytochrome P-450LM.

The aerobic NADPH reduction of the Triton N-101 disintegrated cytochrome P-450LM system has been studied. At this organization level--the components being dispersed in solution--a first-order monophasic reaction is exhibited. Neither the complex formation of cytochrome and reductase, respectively, nor preceding diffusion is rate limiting. The initial rate follows the ratio reductase/P-450 (mole/mole) thus indicating a Michaelis-Menten type enzyme mechanism. A model treatment of the reaction fits the systems behaviour as a whole. A multiparameter equilibrium state and different specified time function equations were developed for the determination of individual step rate constants and other system parameters as well.     

Structure-function relationships of human aromatase cytochrome P-450 using molecular modeling and site-directed mutagenesis

The conversion of androgens to estrogens is catalyzed by an enzyme complex named aromatase, which consists of a form of cytochrome P-450, aromatase cytochrome P-450 (cytochrome P-450AROM), and the flavoprotein, NADPH-cytochrome P-450 reductase. As a first step toward investigation of the structure-function relationships of cytochrome P-450AROM, we have used computer modeling to align the amino acid sequence of cytochrome P-450AROM with that of cytochrome P-450CAM from Pseudomonas putida and thus create a substrate pocket using the heme-binding region and the I-helix of cytochrome P-450CAM as the template. Site-directed mutagenesis was then carried out at two sites: one at a region that aligns with the bend in the I-helix of cytochrome P-450CAM and the other at a glutamate (Glu302) just N-terminal of this bend, which is predicted to be in close proximity to the C2-position of the androstenedione substrate. To determine the importance of the former region, three mutants were constructed: A307G (Ala307----Gly), P308V (Pro308----Val), and GAGV, which changed -Ile305-Ala306-Ala307-Pro308- to -Gly-Ala-Gly-Val- (the corresponding sequence found in 17 alpha-hydroxylase cytochrome P-450). When these proteins were expressed in COS-1 cells, it was found that the activity of P308V was approximately one-third that of the wild type. These observations are consistent with the concept that Pro308 causes a bend in the I-helix of cytochrome P-450AROM, similar to that observed in cytochrome P-450CAM, which is believed to be important in forming the substrate-binding pocket. The next set of mutants were designed to determine the importance of Glu302 in catalysis. Four mutants were prepared in which Glu302 was changed either to Ala, Val, Gln, or Asp, and the activities of the expressed proteins were examined. It was found that mutations in which the carboxylic acid was replaced were essentially devoid of activity. On the other hand, changing Glu302 to Asp resulted in a two-thirds reduction in the apparent Vmax. These results support the role of a carboxylic acid residue at position 302 in the catalytic activity of cytochrome P-450AROM.     

The participation of a second molecule of adrenodoxin in cytochrome P-450-catalyzed 11beta hydroxylation.

We have utilized 11beta-hydroxylase activity and visible absorption spectrophotometry to detect possible complex formation among adrenodoxin reductase, adrenodoxin, and cytochrome P-450(11)beta. At low ionic strength, a 1:1 complex between adrenodoxin reductase and adrenodoxin occurs but does not support maximal rates of 11beta hydroxylation; at least 1 additional molecule of adrenodoxin in excess of the 1:1 complex is required for full hydroxylase activity. Spectrophotometric titration of a mixture of adrenodoxin reductase and cytochrome P-450(11)beta with adrenodoxin indicates sequential formation of 1:1 complexes between adrenodoxin reductase and adrenodoxin and then between a second adrenodoxin and cytochrome P-450(11beta; the adrenodoxin-cytochrome P-450(11)beta complex is only detectable when the concentration of adrenodoxin exceeds that of adrenodoxin reductase.     

Microsomal cytochrome P-450 from neonatal pig testis. Purification and properties of A C21 steroid side-chain cleavage system (17 alpha-hydroxylase-C17,20 lyase)

A cytochrome P-450 from neonatal pig testicular microsomes was purified to homogeneity as judged by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels and by double diffusion on agar against antiserum raised in rabbits against the protein. The enzyme shows both 17 alpha-hydroxylase (Vmax = 4.6 nmol of product/min/nmol of P-450, Km = 1.5 microM) and C17,20 lyase (Vmax = 2.6 nmol of product/min/nmol of P-450, Km = 2.4 microM) activities. Both activities require NADPH and a flavoprotein P-450 reductase; microsomal P-450 reductase from pig and rat livers was used in these studies. The enzyme possesses a single subunit of molecular weight 59,000 +/- 1,000 as determined by electrophoresis on polyacrylamide with sodium dodecyl sulfate and by chromatography on sodium dodecyl sulfate-Sephadex. The enzyme is a glycoprotein and contains 8 nmol of heme/mg of protein and 40 nmol of phospholipid/mg of protein. All heme detected by pyridine hemochromogen is accounted for as P-450 by difference spectroscopy of the reduced P-450.carbon monoxide complex. This complex shows an absorbance maximum at 448 nm with no evidence of P-420. These studies raise the possibility that one microsomal protein (cytochrome P-450) may possess two enzymatic activities (hydroxylase and lyase).     

Protein-protein and lipid-protein interactions in a reconstituted cytochrome P-450 dependent microsomal monooxygenase

NADPH-cytochrome P-450 reductase and cytochrome P-450, both purified from liver microsomes of phenobarbital-treated rabbits, were incorporated into dimyristoylphosphatidylcholine vesicles. The reduction of cytochrome P-450 by NADPH in the reconstituted vesicles proceeded in a biphasic fashion, and 70-80% of the absorbance change was associated with the fast phase. The Arrhenius plot of the apparent first-order rate constant of the fast-phase reduction showed a marked discontinuity around the phase transition temperature of the synthetic phospholipid; an almost 10-fold change in rate constant was associated with this discontinuity. It was, therefore, suggested that the reduction of cytochrome P-450 by reductase in this system was a diffusion-limited reaction controlled by the viscosity of the phospholipid membrane. The Arrhenius plot of overall drug monooxygenase activity catalyzed by the reconstituted vesicles showed a break but in a different way from that observed for the reduction of cytochrome P-450. This break was accompanied only by a change of the slope of the plot but not by a change in reaction rate. This difference in the two Arrhenius plots was attributed to that in the rate-limiting step of the two reactions. NADPH-cytochrome c reductase activity of the reconstituted vesicles, an activity catalyzed by the reductase alone, and cumene hydroperoxide dependent N-methylaniline demethylation activity catalyzed by cytochrome P-450 alone did not show any break in the Arrhenius plots.     

Involvement of FMN and phenobarbital cytochrome P-450 in stimulating a one-electron reductive denitrosation of 1-(2-chloroethyl)-3-(cyclohexyl)-1-nitrosourea catalyzed by NADPH-cytochrome P-450 reductase

Purified hepatic NADPH-cytochrome P-450 reductase, which was reconstituted with dilauroylphosphatidylcholine, catalyzed a one-electron reductive denitrosation of 1-(2-[14C]-chloroethyl)-3-(cyclohexyl)-1-nitrosourea ([14C]CCNU) to give 1-(2-[14C]-chloroethyl)-3-(cyclohexyl)urea at the expense of NADPH. Ambient oxygen or anoxic conditions did not alter the rates of [14C]CCNU denitrosation catalyzed by NADPH-cytochrome P-450 reductase with NADPH. Electron equivalents for reduction could be supplied by NADPH or sodium dithionite. However, the turnover number with NADPH was slightly greater than with sodium dithionite. Enzymatic denitrosation with sodium dithionite or NADPH was observed in anaerobic incubation mixtures which contained NADPH-cytochrome P-450 reductase with or without cytochrome P-450 purified from livers of phenobarbital (PB)-treated rats; PB cytochrome P-450 alone did not support catalysis. PB cytochrome P-450 stimulated reductase activity at molar concentrations approximately equal to or less than NADPH-cytochrome P-450 reductase concentration, but PB cytochrome P-450 concentrations greater than NADPH-cytochrome P-450 reductase inhibited catalytic denitrosation. Cytochrome c, FMN, and riboflavin demonstrated different degrees of stimulation of NADPH-cytochrome P-450 reductase-dependent denitrosation. Of the flavins tested, FMN demonstrated greater stimulation than riboflavin and FAD had no observable effect. A 3-fold stimulation by FMN was not observed in the absence of NADPH-cytochrome P-450 reductase. These studies provided evidence which establish NADPH-cytochrome P-450 reductase rather than PB cytochrome P-450 as the enzyme in the hepatic endoplasmic reticulum responsible for CCNU reductive metabolism.     

Role of phospholipids in reconstituted cytochrome P450 3A form and mechanism of their activation of catalytic activity.

Cytochrome P-450 coded for by the 3A gene family requires specific conditions in a reconstituted system, if its catalytic activity is to be efficient. We investigated the mechanism of activation of the catalytic activity of cytochrome P450 3A by phospholipids. Rat P450 PB-1 (3A2), human P450NF (3A4), and rabbit P450 3c (3A6) were used. They had low activity in a reconstituted system (system I) with dilauroylphosphatidylcholine (DLPC) but had high activity with a mixture of phospholipids (DLPC, dioleoylphosphatidylcholine, and phosphatidylserine) and sodium cholate (system II). P450 3A forms are cationic (having a high content of lysine residues) and needed the anionic phospholipid phosphatidylserine to have sufficient activity. Double-reciprocal plots of the metabolic rate of cytochrome P-450 versus the concentration of NADPH-cytochrome P-450 reductase showed that cytochrome P-450 and the reductase interacted more in system II than in system I. P450 PB-1 did not absorb at 450 nm in the presence of reductase, CO, DLPC, and NADPH, although other cytochrome P-450s absorbed at around 450 nm in such a mixture. However, P450 PB-1 was reduced in the presence of the phospholipid mixture and sodium cholate instead of DLPC. These results suggested that the stimulation of catalytic activity by phospholipids involved increased interaction between cytochrome P-450 and the reductase. Studies of proteolytic digestion and chemical cross-linking in systems I and II showed that a P450 3A form needed disaggregation of cytochrome P-450 and/or the reductase, not the formation of an aggregated complex necessary for the catalytic activity of other cytochrome P-450s.     

The distribution of the components of mixed-function oxidase between the rough and the smooth endoplasmic reticulum of liver cells

Mixed-function oxidase activity, when measured by the N-demethylation of ethylmorphine or the hydroxylation of aniline, is significantly higher in the smooth hepatic endoplasmic reticulum than in the rough. In the rabbit the smooth membrane/rough membrane activity ratios are significantly greater than 1 whether the activities are expressed per g. of liver (ratio 5), per mg. of protein (ratio 3-5), per mug. of phospholipid phosphorus (ratio 2), per unit of cytochrome P-450 (ratio 1.7) or per unit of NADPH-cytochrome c reductase activity (ratio 2). On the other hand, if the activities are normalized to the NADPH-cytochrome P-450 reductase, there is no significant difference between the rough and smooth membranes. These results suggest that, in the rabbit, the rate-limiting step is the reduction of cytochrome P-450. In contrast, in the rat the difference in activities can be explained by differences in the concentration of cytochrome P-450.     

Sex- and strain-dependent hepatic microsomal ethylmorphine N-demethylation in mice: the roles of type I binding and NADPH-cytochrome P-450 reductase

The roles of type I binding and NADPH-cytochrome P-450 reductase in ethylmorphine demethylation were investigated in two strains of mice, using sex differences in these activities as a tool. In the CPB-SE strain, females metabolize ethylmorphine faster than males. Sex differences in cytochrome P-450 content and endogenous NADPH-cytochrome P-450 reductase activity were too small to account for this. On the other hand, the differences in the magnitudes of type I spectra and ethylmorphine-induced enhancement of cytochrome P-450 reduction were considerable larger than those in the rates of demethylation. All parameters, except endogenous cytochrome P-450 reduction, were modified in a similar way by testosterone pretreatment: in females they were depressed to the male level, whereas in males they remained unchanged. Castration had no effect in females and enhanced the activities in males. The CPB-V strain exhibited little or no sex differences in ethylmorphine demethylation, cytochrome P-450 content and endogenous cytochrome P-450 reduction. Testosterone pretreatment had little or no influence on these activities. Type I binding and reductase stimulation, however, showed sex differences, comparable to those observed in the CPB-SE strain, which were also abolished by testosterone. A relationship between reductase stimulation and type I binding was observed, which was, apparently, independent of sex or strain. It is concluded that androgen primarily influences the amount of cytochrome P-450-substrate complex formed, but that the reduction of this complex is not rate-limiting in the demethylation of ethylmorphine.     

Comparative study of the reaction kinetics of cytochrome P-450 reduction by NADPH-cytochrome P-450 reductase and dithionite

The reactions of NADPH- or dithionite-dependent reduction of cytochrome P-450 were studied using a stopped flow technique. It was found that the kinetic curves for both reactions may be fitted by a sum of the two exponents. The arrhenius plots for the fast phase rate constants are linear for both reactions. On the contrary, the breaks on the corresponding plots for the slow phase rate constants are observed at 22 and 33 degrees C for cytochrome P-450 reduction by dithionite and at 31 degrees C for NADPH-dependent reduction of cytochrome P-450. The coincidence of the values of the rate constants and activation energy (56 +/- 5 kJ/mol) for the fast phase of NADPH-dependent reduction of cytochrome P-450 with values of catalytic constants and activation energy for demethylation of tertiary amines suggests that the first electron transfer process from NADPH-cytochrome P-450 reductase to cytochrome P-450 may be the rate-limiting step. A diverse character of the kinetic parameters for the two cytochrome P-450 reduction reactions is indicative of different nature of biphasity of these processes.     

Studies on the rate-determining factor in testosterone hydroxylation by rat liver microsomal cytochrome P-450: evidence against cytochrome P-450 isozyme:isozyme interactions

The aim of the present study was to examine a recent proposal that inhibitory isozyme:isozyme interactions explain why membrane-bound isozymes of rat liver microsomal cytochrome P-450 exert only a fraction of the catalytic activity they express when purified and reconstituted with saturating amounts of NADPH-cytochrome P-450 reductase and optimal amounts of dilauroylphosphatidylcholine. The different pathways of testosterone hydroxylation catalyzed by cytochromes P-450a (7 alpha-hydroxylation), P-450b (16 beta-hydroxylation), and P-450c (6 beta-hydroxylation) enabled possible inhibitory interactions between these isozymes to be investigated simultaneously with a single substrate. No loss of catalytic activity was observed when purified cytochromes P-450a, P-450b, or P-450c were reconstituted in binary or ternary mixtures under a variety of incubation conditions. When purified cytochromes P-450a, P-450b, and P-450c were reconstituted under conditions that mimicked a microsomal system (with respect to the absolute concentration of both the individual cytochrome P-450 isozyme and NADPH-cytochrome P-450 reductase), their catalytic activity was actually less (69-81%) than that of the microsomal isozymes. These results established that cytochromes P-450a, P-450b, and P-450c were not inhibited by each other, nor by any of the other isozymes in the liver microsomal preparation. Incorporation of purified NADPH-cytochrome P-450 reductase into liver microsomes from Aroclor 1254-induced rats stimulated the catalytic activity of cytochromes P-450a, P-450b, and P-450c. Similarly, purified cytochromes P-450a, P-450b, and P-450c expressed increased catalytic activity in a reconstituted system only when the ratio of NADPH-cytochrome P-450 reductase to cytochrome P-450 exceeded that normally found in liver microsomes. These results indicate that the inhibitory cytochrome P-450 isozyme:isozyme interactions described for warfarin hydroxylation were not observed when testosterone was the substrate. In addition to establishing that inhibitory interactions between different cytochrome P-450 isozymes is not a general phenomenon, the results of the present study support a simple mass action model for the interaction between membrane-bound or purified cytochrome P-450 and NADPH-cytochrome P-450 reductase during the hydroxylation of testosterone.