Kinetic isotope effects on cytochrome P-450-catalyzed oxidation reactions. The oxidative O-dealkylation of 7-ethoxycoumarin

The primary deuterium and tritium isotope effects on Vm/Km and on Vm have been measured for the O-deethylation of 7-ethoxycoumarin catalyzed by two purified isozymes of cytochrome P-450. From these data the intrinsic isotope effects have been calculated as described by D. B. Northrop (Biochemistry (1975) 14, 2644-2651). The observed deuterium isotope effects on Vm/Km are 3.79 and 1.90 for the isozymes isolated from the livers of rats induced by phenobarbital and 3-methylcholanthrene, respectively. The calculated intrinsic isotope effects, however, are similar and much larger (kH/kD = 12.8 to 14.0) than the observed isotope effects on Vm/Km for the two enzymes. This demonstrates that the intrinsic isotope effects are attenuated by various steps preceding the isotopically sensitive C-H bond cleavage step resulting in the low values for the observed isotope effects. Thus, the observed isotope effects do not accurately reflect the magnitude of the intrinsic isotope effect associated with this reaction. No incorporation of 18O into the 7-hydroxycoumarin product was observed in studies employing H218O or 18O2 demonstrating that the phenolic oxygen arises exclusively from the substrate. Taken together, these data provide compelling evidence that both cytochrome P-450 isozymes catalyze the O-dealkylation of this substrate by an identical radical recombination mechanism during the obligatory formation of a hemiacetal intermediate.     

Interaction between cytochrome P-448 and NADP-cytochrome P-450 reductase in reconstituted microsomal membranes

The regularities of changes in the functional activity of the microsomal monooxygenase system reconstituted by self-assembly from intact rat liver microsomes solubilized with 4% sodium cholate were studied at variable levels of NADPH-cytochrome P-450 reductase and the 3-methylcholanthrene-induced form of cytochrome P-450. Using antibodies against cytochrome P-448, the role of cytochrome P-448 in the overall reaction of benzopyrene hydroxylation induced in the microsomal membrane by a set of molecular forms of cytochrome P-450 was investigated. The effect of NADPH-cytochrome P-450 reductase and cytochrome P-448 incorporation into reconstituted microsomal membranes on benzpyrene metabolism suggests that in intact microsomal membranes benzopyrene metabolism induced by different forms of cytochrome P-450, with the exception of P-448, is limited by reductase is not the limiting component; however, cytochrome P-448 reveals its maximum activity at the cytochrome to reductase optimal molar ratio of 5:1; above this level, the catalytic activity of cytochrome P-448 is lowered.     

Purification and characterization of seven distinct forms of liver microsomal cytochrome P-450 from untreated and inducer-treated male Wistar rats

A total of nine forms of cytochrome P-450 were purified to homogeneity from liver microsomes of male Wistar rats. They were P-451 I and P-451 II from untreated rats, P-450 II and P-450 III from phenobarbital-treated rats, MC-P-448 L and MC-P-448 H from 3-methylcholanthrene-treated rats, and P-452, P-448 L, and P-448 H from 3,4,5,3',4'-pentachlorobiphenyl-treated rats. Among them, MC-P-448 L and MC-P-448 H were indistinguishable from P-448 L and P-448 H, respectively, with regard to electrophoretic, spectral, catalytic and immunochemical properties, and thus seven forms were distinct hemoproteins. The minimal molecular weight of each form was as follows: P-451 I (49,000), P-451 II (52,000), P-450 II (52,000), P-450 III (53,500), P-452 (48,000), P-448 L (56,000), P-448 H (54,000). Judging from the oxidized absolute spectra, P-448 H was a high-spin form and the others were of low-spin type. In a reconstituted system, N-demethylations of benzphetamine and aminopyrine were catalyzed by most of the forms at comparable rates. On the other hand, the activities for the oxidations of benzo[a]pyrene, 7-ethoxycoumarin, biphenyl, and estradiol-17 beta varied greatly among the forms of cytochrome P-450. The most efficient catalysts were as follows: P-448 L and P-451 II for benzo[a]pyrene 3-hydroxylation; P-448 L for 7-ethoxycoumarin O-deethylation; P-448 L, P-451 II, and P-448 H for biphenyl 4-hydroxylation; P-448 L and P-448 H for biphenyl 2-hydroxylation; and P-451 II and P-448 H for estradiol 2-hydroxylation. P-451 I, P-450 II, and P-450 III were somewhat poorer catalysts in metabolizing all the substrates except for benzphetamine and aminopyrine, but their substrate specificities were still distinguishable from one another. Of all the purified cytochrome P-450's, P-452 showed the least ability to metabolize all the substrates. Judging from the properties, it appears that six forms in male Wistar rats correspond to the distinct forms of cytochrome P-450 in Long-Evans and/or Sprague-Dawley rats reported by other workers, but P-451 I is a new constitutive isozyme in Wistar rats.     

Triple isotopic labeling and kinetic isotope effects: exposing H-transfer steps in enzymatic systems

Kinetic isotope effect (KIE) studies can provide insight into the mechanism and kinetics of specific chemical steps in complex catalytic cascades. Recent results from hydrogen KIE measurements have examined correlations between enzyme dynamics and catalytic function, leading to a surge of studies in this area. Unfortunately, most enzymatic H-transfer reactions are not rate limiting, and the observed KIEs do not reliably reflect the intrinsic KIEs on the chemical step of interest. Given their importance to understanding the chemical step under study, accurate determination of the intrinsic KIE from the observed data is essential. In 1975, Northrop developed an elegant method to assess intrinsic KIEs from their observed values [Northrop, D. B. (1975) Steady-state analysis of kinetic isotope effects in enzymic reactions. Biochemistry 14, 2644-2651]. The Northrop method involves KIE measurements using all three hydrogen isotopes, where one of them serves as the reference isotope. This method has been successfully used with different combinations of observed KIEs over the years, but criteria for a rational choice of reference isotope have never before been experimentally determined. Here we compare different reference isotopes (and hence distinct experimental designs) using the reduction of dihydrofolate and dihydrobiopterin by two dissimilar enzymes as model reactions. A number of isotopic labeling patterns have been applied to facilitate the comparative study of reference isotopes. The results demonstrate the versatility of the Northrop method and that such experiments are limited only by synthetic techniques, availability of starting materials, and the experimental error associated with the use of distinct combinations of isotopologues.     

Isolation and characterization of multiple forms of cytochrome P-450 from liver microsomes of 3-methylcholanthrene-induced Wistar rats

Chromatography on 1.8-diaminooctyl-Sepharose and DEAE-Sephacel resulted in 4 fractions of cytochrome P-450 from liver microsomes of 3-methylcholanthrene-induced Wistar rats. All the four fractions differed in terms of their absorption maxima in the CO-reduced state, Mr and catalytic activity. Only one cytochrome fraction (cytochrome P-450 C) possessed a high activity upon benz(a)pyrene hydroxylation. All cytochrome P-450 forms were characterized by a low rate of aminopyrine N-demethylation. Antibodies against cytochrome P-450 C (P-448) (anti-P-448) were raised. Cytochromes of fractions A, B1 and B2 in the Ouchterlony reaction of double immunodiffusion did not give precipitation bands with anti-P-448. Neither of the four cytochrome P-450 forms interacted with the antibodies raised against cytochrome P-450 isolated from liver microsomes of rats induced with phenobarbital. The procedure developed is applicable to the isolation of multiple forms of cytochrome P-450 from liver microsomes of 3-methylcholanthrene-induced rats. Using rocket immunoelectrophoresis, cytochrome P-450 C possessing a high (as compared to benz(a)pyrene metabolism) activity (18 nmol/min/nmol cytochrome) and a high (60-70%) content in 3-methylcholanthrene-induced rat liver microsomes was shown to give a relatively high yield.     

Use of multiple isotope effects to study the mechanism of 6-phosphogluconate dehydrogenase

The multiple isotope effect method of Hermes et al. [Hermes, J. D., Roeske, C. A., O'Leary, M. H., & Cleland, W. W. (1982) Biochemistry 21, 5106-5114] has been used to study the mechanism of the oxidative decarboxylation catalyzed by 6-phosphogluconate dehydrogenase from yeast. 13C kinetic isotope effects of 1.0096 and 1.0081 with unlabeled or 3-deuterated 6-phosphogluconate, plus a 13C equilibrium isotope effect of 0.996 and a deuterium isotope effect on V/K of 1.54, show that the chemical reaction after the substrates have bound is stepwise, with hydride transfer preceding decarboxylation. The kinetic mechanism of substrate addition is random at pH 8, since the deuterium isotope effect is the same when either NADP or 6-phosphogluconate or 6-phosphogluconate-3-d is varied at fixed saturating levels of the other substrate. Deuterium isotope effects on V and V/K decrease toward unity at high pH at the same time that V and V/K are decreasing, suggesting that proton removal from the 3-hydroxyl may precede dehydrogenation. Comparison of the tritium effect of 2.05 with the other measured isotope effects gives limits of 3-4 on the intrinsic deuterium and of 1.01-1.05 for the intrinsic 13C isotope effect for C-C bond breakage in the forward direction and suggests that reverse hydride transfer is 1-4 times faster than decarboxylation.     

Comparison of spectral properties of 3-MC induced cytochrome P-448 from rabbits and rats.

EPR and optical spectral properties of cytochrome P-488 from 3-methylcholanthrene-induced rabbits are compared with those of rats. In the EPR spectra at 77K and in the optical absorption spectra at room temperature a considerable temperature independent high spin content of the rabbit cytochrome is observed which has been estimated to about 55% by titration with n-octylamine. On the other hand the high spin content of the rat cytochrome depends strongly on temperature and amounts to about 6% at 5 degrees C and to about 35% at 34 degrees C. The binding of substrates and ligands to the rabbit cytochrome as well as its reduction by sodium dithionite are slower as compared with the rat cytochrome but also with phenobarbital-induced cytochrome P-450 from rats and rabbits. Contrary to the 3-methylcholanthrene induced cytochrome P-448 from rats, that from rabbits does not bind 3-methylcholanthrene. A particular protein structure establishing the high spin state and an absent binding site for type I substrates is assumed to be responsible for these and other peculiarities of cytochrome P-448 from 3-methylcholanthrene-induced rabbits.     

Calculations of intrinsic isotope effects and the detection of tunneling in enzyme-catalyzed reactions

The equation of Northrop [1975, Biochemistry, 14, 2644] for calculating intrinsic isotope effects from observed deuterium and tritium isotope effects of V/K, in which hydrogen is the reference isotope, has been extended to experimental designs using either deuterium or tritium as a reference. Partial derivatives of the intrinsic equations allow calculation of the relative precision of the three referenced isotope effects and these favor the order deuterium > tritium > hydrogen. In comparisons of observed and calculated isotope effects when hydrogen tunneling is present, both the precision and the magnitude of the difference was greater for intrinsic calculations than for exponentiations based upon a breakdown in the Swain-Schaad relationship.     

Kinetic deuterium isotope effects on the N-demethylation of tertiary amides by cytochrome P-450

Liver microsomal cytochrome P-450 readily N-dealkylates N,N-dimethylamides. N-Methyl-N-hydroxymethyl amides were isolated as intermediates and characterized by gas chromatography-mass spectrometry as their trimethylsilyl ethers. Intramolecular kinetic deuterium isotope effects measured for the enzymic N-demethylation of a series of 12 aromatic and aliphatic N-methyl-N-trideuteriomethyl amides, RCON(CH3)CD3, varied from 3.6 to 6.9 but were independent of both amide bond rotation rate and substrate oxidation potential. These values, which represent a lower limit to the intrinsic isotope effect (Dkintrinsic), are significantly larger than those observed for anodic N-demethylation and are consistent with a mechanism involving hydrogen atom abstraction. On the other hand, with N,N-dimethylbenzamide the intermolecular kinetic deuterium isotope effects on Vmax and Vmax/Km were found to be much smaller (1.23 and 1.75, respectively) indicating substantial suppression of the intrinsic isotope effect. Such suppression indicates the occurrence of a rate-limiting step other than the isotopically sensitive step together with a strong commitment to catalysis.     

The onset of the deuterium isotope effect in cytochrome c oxidase

We have investigated the dynamics of proton equilibration within the proton-transfer pathway of cytochrome c oxidase from bovine heart that is used for the transfer of both substrate and pumped protons during reaction of the reduced enzyme with oxygen (D-pathway). The kinetics of the slowest phase in the oxidation of the enzyme (the oxo-ferryl --> oxidized transition, F --> O), which is associated with proton uptake, were studied by monitoring absorbance changes at 445 nm. The rate constant of this transition, which is 800 s(-)(1) in H(2)O (at pH approximately 7.5), displayed a kinetic deuterium isotope effect of approximately 4 (i.e., the rate was approximately 200 s(-)(1) in 100% D(2)O). To investigate the kinetics of the onset of the deuterium isotope effect, fully reduced, solubilized CO-bound cytochrome c oxidase in H(2)O was mixed rapidly at a ratio of 1:5 with a D(2)O buffer saturated with oxygen. After a well-defined time period, CO was flashed off using a short laser flash. The time between mixing and flashing off CO was varied within the range 0. 04-10 s. The results show that for the bovine enzyme, the onset of the deuterium isotope effect takes place within two time windows of O transition is internal proton transfer from a site, proposed to be Glu (I-286) (R. sphaeroides amino acid residue numbering), to the binuclear center. The spontaneous equilibration of protons/deuterons with this site in the interior of the protein is slow (approximately 1 s).     

Magnitude of intrinsic isotope effects in the dopamine beta-monooxygenase reaction

Intrinsic primary hydrogen isotope effects (kH/kD) have been obtained for the carbon-hydrogen bond cleavage step catalyzed by dopamine beta-monooxygenase. Irreversibility of this step is inferred from the failure to observe back-exchange of tritium from TOH into substrate under conditions of dopamine turnover; this result cannot be due to solvent inaccessibility at the enzyme active site, since we will demonstrate [Ahn, N., & Klinman, J. P. (1983) Biochemistry (following paper in this issue)] that a solvent-derived proton or triton must be at the enzyme active site prior to substrate activation. As shown by Northrop [Northrop, D. B. (1975) Biochemistry 14, 2644], for enzymatic reactions in which the carbon-hydrogen bond cleavage step is irreversible, comparison of D(V/K) to T(V/K) allows an explicit solution for kH/kD. Employing a double-label tracer method, we have been able to measure deuterium isotope effects on Vmax/Km with high precision, D(V/K) = 2.756 +/- 0.054 at pH 6.0. The magnitude of the tritium isotope effect under comparable experimental conditions is T(V/K) = 6.079 +/- 0.220, yielding kH/kD = 9.4 +/- 1.3. This result was obtained in the presence of saturating concentrations of the anion activator fumarate. Elimination of fumarate from the reaction mixture leads to high observed values for isotope effects on Vmax/Km, together with an essentially invariant value for kH/kD = 10.9 +/- 1.9. Thus, the large disparity between isotope effects, plus or minus fumarate, cannot be accounted for by a change in kH/kD, and we conclude a role for fumarate in the modulation of the partitioning of enzyme-substrate complex between catalysis and substrate dissociation. On the basis of literature correlations of primary hydrogen isotope effects and the thermodynamic properties of hydrogen transfer reactions, the very large magnitude of kH/kD = 9.4-10.9 for dopamine beta-monooxygenase suggests an equilibrium constant not very far from unity for the carbon-hydrogen bond cleavage step. This feature, together with the failure to observe re-formation of dopamine from enzyme-bound intermediate or product and overall rate limitation of enzyme turnover by product release, leads us to propose a stepwise mechanism for norepinephrine formation from dopamine in which carbon-hydrogen bond cleavage is uncoupled from the oxygen insertion step.     

Separation of two forms of cytochrome P-450 with aryl hydrocarbon hydroxylase activity from intestinal mucosa microsomes of rabbits treated with 3-methylcholanthrene

Two forms of cytochrome P-450, designated P-448a and P-448b, were purified from intestinal mucosa microsomes of rabbits treated with 3-methylcholanthrene. Both the cytochromes had absorption maxima at 448 nm in the carbon monoxide-reduced difference spectra. They exhibited comparable catalytic activities with benzo(a)pyrene, 7-ethoxycoumarin, and 7-ethoxyresorufin, when reconstituted with hepatic NADPH-cytochrome c reductase and phosphatidylserine. P-448a was apparently homogeneous on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and its monomeric molecular weight was estimated to be 58,000. The oxidized form had absorption maxima at 416, 512 and 571 nm, indicative of the low spin state. Thus P-448a appeared to be similar to one form of P-450, which was induced in rabbit liver by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). On the other hand, SDS-PAGE of P-448b gave a single major protein band with a monomeric molecular weight of 55,500, indicating that P-448b can be distinguished from P-448a.     

Determination of the rate-limiting steps for malic enzyme by the use of isotope effects and other kinetic studies.

Isotope effects have been measured with Mg2+ as the activator and L-malate labeled with deuterium or tritium at carbon 2 as the substrate over the pH range 4-10. Comparison of the nearly pH-independent deuterium-isotope effect on V/Kmalate of 1.5 with the tritium effect of 2.0 by the method of Northrop (Northrop, D.B. (1975), Biochemistry 14, 2644) gives limits on the true effect of deuterium substitution on the bond-breaking step of 5-8 in the forward reaction and 4-6.5 in the reverse direction. Comparison of the deuterium effect on V/K with the 13C-isotope effect of 1.031 reported by Schimerlik et al. (Schimerlik, M.I., Rife, J.E., and Cleland, W.W. (1975), Biochemistry 14, 5347) allows the deduction that at pH 8 reverse hydride transfer is six to eight times faster than decarboxylation, which is thus largely rate limiting for the catalytic reaction. The absence of a deuterium-isotope effect on V at pH 7-8 and comparison of the Ki of pyruvate as an uncompetitive inhibitor of the forward reaction and a substrate for the reverse reaction indicate that at neutral pH the release of TPNH from enzyme-reduced triphosphopyridine nucleotide (E-TPNH) is the rate-limiting step in the forward direction. The observation of a deuterium effect on V that approaches 3 at pH 4 and 10 shows, however, that, at very low and very high pH, hydride transfer may become partly rate limiting. In the reverse reaction, the probable rate-limiting step at pH 7 is the isomerization of E-TPNH, while at pH 8.5 and above V becomes too large to measure and appears infinite. Substitution of Co2+, Ni2+, or low levels of Mn2+ for Mg2+ gives similar deuterium-isotope effects, although the values of V and Kmalate vary considerably with metal. The kinetics of Mn2+ show pronounced negative cooperativity, with Km values of 7 muM and 5 mM for concentration ranges from 4 to 100 muM and over 1 mM.     

Cytochrome P-448 induction in liver microsomes of mice of inbred strains after administration of xenobiotics of MC-type

Using immunochemical methods, the identity of cytochrome P-448 from liver microsomes of mice of "inducible" and "non-inducible" lines during induction by xenobiotics of MX-type (3-methylcholanthrene, 3,4-benzpyrene, 2,3,7,8-tetrachlorodibenzodioxin) was established. This hemoprotein form was shown to play a role in 3,4-benzpyrene metabolism. Monospecific antibodies to purified cytochromes P-448 and P-450 were obtained; the cytochrome P-448 content in microsomes was measured by rocket immunoelectrophoresis. The content of cytochrome P-448 in control and phenobarbital-induced microsomes makes up to 10-15% of the total hemoprotein content determinable from the CO-spectra. 3-Methylcholanthrene and 3,4-benzpyrene injected into "non-inducible" mice cause no increase in the content of this hemprotein form, whereas in mice induced with 2,3,7,8-tetrachlorodibenzodioxin it rises to 50%. Under these conditions, an almost 100% inhibition of 3,4-benzpyrene metabolism by antibodies to cytochrome P-448 is observed. Antibodies against cytochrome P-448 obtained from liver microsomes of 3-methylcholanthrene-induced mice cause a 90% inhibition of 3,4-benzpyrene in microsomes induced with 3-methylcholanthrene and 2,3,7,8-tetrachlorodibenzodioxin.     

Unusual four-bond secondary H/D isotope effect supports a short-strong hydrogen bond between phospholipase A2 and a transition state analogue inhibitor

A prominent secondary four-bond hydrogen/deuterium isotope effect was observed from proton NMR at the active site histidine imidazole ring of bovine pancreatic sPLA(2) in the presence of a phosphonate transition state analogue. The cross-modulation of H(epsilon2)/H48 and H(delta1)/H48 resonances was confirmed by line shape simulation that follows the McConnell equation with fractionation factors incorporated to account for the change in the signal magnitude as well as the resonance line shape at various H(2)O/D(2)O solvent mixtures. While the downfield shift of each individual proton upon deuteration on the opposite site can be attributed to the proton-relay system of the H48-D99 catalytic dyad in sPLA(2), the observation that H(delta1)/H48 induces a 3-fold larger H/D secondary isotope effect ( approximately 0.15 ppm) on H(epsilon2)/H48 than vice versa ( approximately 0.05 ppm) is interpreted as additional spectroscopic evidence for the previously proposed short-strong hydrogen bond formed between the donor N(delta1)/H48 and a nonbridging phosphonate oxygen atom of the transition state analogue. These results provide additional details for the catalytic mechanism of sPLA(2) and demonstrate that the intrinsic H/D secondary isotope effect is a useful tool to probe hydrogen bond strength.     

Induction of the hepatic microsomal and nuclear cytochrome P-450 system by hexachlorobenzene, pentachlorophenol and trichlorophenol.

The application of hexachlorobenzene (HCB), pentachlorophenol (PCP) and 2,4,5-trichlorophenol (TCP) to female rats led to an induction of both the microsomal and the nuclear cytochrome P-450 system in the liver. The increase of th mixed-function hydroxylase activities examined (7-ethoxycoumarin deethylase, 7-ethoxyresorufin deethylase, NADPH-dependent cytochrome c reductase, aminopyrine demethylase, benzpyrene hydroxylase) did not correlate strictly with the cytochrome P-450 content. Depending on the inducers and the substrates used, the content and the activity of the cytochrome P-450 were essentially smaller in the nuclei than in the microsomes. It was striking that in the nuclei those activities (benzpyrene hydroxylase, 7-ethoxyresorufin deethylase, 7-ethoxycoumarin deethylase) were preferably induced which can be attributed to the methyl-cholanthrene-induced form of the cytochrome P-450 (cytochrome P-448). These results suggest, also in the light of findings of other authors, the induction of different species of cytochrome P-450 in the nuclei and microsomes.     

Purification and characterization of two forms of 2,3,4,7,8-pentachlorodibenzofuran-inducible cytochrome P-450 in hamster liver

Two forms of cytochrome P-450 (P-450) from liver microsomes of hamsters treated with 2,3,4,7,8-pentachlorodibenzofuran (PenCDF), which possesses the potent acute toxicity and 3-methylcholanthrene (MC)-type inducing ability of liver microsomal monooxygenases in animals, were purified and characterized. These P-450 forms, designated as hamster P-450H and hamster P-450L, had the molecular masses of 52 and 50 kDa, respectively, and showed the absorption maximum of CO-reduced difference spectra at 446 nm. The absolute spectra of their oxidized forms indicated that hamster P-450H was in high-spin state and hamster P-450L was in low-spin state. A part of PenCDF injected into hamster was tightly bound to purified hamster P-450H at a ratio of 0.107 nmol PenCDF/nmol P-450. In a reconstituted system, both hamster P-450H and hamster P-450L showed relatively low catalytic activities for 3-hydroxylation of benzo[a]pyrene and O-deethylations of both 7-ethoxyresorufin and 7-ethoxycoumarin, while they both catalyzed 7 alpha- and 2 alpha-hydroxylations of testosterone effectively to a similar extent. Addition of cytochrome b5-to a reconstituted system accelerated the formation of 7 alpha-hydroxytestosterone 5.3-fold with hamster P-450L and 2.2-fold with hamster P-450H. In addition, hamster P-450H catalyzed estradiol 2-hydroxylation at a high rate but hamster P-450L did not.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunohistochemical study of temporal variations in cytochrome P-450 isozymes in rat testis and their modifications by the inductive effects of cadinenes

Temporal variations in cytochrome P-450 isozymes of rat testis, PB-P-450 (forms of cytochrome P-450 strongly induced by phenobarbital) and MC-P-448 (forms of cytochrome P-450 strongly induced by 3-methylcholanthrene), were investigated immunohistochemically by the avidin-biotin-complex method using specific antibodies against PB-P-450 and MC-P-448 isozymes. Immunoreactivity to both PB-P-450 and MC-P-448 isozymes was observed in Leydig cells. The number of PB-P-450 positive Leydig cells was found to undergo significant time-of-day variation with a peak time of 0000 hours (light phase from 0800 to 2000 hours). Injection of cadinenes (300 mg/kg per day intraperitoneally at 48 and 96 h before sacrifice) induced PB-P-450 isozyme but did not induce MC-P-448 isozyme. The induction of PB-P-450 isozyme by cadinenes was time dependent, and the early dark phase (2000 and 0000 hours) was most sensitive. These results suggest that temporal variation of cytochrome P-450 isozymes is one of the important physiological variations in detoxification and activation of various xenobiotics and chemicals in the testis.     

Branchpoint for heme alkylation and metabolite formation in the oxidation of arylacetylenes by cytochrome P-450

Phenylacetylene and biphenylacetylene are oxidized by cytochrome P-450 to the corresponding arylacetic acids. The acetylenic hydrogen shifts to the adjacent carbon and one atom of molecular oxygen is incorporated into the carboxylic acid group in these transformations, which are subject to a large kinetic isotope effect when the acetylenic hydrogen is replaced by deuterium. The same products and isotope effects are observed when the two arylacetylenes are oxidized by m-chloroperbenzoic acid rather than by the enzyme. In contrast, the inactivation of cytochrome P-450 that occurs during the oxidation of phenylacetylene is insensitive to deuterium substitution. The partition ratio between metabolite formation and enzyme inactivation consequently changes from 26 to 15 in going from phenylacetylene to the deuterated analogue. Metabolite formation therefore diverges from heme alkylation very early in the catalytic process.     

Metabolic conditions determining the composition and catalytic activity of cytochrome P-450 monooxygenases in Candida tropicalis.

In the microsomal fraction of Candida tropicalis cells, two distinct monooxygenases were detected, depending on the growth conditions. The distinction of the two monooxygenases was evident from: (i) the absorption maxima in the reduced CO difference spectra of the terminal oxidases (cytochromes P-450 and P-448); (ii) the contents of the monooxygenase components (cytochromes P-450/P-448, NADPH-cytochrome c (P-450) reductase, and cytochrome b5) and (iii) the catalytic activity of the complete system (aliphatic hydroxylation and N-demethylation activity). The occurrence of the respective monooxygenases could be related to the carbon source (n-alkanes or glucose). Oxygen limitation led to a significant increase of cytochrome P-450/P-448 content, independent of the carbon source utilized by the cells. An improved method for the isolation of microsomes enabled us to demonstrate the presence of cytochrome P-448 in glucose-grown cells.