Rate-Limiting Step in the Reconstituted Microsomal Drug

Transcription

1 J. Biochem. 82, (1977) Rate-Limiting Step in the Reconstituted Microsomal Drug Hydroxylase System1 Yoshio IMAI,* Ryo SATO,* and Takashi IYANAGI** *Institute for Protein Research, Osaka University, Suita, Osaka 565, and **Division of Biochemistry, Tsukuba University School of Medicine, Sakura, Niihari, Ibaraki Received for publication, May 30, 1977 Stopped-flow spectrophotometric measurements were carried out by the rate of cytochrome P-450 reduction by NADPH in a reconstituted system consisting of NADPH-cytochrome C (P-450) reductase and cytochrome P-450 purified from phenobarbital-induced rat and rabbit liver microsomes, respectively. The reduction rate was increased by the addition of hydroxyl atable substrates and the apparent first-order rate constant of the reduction in the presence of 1 mm benzphetamine was determined to be 4-12 s-1, a value which corresponds to a potential rate of nmol per min per nmol of cytochrome P-450. The rate constant of the binding of benzphetamine to cytochrome P-450 was also measured by stopped-flow spectrophotometry and found to be of the order of 50 s-1. The rate constants determined for the two partial reactions are much higher than the overall rate of benzphetaminedependent NADPH oxidation of nmol per min per nmol of cytochrome P-450. A difference spectrum attributable to an oxygenated form of ferrous cytochrome P-450 was observed during the steady state of the benzphetaminedependent NADPH oxidation. It is concluded from these and other lines of evidence that the rate-limiting step of benzphetamine N-demethylation by the reconstituted system is at or after the introduction of the second electron. Although the reduction of cytochrome P-450 is not rate-limiting, it was affected in parallel with the overall NADPH oxidation in response to various substrates, ph, and benzphetamine concentration, suggesting that substrate binding to cytochrome P-450 causes parallel enhancement of the cytochrome reduction and the rate-limiting step of the overall process. Hepatic microsomal cytochrome P-450, in col laboration with NADPH-cytochrome c (P-450) reductase, acts as a monooxygenase which cata lyzes NADPH-dependent hydroxylations of various lipophilic compounds such as drugs, alkanes, and steroids. It has been proposed that this monooxy 1 This work was supported in part by a Scientific Research Grant (No ) from the Ministry of Education, Science and Culture of Japan. genase reaction proceeds as follows (1, 2). The first step of the reaction is the formation of a fer ric cytochrome P-450-substrate complex by a reversible interaction of the substrate with the oxidized hemoprotein. Subsequently, the complex accepts one electron from NADPH via the re ductase to give a ferrous cytochrome P-450-sub strate complex, which then combines with mo lecular oxygen to yield an oxygenated form. On introduction of a second electron from NADPH to Vol. 82, No. 5,

2 1238 Y. IMAI, R. SATO, and T. IYANAGI the oxygenated form, an internal electronic rearrangement takes place and the complex undergoes decomposition, via an intermediate analogous to Compound I of catalase and peroxidase, leading to liberation of the hydroxylated product and regeneration of ferric cytochrome P-450. Guen gerich et al. (3), on the other hand, proposed a mechanism in which two electrons are introduced simultaneously into the ferric cytochrome P-450- substrate complex. On the basis of the observation that substrates causing Type I spectral changes accelerate the reduction of cytochrome P-450 by NADPH in intact microsomes, it has been suggested that the reduction of the cytochromesubstrate complex is the rate-limiting step of the overall reaction (4). In disagreement with this suggestion, however, a spectral intermediate having characteristics of an oxygenated form of ferrous cytochrome P-450 has been observed during the aerobic steady state in the presence of a Type I substrate (5). Matsubara et al. (6) have further reported that the rate of cytochrome P-450 reduction in liver microsomes of phenobarbital-treated rats in the presence of ethylmorphine, a Type I substrate, is faster than the overall rate of N-demethylation of the substrate and concluded that the introduction of the first electron to cytochrome P-450 cannot be the ratelimiting step of the overall demethylation reaction. This paper reports stopped-flow spectropho tometric studies of the rate of cytochrome P-450 reduction by NADPH in a reconstituted system consisting of purified preparations of cytochrome P-450 and NADPH-cytochrome c reductase. The results obtained indicate that the apparent firstorder rate constant of the cytochrome reduction varies in parallel with the rate of substrate-dependent NADPH oxidation in response to the addition of various substrates, but the reduction rate is always too high for reduction of the cyto chrome-substrate complex to be the rate-limiting step of the overall process, in agreement with the conclusion reached with intact microsomes (6). MATERIALS AND METHODS A homogeneous preparation of cytochrome P-450 was obtained from liver microsomes of phenobarbitaltreated rabbits as described previously (7). Two high-spin forms of cytochrome P-450 (cyto chrome P-448) were purified from 3-methylcho lanthrene-treated (8) and phenobarbital-treated rabbit liver microsomes (9). NADPH-cytochrome c reductase was partially purified (to a specific activity of units/mg protein) from pheno barbital-treated rat liver microsomes (10); this preparation was free from NADH-cytochrome b5 reductase, cytochrome b5, and cytochrome P-450. The reductase preparation was dialyzed overnight at 4 Ž against 100 volumes of 50 mm potassium phosphate buffer, ph 7.25, containing 20% (v/v) glycerol and 1 mm EDTA and then against 100 volumes of potassium phosphate buffer, ph 7.25, containing I mm EDTA. NADP+ and NADPH were purchased from Oriental Yeast Co., and glucose-6-phosphate and glucose-6-phosphate de hydrogenase from Sigma Chemical Co. Benz phetamine was kindly supplied by Dr. T. Kamataki of Chiba University. Other chemicals used were of reagent grade. Stopped-flow experiments were performed using a Union Giken RA-401 stopped-flow spec trophotometer equipped with a kinetic data pro cessor (RA-456) and a Yokogawa type 3083 technicorder. The rate of cytochrome P-450 reduction was determined by following the for mation of the CO complex of ferrous cytochrome P-450. In one syringe of the stopped-flow ap paratus was placed a solution containing 0.2 pm cytochrome P-450 (or P-448), NADPH-cytochrome c reductase (0.3 unit/ml), a desired concentration of a substrate, and 0.1 M potassium phosphate buffer, ph 7.25; the cytochrome and the reductase were preincubated for 2-3 min before dilution with the buffer, and the whole solution was saturated with CO by thorough bubbling. Neither detergents nor phospholipids were added to this mixture, because the reductase preparations used contained a suitable concentration of detergents (or phospholipids) for reconstitution of the monooxygenase system. In the other syringe of the apparatus was placed a CO-saturated solution containing 0.2 mm NADPH, the substrate (at the same concentration as above), and 0.1 M potassium phosphate buffer, ph Equal volumes of the two solutions were mixed in the mixing chamber and the increase in absorbance at 450 (or 448) nm was recorded. The rate of benzphetamine binding to ferric cytochrome P-450 was determined by recording the increasse in ab sorbance at 383 nm or the decrease at 420 nm onj. Biochem.

3 RECONSTITUTED MICROSOMAL DRUG HYDROXYLASE SYSTEM 1239 mixing equal volumes of 0.1 M potassium phosphate buffer, ph 7.25, one containing 0.4 um cytochrome P-450 and the other containing 1 mm benz phetamine. NADPH oxidation was measured by following the decrease in absorbance at 340 nm in a Cary 14 or Shimadzu UV-200 spectrophotometer, taking a millimolar extinction coefficient of 6.2 mm-1-cm-1 for NADPH. The reaction mixture contained 0.1 um cytochrome P-450, NADPHcytochrome c reductase (0.15 unit/ml), 0.1 M potassium phosphate buffer, ph 7.25, 0.15 mm NADPH, and an appropriate concentration of a substrate. The cytochrome and the reductase were mixed first, the mixture was preincubated for a few min, and the remaining components were added in the order given. The difference absorption spectra were mea sured in a Cary 14 spectrophotometer. tracings thus obtained in the absence and presence of 1 mm benzphetamine. As can be seen, the reduction of cytochrome P-450 could occur even in the absence of added substrate, but the reduction rate was increased about 10-fold in the presence of benzphetamine. In both the absence and pres ence of the substrate, the reaction was clearly biphasic consisting of two first-order processes as illustrated in Fig. 2, from which the apparent RESULTS Kinetics of Cytochrome P-450 Reduction-The time course of cytochrome P-450 reduction by NADPH in the reconstituted system was studied in the stopped-flow spectrophotometer by following the formation of the CO complex of the reduced cytochrome at 450 run. Figure 1 shows typical Fig. 2. First-order plots of the rate of cytochrore P-450 reduction against time (t). (A:-Amax)/Amax is plotted against t, using the data shown in Fig. 1, where At and Amax are the changes in absorbance at time t and at the completion of reduction, respectively. A, In the pres ence of 1 mm benzphetamine; B, in the absence of benz phetamine. Plots for the fast phase (a and b) were obtained by subtracting the values for the slow phase from the uncorrected fast phase portions of the curves in A and B. TABLE I. Kinetic parameters determined for the reduction of cytochrome P-450 by NADPH in the reconstituted system. The data were estimated from Figs. I and 2. The half-time (t1/2) was obtained from the tracings in Fig. 1 and the mean value of the rate constants of the fast and slow phases (kapp) was calculat ed using the equation: t1/2-in (2/kapp). Fig. 1. Time courses of the reduction of cytochrome P-450 by NADPH in the absence and presence of 1 mm benzphetamine. The experiments were performed in a stopped-flow spectrophotometer as described in " MA- TERIALS AND METHODS." The dotted line indicates the final level of absorbance. A, In the presence of benzphetamine; tamine. B, in the absence of benzphe Vol. 82, No. 5, 1977

4 1240 Y. IMAI, R. SATO, and T. IYANAGI first-order rate constants for the fast and slow phases could be estimated (Table I). Their values were variable depending on the NADPH-cyto chrome c reductase preparation used and the precise experimental conditions adopted, but the rate constants for both phases were always 6-12 times higher in the presence of benzphetamine than in its absence. The amount of cytochrome P-450 reducible in the fast phase was also variable, but amounted to roughly 50% of the total cytochrome present and tended to increase on addition of benzphetamine. In the present study, a mean value of the rate constants for both phases was determined from the half-time (t1/2) of the cyto chrome reduction and used for comparison with the other kinetic data because of our insufficient knowledge of the significance of each phase. It should be noted that essentially similar conclusions could be reached if only the rate constant for the fast phase was considered. At any rate, the rate constant for the reduction of cytochrome P-450 under the conditions employed was calculated to be 4-12 s'1 in the presence of 1 mm benzphetamine, accounting for a potential reduction rate of nmol per min per nmol of cytochrome P-450. On the other hand, the rate of benzphetaminedependent oxidation of NADPH measured under identical conditions, except for the presence of air instead of CO, was nmol per min per nmol of cytochrome P-450. This indicates that the ratelimiting step of benzphetamine-dependent NADPH oxidation cannot be the reduction of cytochrome P-450, at least under the experimental conditions employed. Benzphetamine Binding to Ferric Cytochrome P-450-As shown in Fig. 3, a difference spectrum having a peak at 383 nm and a trough at 420 nm was observed in the Soret region on addition of benzphetamine to ferric cytochrome P-450. It is generally accepted that this type of spectral change, classified as Type I (11), is due to the binding of a substrate to ferric cytochrome P-450 and that this binding is followed by the introduction of one electron to the heme iron in the general mechanism of the monooxygenase reaction (1, 4). It has been reported that substrate binding to cytochrome P-450 is an extremely rapid reaction in intact microsomes (12). In the present study, an attempt was made to measure the time course of spectral change induced by mixing benzphetamine with purified cytochrome P-450 in the stopped-flow apparatus. However, it was not possible to analyze the results accurately because of the very high rate of the reaction and the small magnitude of the spectral change. Another difficulty was the limited solubility of benzphetamine at ph It was, however, possible to trace the benzphetamine binding reaction satisfactorily by reducing the benzphetamine concentration to 0.5 mm and increasing the cytochrome P-450 concentration two-fold. Thus, the half-time of the spectral change was determined to be ms, corre sponding to a rate constant of about 50 s-1. This value is much higher than that of the reduction of cytochrome P-450 determined above. Fig. 3. Benzphetamine-induced difference spectrum of ferric cytochrome P-450. The sample and reference cuvettes contained 0.1 t1m cytochrome P-450 in 0.1 M potassium phosphate buffer, ph The difference spectrum was measured in cuvettes with an optical path of 5 cm after addition of 1 mm benzphetamine to the sample cuvette. Fig. 4. Effect of benzphetamine concentration on the rate of cytochrome P-450 reduction. The experiments were conducted as in Fig. 1, except that the concentra tion of benzphetamine was varied as indicated. The apparent rate constant was estimated from the half-time of the reduction as described in Table I. J. Biochem.

5 RECONSTITUTED MICROSOMAL DRUG HYDROXYLASE SYSTEM 1241 Effect of Benzphetamine Concentration-Figure 4 shows the effect of benzphetamine concentration on the rate of reduction of cytochrome P-450 by NADPH in the standard reconstituted system. It can be seen that the apparent rate constant of cytochrome reduction increased as the benz phetamine concentration was increased. From the double-reciprocal plots of these results, it was estimated that the half-maximal effect was ob servable at about 0.1 mm. This value is approximately equal to the equilibrium dissociation constant of the ferric cytochrome P-450-benz phetamine complex determined from the benz phetamine-induced spectral change, but is slightly larger than the Michaelis constant for benz phetamine in benzphetamine-dependent NADPH oxidation (Imai, Y. & Sato, R., unpublished re sults). To compare the benzphetamine depend ences of these three processes in more detail, the magnitude of benzyphetamine-induced spectral change and the rate of benzphetamine-dependent NADPH oxidation were examined in the presence of various concentrations of benzphetamine under conditions similar to those for the cytochrome reduction assay. It can be seen in Fig. 5 that the rate of cytochrome P-450 reduction changed in response to benzphetamine concentration in the same way as did the magnitude of spectral change, and hence the amount of the cytochrome-benz phetamine complex formed. The rate of NADPH oxidation was also affected by benzphetamine in parallel with the other two processes but at a slightly lower concentration of benzphetamine. Difference Spectrum in Steady State-In view of the finding described above, that the rate of cytochrome P-450 reduction by NADPH in the presence of benzphetamine is much higher than that of the overall NADPH oxidation and of the very rapid binding of oxygen to ferrous cytochrome Fig. 6. Difference spectra of the reconstituted system in the aerobic and anaerobic steady states. The sample and reference cuvettes contained 0.1 Đm cytochrome P-450, NADPH-cytochrome c reductase (0.17 unit/ml), Fig. 5. Effects of benzphetamine concentration on the benzphetamine-induced spectral change, the rate of cytochrome P-450 reduction by NADPH, and the rate of benzphetamine-dependent NADPH oxidation. The absorbance increment between 383 and 420 nm induced by the addition of benzphetamine to ferric cytochrome P-450 was determined as described in Fig. 3. The data in Fig. 4 were used for the rate of cytochrome P-450 reduction. The rate of NADPH oxidation was measured as described in " MATERIALS AND METHODS," except that the benzphetamine concentration was varied as indicated. Maximal values of the absorbance incre ment and the rates of the two reactions were calculated from double-reciprocal plots. The ordinate shows the values relative to the maxima thus determined. Curve A, NADPH oxidation; Curve B, cytochrome P-450 reduction; Curve C, spectral change. 0.1 M potassium phosphate buffer, ph 7.25, 0.2 mm glucose-6-phosphate, glucose-6-phosphate dehydroge nase (1 unit/ml), 1 MM MgCl2, and 1 mm benzphetamine. The cytochrome and reductase preparations were mixed first and after preincubation for a few min the other com ponents were added in the order given. The reaction was started by adding mm NADPH to the sample cuvette. The spectrum was recorded in cuvettes with an optical path of 10 cm either aerobically (Curve A) or under a nitrogen atmosphere (Curve B). Even when glucose-6-phosphate, glucose-6-phosphate dehydrogen ase, and MgCl2 were omitted from both cuvettes, a spec trum identical with that shown in Curve A was observed immediately after aerobic addition of NADPH. The NADPH in the sample cuvette was, however, exhausted after a while as indicated by the decrease in absorbance at 340 run. After complete exhaustion of NADPH, the difference spectrum was again recorded (Curve C). Vol. 82, No. 5, 1977

6 1242 Y. IMAI, R. SATO, and T. IYANAGI P-450 (13-15), it was expected that an oxygenated species of the cytochrome might be detected during the aerobic steady state of the benzphetamine dependent oxidation of NADPH by the recon stituted system. Since a linear relationship be tween the rate of NADPH oxidation and the cytochrome P-450 concentration was observed only at cytochome concentrations lower than TABLE II. Effects of various substrates on the reduc tion of cytochrome P-450 and substrate-dependent NADPH oxidation. The rates of the two reactions were determined as described in " MATERIALS AND ME- THODS," except that the indicated substrate was added at the indicated concentration. The apparent rate con stant of cytochrome P-450 reduction was estimated from the half-time of reduction as described in Table I. 0.2 Đm, a cytochrome P-450 concentration of 0.1 Đm was chosen for this purpose and the dif ference spectrum was measured by using cuvettes with an optical path of 10 cm. As shown in Fig. 6, a difference spectrum having a peak at about 440 nm and a trough at about 410 nm was observed under aerobic conditions between the NADPHtreated and non-treated systems both containing benzphetamine. This difference spectrum is very similar to that reported for an oxygenated form of cytochrome P-450 in intact liver microsomes (5, 15). It disappeared when all the NADPH added was exhausted, as indicated by the cessation of decrease in the absorbance at 340 nm. When the same experiment was carried out under anaerobic conditions, the difference spectrum observed was almost identical with that reported between the dithionite-reduced and oxidized forms of purified cytochrome P-450 (16). It is, therefore, suggested that the spectrum observed under aerobic condi tions represented an oxygenated form of ferrous cytochrome P-450. Effects of Various Substrates-As reported previously (10), NADPH was oxidized very slowly by the reconstituted system in the absence of any substrate, although cytochrome P-450 was fully reducible by NADPH. The addition of various substrates to this system caused stimulation of NADPH oxidation to various extents (Imai, Y. & Sato, R., unpublished results). Among the sub strates tested, benzphetamine caused the highest (about 10-fold) stimulation, whereas aminopyrine was least effective, causing only about 1.5-fold enhancement (Table II). Under the same condi tions, except for the presence of CO instead of air, these substrates also enhanced the rate of cytochrome P-450 reduction by NADPH. As can be clearly seen from Table II, the rate constant for the cytochrome reduction was affected by the substrates closely in parallel with the rate of NADPH oxidation. However, the potential rate of cytochrome P-450 reduction estimated from the rate constant was about 10 times higher than the actual rate of NADPH oxidation, regardless of the kind of substrate used. Effect of ph-the benzphetamine-dependent NADPH oxidation activity of the reconstituted system had an optimal ph at about 7.5 (Imai, Y. & Sato, R., unpublished results). Since benzphet amine is insoluble in alkaline media, the rate of cytochrome P-450 reduction by NADPH in the absence and presence of benzphetamine was measured as a function of ph in the range from ph 6.25 to As shown in Fig. 7, the reduction rate in the absence of benzphetamine decreased as the ph was decreased. At any ph, addition of benzphetamine caused 7- to 10-fold stimulation of the reduction rate and the ph profile was similar to that for NADPH oxidation. Here again, the rate constant of cytochrome reduction can account for the potential rate of the cytochrome reduction, which is several times higher than the actual rate of NADPH oxidation over the entire ph range examined. Rate of Cytochrome P-448 Reduction-It is now established that multiple forms of cytochrome P-450 exist in liver microsomes (9,17-21). In addition to the major component in liver micro somes of phenobarbital-treated rabbits (7, 22), which was used in the experiments described above, J. Biochem

7 RECONSTITUTED MICROSOMAL DRUG HYDROXYLASE SYSTEM 1243 TABLE III. The rates of cytochrome P-448 reduction and NADPH oxidation in the reconstituted system con taining cytochrome P-448, instead of cytochrome P-450. The cytochrome reduction was measured as described in "MATERIALS AND METHODS," except that cyto chrome P-450 was replaced by cytochrome P-448 as in dicated, and kapp was estimated as in Table I. NADPH oxidation was determined in the system described in " MATERIALS AND METHODS." Where indicated, 1 mm benzphetamine was added. Fig. 7. Effects of ph on the rates of cytochrome P-450 reduction and benzphetamine-dependent NADPH oxi dation. The experiments were performed as described in Fig. I (cytochrome P-450 reduction) and in " MA- TERIALS AND METHODS," except that phosphate buffer of the indicated ph was used. The apparent rate constant of cytochrome reduction was estimated from the half-time as described in Table I. Curve A, cytochrome P-450 reduction in the presence of benzphetamine; Curve B, cytochrome P-450 reduction in the absence of benzphetamine; Curve C, benzphetamine (I mm)- dependent NADPH oxidation. at least two cytochrome P-450 preparations have been purified to homogeneity in our laboratory; one from liver microsomes of 3-methylcho lanthrene-treated rabbits (8) and the other from phenobarbital-treated rabbits (9). These two cyto chromes show a Soret absorption peak at 448 nm, instead of at 450 nm, in their reduced CO complex form and, therefore, belong to the class called cytochrome P-448. They are practically incapable of catalyzing benzphetamine-dependent NADPH oxidation in the presence of NADPH-cytochrome c reductase, though they are fully reducible by NADPH. In agreement with this property, ad dition of benzphetamine to the reconstituted system containing either of these cytochrome P-448's, instead of cytochrome P-450, caused no stimulation of cytochrome reduction, the rate constant of which was nearly equal to that of cytochrome MC, 3-Methylcholanthrene; PB, phenobarbital. P-450 reduction in the absence of the substrate (Table III). As will be reported elsewhere, a third form of cytochrome P-450 has been partially purified from liver microsomes of phenobarbitaltreated rabbits. This cytochrome is very similar in spectral properties to the phenobarbital-inducible one mainly used in this study but is clearly dif ferent from it in catalytic activity and ESR signals. The reduction of this cytochrome by NADPH in the reconstituted system was not stimulated by the addition of benzphetamine (data not shown). DISCUSSION Recent studies have provided evidence that the NADPH-dependent monooxygenase reaction cata lyzed by liver microsomes involves sequential transitions of cytochrome P-450, as shown in Fig. 8 (1, 2). In this scheme, it is assumed that the two electrons required for the overall process are separately introduced from NADPH (via NADPHcytochrome c reductase) to the cytochrome P-450 substrate complex before and after the binding of molecular oxygen. On the other hand, Guengerich et al. (3) have proposed that the two electrons are simultaneously donated to the ferric cytochrome P-450-substrate complex before oxygen binding. In the latter mechanism the second electron is Vol. 82, No. 5, 1977

8 1244 Y. IMAI, R. SATO, and T. IYANAGI TABLE IV. Kinetic parameters of the benzphetamine N-demethylation reaction and its partial reactions cata lyzed by the reconstituted system. Fig. 8. Proposed mechanism for the hydroxylation reaction catalyzed by cytochrome P-450. S and SO represent the substrate and the hydroxylated product, respectively. thought to be bound by an unknown site of the ferrous cytochrome and to appear at the heme site only after oxygen binding (3). However, their results, obtained by equilibrium experiments, do not necessarily exclude the possibility of separate introduction of the electrons. In the present study, the rate constants of substrate binding to ferric cytochrome P-450 and the reduction of the cytochrome-substrate com plex by NADPH, the first and second steps, re spectively, of the monooxygenase reaction, were measured in a system reconstituted from purified cytochrome P-450 and NADPH-cytochrome c reductase, and the potential rates of these partial reactions were compared with the actual rate of the substrate-dependent NADPH oxidation, a measure of the overall monooxygenase reaction. The results obtained indicate that both partial reactions, when measured with benzphetamine as a substrate, are 10 times or more faster than the NADPH oxidation and, therefore, cannot be ratelimiting steps of the whole process. The third step in the reaction sequence, i.e. the binding of oxygen to the cytochrome-substrate complex, must be extremely rapid because the formation of a spectral intermediate called Complex I in the reaction of oxygen with cytochrome P-450 has been shown to be completed in the dead time of the stopped-flow apparatus used by Guengerich et al. (14). Moreover, it has been reported that the second-order rate constant of the reaction between the reduced form of bacterial cytochrome P-450cam and oxygen to yield the oxygenated form is 7.7 x 10, M-1 s-1 at 4 Ž (13), a value which corresponds a Calculated from Ref. 14. b Calculated from Ref. 15, c Calculated from Ref. 23. d Calculated from the data of Imai, Y. & Sato, R. (unpublished results). to an apparent first-order rate constant of about 200 s-1, in a medium saturated with air at atmos pheric pressure. In Fig. 8 it is assumed that the final step of the monooxygenase reaction is the decomposition of the intermediate, a monooxy-ferric cytochrome P-450-substrate complex which is analogous to Compound I of catalase and peroxidase, to release the hydroxylated product with concomitant regeneration of ferric cytochrome P-450. Since this step is also involved in the mechanism proposed for hydroperoxide-dependent hydroxylation catalyzed by cytochrome P-450 in the absence of NADPH and molecular oxygen (2, 23), a minimal rate for this step may be estimated from the hy drogen peroxide-dependent N-demethylation of benzphetamine by purified cytochrome P-450. The maximal velocity (Vmax) of this reaction has been estimated to be 140 nmol per min per nmol of cytochrome P-450 (23), a value which is higher than the rate of the overall monooxygenation process. Table IV summarizes the rate constants and related kinetic parameters for the partial reactions discussed above as well as the overall rates of NADPH oxidation and formaldehyde formation in the N-demethylation of benzphetamine cata lyzed by the reconstituted system. It can be seen that only about 60% of the NADPH oxidized in response to the addition of benzphetamine is accompanied by the formation of formaldehyde J. Biochem.

9 RECONSTITUTED MICROSOMAL DRUG HYDROXYLASE SYSTEM 1245 from the substrate, as already reported (24). It is likely that this uncoupled oxidation is due to the auto-decomposition of an oxygenated form(s) of cytochrome P-450, as in the case of bacterial cyto chrome P-450cam (12, 25). At any rate, the data in Table IV indicate that the potential rates of all the partial reactions considered are higher than the actual rate of overall benzphetamine N-demethylation. Although the reduction of cytochrome P-450 was followed in the present study by measuring the formation of the CO complex, the binding of CO to ferrous cytochrome P-450 is too fast to limit the entire process. Thus, the second-order rate constant of CO binding has been determined by flash photol ysis experiments to be 2.4 x lob M-1 s-1 at 25 Ž (15). This value gives an apparent first-order rate constant of 200s-1, when the reaction mixture is saturated with CO at atmospheric pressure, a value which is much higher than that obtained for the cytochrome reduction. Therefore, it can be concluded that the overall rate of benzphetamine N-demethylation is limited by a certain step be tween the introduction of the second electron to the oxygenated ferrous cytochrome-substrate com plex and the liberation of water with concomitant formation of the postulated monooxy-cytochrome substrate complex. This conclusion is consistent with the observation that a difference spectrum closely resembling that reported for an oxygenated form of cytochrome P-450 in intact liver micro somes (5, 15) appears during the aerobic steady state. Guengerich et al. (14) have also demon strated that the rate-limiting step of the reaction of oxygen with the ferrous cytochrome P-450-substrate complex to form the hydroxylated product is the decomposition of an intermediate termed Com plex II. Although the reduction of the cytochrome substrate complex is not the rate-limiting step of the overall reaction, as discussed above, the results obtained in this study indicate that the addition of various substrates to the reconstituted system stimulates both the reduction of the cytochrome substrate complex and the substrate-dependent NADPH oxidation in a closely parallel fashion. The two processes are also similarly dependent on both reactions are affected in parallel by ph, at least on the acidic side of the optimum. These findings suggest that the binding of a substrate to cytochrome P-450 increases not only the rate of cytochrome P-450 reduction but also that of the rate-limiting step of the overall reaction in a simi lar manner. It is likely that the other partial reactions which are not limiting the overall rate are also similarly affected by the substrate binding. Nothing, however, is known of the mechanism by which the substrate binding induces these interrelated effects. In the intact microsomal membrane both cytochrome P-450 and NADPH-cytochrome c reductase are thought to be inserted into the fluid lipid bilayer. In the reconstituted system, on the other hand, the two amphipathic proteins solu bilized and purified with the aid of detergents are mixed in an aqueous medium in the presence of low concentrations of detergents, though it is not yet clear whether or not the two proteins are integrated into a functional complex. In the two systems, therefore, the circumstances in which the reaction takes place appear to be rather different. Nevertheless, the rate constants reported for the substrate binding to cytochrome P-450 and the reduction of the cytochrome P-450-substrate com plex in intact microsomes fall in the same order of magnitude as those determined in this study for the reconstituted system. Thus, the rate constant of hexobarbital binding in liver microsomes has been reported to be 35 s'1 (12) and that of the re duction of cytochrome P-450 in the presence of ethylmorphine to be 1.7 s-1 (6). As mentioned earlier, it has also been reported that the ratelimiting step of the overall drug hydroxylation reaction by liver microsomes occurs after sub strate binding to cytochrome P-450 (6). It may, therefore, be concluded that the reaction proceeds by a very similar mechanism in both the intact microsomal and reconstituted systems. Finally, the reason for the biphasic kinetics of cytochrome P-450 reduction in the reconstituted system is unclear at present, but it should be noted that the reduction of cytochrome P-450 in intact liver microsomes has also been shown to proceed in a biphasic fashion (6). the benzphetamine concentration in proportion to the amount of the cytochrome P-450-benzphet amine complex formed. Moreover, the rates of Vol. 82, No. 5, 1977

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