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The Rate-determining Step in P450 C21-catalyzing Reactions in a Membrane-reconstituted System

2001; Elsevier BV; Volume: 276; Issue: 14 Linguagem: Inglês

10.1074/jbc.m006043200

1083-351X

Shiro Kominami, Akiko Owaki, Tsuyoshi Iwanaga, Hiroko Tagashira-Ikushiro, Takeshi Yamazaki,

Estrogen and related hormone effects

Adrenal cytochrome P450 C21 in a membrane-reconstituted system catalyzed 21-hydroxylation of 17α-hydroxyprogesterone at a rate higher than that for progesterone in the steady state at 37 °C. The rate of product formation in the steady state increased with the concentration of the complex between P450 C21 and the reductase in the membranes. The complex formation was independent of the volume of the reaction, showing that the effective concentrations of the membrane proteins should be defined with the volume of the lipid phase. The rates of conversion of progesterone and 17α-hydroxyprogesterone to the product in a single cycle of the P450 C21 reaction were measured with a reaction rapid quenching device. The first-order rate constant for the conversion of progesterone by P450 C21 was 4.3 ± 0.7 s−1, and that for 17α-hydroxyprogesterone was 1.8 ± 0.5 s−1 at 37 °C. It was found from the analysis of kinetic data that the rate-determining step in 21-hydroxylation of progesterone in the steady state was the dissociation of product from P450 C21, whereas the conversion to deoxycortisol was the rate-determining step in the reaction of 17α-hydroxyprogesterone. The difference in the rate-determining steps in the reactions for the two substrates was clearly demonstrated in the pre-steady-state kinetics. Adrenal cytochrome P450 C21 in a membrane-reconstituted system catalyzed 21-hydroxylation of 17α-hydroxyprogesterone at a rate higher than that for progesterone in the steady state at 37 °C. The rate of product formation in the steady state increased with the concentration of the complex between P450 C21 and the reductase in the membranes. The complex formation was independent of the volume of the reaction, showing that the effective concentrations of the membrane proteins should be defined with the volume of the lipid phase. The rates of conversion of progesterone and 17α-hydroxyprogesterone to the product in a single cycle of the P450 C21 reaction were measured with a reaction rapid quenching device. The first-order rate constant for the conversion of progesterone by P450 C21 was 4.3 ± 0.7 s−1, and that for 17α-hydroxyprogesterone was 1.8 ± 0.5 s−1 at 37 °C. It was found from the analysis of kinetic data that the rate-determining step in 21-hydroxylation of progesterone in the steady state was the dissociation of product from P450 C21, whereas the conversion to deoxycortisol was the rate-determining step in the reaction of 17α-hydroxyprogesterone. The difference in the rate-determining steps in the reactions for the two substrates was clearly demonstrated in the pre-steady-state kinetics. cytochrome P450 having ethanol oxidation activity cytochrome P450 having steroid 21-hydroxylase activity cytochrome P450 having steroid 11β-hydroxylase activity cytochrome P450 having steroid 17α-hydroxylase activity high performance liquid chromatography Microsomal cytochrome P450 isozymes are integral membrane proteins responsible for the metabolism of various exogenous and endogenous compounds, including steroid hormones. The monooxygenase reaction of P450 requires two electrons and an oxygen molecule. NADPH-cytochrome P450 reductase in the microsomal membranes supplies the first and the second electrons to P450s in the same membrane. Although the supply of the second electron to P450 is often the rate-limiting step in P450 reactions, results of recent experiments have revealed that some P450 reactions have rate-determining steps other than in the electron supply (1Bell L.C. Guengerich F.P. J. Biol. Chem. 1997; 272: 29643-29651Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 2Bell-Parikh L.C. Guengerich F.P. J. Biol. Chem. 1999; 274: 23833-23840Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 3Imai T. Yamazaki T. Kominami S. Biochemistry. 1998; 37: 8097-8104Crossref PubMed Scopus (30) Google Scholar, 4Yamazaki T. Ohno T. Sakaki T. Akiyoshi-Shibata M. Yabusaki Y. Imai T. Kominami S. Biochemistry. 1998; 37: 2800-2806Crossref PubMed Scopus (43) Google Scholar, 5Tagashira H. Kominami S. Takemori S. Biochemistry. 1995; 34: 10939-10945Crossref PubMed Scopus (25) Google Scholar). Bell and Guengerich (1Bell L.C. Guengerich F.P. J. Biol. Chem. 1997; 272: 29643-29651Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar) clearly showed in the oxidation of ethanol catalyzed by P450 2E11 that the kinetic deuterium isotope effect on Km with no effect onkcat was attributed to the rate-limiting product release. P450 2E1 catalyzes the oxidation of ethanol to acetic acid via acetaldehyde, in which about 90% of the intermediate acetaldehyde is directly converted to acetic acid without dissociation from the active site of the enzyme (2Bell-Parikh L.C. Guengerich F.P. J. Biol. Chem. 1999; 274: 23833-23840Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). They showed for the first time in P450 reactions that the reactions for two substrates catalyzed by one molecular species of P450 could have different rate-determining steps (1Bell L.C. Guengerich F.P. J. Biol. Chem. 1997; 272: 29643-29651Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). We demonstrated in studies on the successive reactions of P450 11β and P450 17α that the rates of product formation in the steady state were regulated by the rates of product release from the enzymes (3Imai T. Yamazaki T. Kominami S. Biochemistry. 1998; 37: 8097-8104Crossref PubMed Scopus (30) Google Scholar, 4Yamazaki T. Ohno T. Sakaki T. Akiyoshi-Shibata M. Yabusaki Y. Imai T. Kominami S. Biochemistry. 1998; 37: 2800-2806Crossref PubMed Scopus (43) Google Scholar, 5Tagashira H. Kominami S. Takemori S. Biochemistry. 1995; 34: 10939-10945Crossref PubMed Scopus (25) Google Scholar). P450 17α and P450 11β catalyze multistep reactions for the formation of androgens and aldosterone, respectively, where the final products are formed from fractions of the intermediates that do not dissociate from the enzymes (3Imai T. Yamazaki T. Kominami S. Biochemistry. 1998; 37: 8097-8104Crossref PubMed Scopus (30) Google Scholar, 4Yamazaki T. Ohno T. Sakaki T. Akiyoshi-Shibata M. Yabusaki Y. Imai T. Kominami S. Biochemistry. 1998; 37: 2800-2806Crossref PubMed Scopus (43) Google Scholar, 5Tagashira H. Kominami S. Takemori S. Biochemistry. 1995; 34: 10939-10945Crossref PubMed Scopus (25) Google Scholar). The same mechanism for diminishing the rate of dissociation of final products from P450 2E1, P450 11β, and P450 17α might decrease the dissociation rates of the intermediates, facilitating the successive monooxygenase reactions in these P450s without the intermediate metabolites leaving the enzyme. It is of interest to examine whether the product release can be the rate-determining step for P450 that does not catalyze multistep reactions. The interaction between NADPH-cytochrome P450 reductase and P450 has been investigated by several laboratories in the systems reconstituted with detergents (6Kominami S. Hara H. Ogishima T. Takemori S. J. Biol. Chem. 1984; 259: 2991-2999Abstract Full Text PDF PubMed Google Scholar, 7Sevrukova I.F. Kanaeva I.P. Koen Y.M. Samenkova N.F. Bachmanova G.I. Archakov A.I. Arch. Biochem. Biophys. 1994; 311: 133-143Crossref PubMed Scopus (13) Google Scholar) and dilauroylphosphatidylcholine (8French J.S. Guengerich F.P. Coon M.J. J. Biol. Chem. 1980; 255: 4112-4119Abstract Full Text PDF PubMed Google Scholar, 9Oprian D.D. Vatsis K.P. Coon M.J. J. Biol. Chem. 1979; 254: 8895-8902Abstract Full Text PDF PubMed Google Scholar, 10Voznesensky A.I. Schenkman J.B. J. Biol. Chem. 1992; 267: 14669-14676Abstract Full Text PDF PubMed Google Scholar, 11Schen S. Strobel H.W. Biochemistry. 1994; 33: 8807-8812Crossref PubMed Scopus (23) Google Scholar, 12Backes W.L. Eyer C.S. J. Biol. Chem. 1989; 264: 6252-6259Abstract Full Text PDF PubMed Google Scholar, 13Guengerich F.P. Johnson W.W. Biochemistry. 1997; 36: 14741-14750Crossref PubMed Scopus (155) Google Scholar). Miwaet al. (14Miwa G.T. Lu A.Y.H. Arch. Biochem. Biophys. 1984; 234: 161-166Crossref PubMed Scopus (54) Google Scholar, 15Miwa G.T. West S.B. Huang M. Lu A.Y.H. J. Biol. Chem. 1979; 254: 5695-5700Abstract Full Text PDF PubMed Google Scholar), using a phospholipid vesicular membrane system, found that the active species for the P450-catalyzing monooxygenase reaction in the steady state was the binary complex consisting of P450 and the reductase. Kawato and co-workers (16Gut J. Richter C. Cherry R.J. Winterhalter K.H. Kawato S. J. Biol. Chem. 1982; 257: 7030-7036Abstract Full Text PDF PubMed Google Scholar, 17Kawato S. Gut J. Cherry R.J. Winterhalter K.H. Richter C. J. Biol. Chem. 1982; 257: 7023-7029Abstract Full Text PDF PubMed Google Scholar, 18Etter H.U. Richter C. Ohta Y. Winterhalter K.H. Sasabe H. Kawato S. J. Biol. Chem. 1991; 266: 18600-18605Abstract Full Text PDF PubMed Google Scholar, 19Iwase T. Sasaki T. Yabusaki Y. Ohkawa H. Ohta Y. Kawato S. Biochemistry. 1991; 30: 8347-8351Crossref PubMed Scopus (18) Google Scholar, 20Yamada M. Ohta Y. Sasaki T. Yabusaki Y. Ohkawa H. Kawato S. Biochemisty. 1999; 38: 9465-9470Crossref PubMed Scopus (4) Google Scholar) have extensively studied the interaction between P450 and the reductase in phospholipid vesicles by measuring the rotational diffusion of P450s in the membranes, and they suggested that the mode of interaction depends on the individual P450. Rotational diffusion measurement of P450 C21 in liposomal membranes revealed a complex formation with the reductase (21Ohta Y. Kawato S. Tagashira H. Takemori S. Kominami S. Biochemistry. 1992; 31: 12680-12687Crossref PubMed Scopus (46) Google Scholar). In this study, we obtained experimental evidence that the effective concentrations of P450 C21 and NADPH-P450 reductase for the complex formation should be defined with the volume of the lipid phase of the membranes rather than the total volume of the reaction solution. Two types of cytochrome P450 function in the endoplasmic reticulum of the adrenal cortex (22Takemori S. Kominami S. Trends Biochem. Sci. 1984; 9: 393-396Abstract Full Text PDF Scopus (47) Google Scholar, 23Higuchi A. Kominami S. Takemori S. Biochim. Biophys. Acta. 1991; 1084: 240-246Crossref PubMed Scopus (28) Google Scholar): P450 C21, catalyzing steroid 21-hydroxylation (24Kominami S. Ochi H. Kobayashi Y. Takemori S. J. Biol. Chem. 1980; 255: 3386-3394Abstract Full Text PDF PubMed Google Scholar, 25Yoshioka H. Morohashi K. Sogawa K. Yamane M. Kominami S. Takemori S. Okada Y. Omura T. Fujii-Kuriyama Y. J. Biol. Chem. 1986; 261: 4106-4109Abstract Full Text PDF PubMed Google Scholar), and P450 17α, catalyzing steroid 17α-hydroxylation and androgen formation (26Kominami S. Shinzawa K. Takemori S. Biochem. Biophys. Res. Commun. 1982; 109: 916-921Crossref PubMed Scopus (94) Google Scholar, 27Shinzawa K. Kominami S. Takemori S. Biochim. Biophys. Acta. 1985; 833: 151-160Crossref PubMed Scopus (62) Google Scholar). Some of the progesterone originally produced from cholesterol is hydroxylated to 17α-hydroxyprogesterone, a part of which is further metabolized to androstenedione without dissociation from P450 17α (5Tagashira H. Kominami S. Takemori S. Biochemistry. 1995; 34: 10939-10945Crossref PubMed Scopus (25) Google Scholar). Progesterone and 17α-hydroxyprogesterone are the physiological substrates for P450 C21 and are converted to deoxycorticosterone and deoxycortisol, respectively. The activity of P450 C21 for the reaction of 17α-hydroxyprogesterone is higher than that for progesterone in bovine and guinea pig microsomes and also in the reconstituted systems (23Higuchi A. Kominami S. Takemori S. Biochim. Biophys. Acta. 1991; 1084: 240-246Crossref PubMed Scopus (28) Google Scholar, 24Kominami S. Ochi H. Kobayashi Y. Takemori S. J. Biol. Chem. 1980; 255: 3386-3394Abstract Full Text PDF PubMed Google Scholar). The difference in the activities of P450 C21 in the reactions of the two substrates has been investigated by various methods. The rate of first electron transfer from the reductase to P450 C21 in the presence of progesterone was not much different from that in the presence of 17α-hydroxyprogesterone, where the high spin content of the P450 C21–17α-hydroxyprogesterone complex was higher than that of the P450 C21-progesterone complex (28Kominami S. Takemori S. Biochim. Biophys. Acta. 1982; 709: 147-153Crossref PubMed Scopus (12) Google Scholar). The rates of substrate binding to P450 C21 in liposomal membranes do not differ much between the two substrates (29Kominami S. Itoh Y. Takemori S. J. Biol. Chem. 1986; 261: 2077-2083Abstract Full Text PDF PubMed Google Scholar). To elucidate why the activity of P450 C21 in the steady state is higher for 17α-hydroxyprogesterone than for progesterone, it is necessary to determine the rate-determining step in 21-hydroxylation reactions of progesterone and 17α-hydroxyprogesterone. We performed kinetic studies on P450 C21-catalyzing hydroxylation reactions of progesterone and 17α-hydroxyprogesterone in the steady state, in single turnover experimental conditions, and in the pre-steady state. Cytochrome P450 C21 and NADPH-cytochrome P450 reductase were purified from bovine adrenocortical and hepatic microsomes, respectively, according to methods described previously (24Kominami S. Ochi H. Kobayashi Y. Takemori S. J. Biol. Chem. 1980; 255: 3386-3394Abstract Full Text PDF PubMed Google Scholar, 30Takemori S. Kominami S. Nozaki M. Yamamoto S. Ishimura Y. Coon M, J. Ernster I. Estabrook R.W. Oxygenase and Oxygen Metabolism. Academic Press, Inc., New York1982: 403-408Google Scholar). The purified P450 C21 was incorporated into unilamellar vesicular membranes by the cholate dialysis method using phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine at a molar ratio of 5:3:1 (31Kominami S. Inoue S. Higuchi A. Takemori S. Biochim. Biophys. Acta. 1989; 985: 293-299Crossref PubMed Scopus (38) Google Scholar). P450 C21 proteoliposomes used in this experiment contained about 0.65 nmol of P450 C21 per mg of the phospholipids, which corresponds to one molecule of P450 C21 in about 2000 molecules of phospholipids. A certain amount of the reductase was incorporated in the P450 C21 proteoliposomes by incubation with the preformed P450 C21 proteoliposomes at 0 °C for 1 h (32Kominami S. Ikushiro S. Takemori S. Biochim. Biophys. Acta. 1987; 905: 143-150Crossref PubMed Scopus (9) Google Scholar). The amount of P450 in the reaction solution was determined from the dithionite-reduced CO difference spectra (33Omura T. Sato R. J. Biol. Chem. 1964; 239: 2370-2378Abstract Full Text PDF PubMed Google Scholar). In some cases, the concentration of P450 in the membrane reconstituted system is expressed in mol of P450/liter of the lipid phase of the membranes under the assumption that 1 g of phospholipids occupies 1 ml (34Huang C. Mason J.T. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 308-310Crossref PubMed Scopus (543) Google Scholar). The rates of 21-hydroxylation of P450 C21 proteoliposomes for progesterone and 17α-hydroxyprogesterone were measured at 37 °C using3H-labeled substrates (31Kominami S. Inoue S. Higuchi A. Takemori S. Biochim. Biophys. Acta. 1989; 985: 293-299Crossref PubMed Scopus (38) Google Scholar). In general, 0.5 ml of 50 mm potassium phosphate buffer (pH 7.2) contained 10 pmol of P450 C21 and various amounts of the reductase (2.5–25 pmol) in the liposomal membranes (0.015 mg of phospholipids), 10 nmol of progesterone or 17α-hydroxyprogesterone with corresponding3H-labeled steroid (0.5 μCi), and 0.1 mmEDTA. The reaction was initiated by the addition of 100 nmol of NADPH and terminated by mixing with 1 ml of chloroform containing14C-labeled androstenedione (0.005 μCi). The radioactivity of 14C-labeled androstenedione was used for the estimation of the recovery of steroids in the assay procedures. The extracted steroids were separated with an HPLC system described previously (23Higuchi A. Kominami S. Takemori S. Biochim. Biophys. Acta. 1991; 1084: 240-246Crossref PubMed Scopus (28) Google Scholar, 31Kominami S. Inoue S. Higuchi A. Takemori S. Biochim. Biophys. Acta. 1989; 985: 293-299Crossref PubMed Scopus (38) Google Scholar). To determine the rate of conversion from progesterone or 17α-hydroxyprogesterone to the 21-hydroxylated product under single turnover reaction conditions, rapid quenching experiments were performed at 37 °C with a rapid quenching device (UNISOKU MX-200) equipped with four cylinders and two mixers (35Iwanaga T. Yamazaki T. Kominami S. Biochemistry. 1999; 38: 16629-16635Crossref PubMed Scopus (28) Google Scholar). Solutions A and B were mixed rapidly with mixer 1 and stored in a reaction coil. The reacted solution was pushed out from the coil with solution C and was mixed rapidly in mixer 2 with solution D, the termination solution. In the single turnover experiment of P450 C21, solution A (100 μl) contained 50 pmol of P450 C21 proteoliposomes with various amounts of the reductase (50–300 pmol) and 2.5 pmol of the 3H-substrate (0.25 μCi) in 50 mm potassium phosphate buffer (pH 7.2) with 0.1 mm EDTA. Solution B (100 μl) contained 100 nmol of NADPH and 100 nmol of the unlabeled substrate in the buffer. Solution C (200 μl) was the buffer solution, and solution D (200 μl) was 1m HCl. Under these experimental conditions, only the reaction of 3H-substrate bound to P450 C21 at the initial stage of the reaction could be detected. 3H-substrate or3H-product dissociated from P450 C21 could not be metabolized again, because of the presence of an excess amount of unlabeled substrate in the reaction solution. The steroids were extracted with chloroform after termination of the reaction and separated by HPLC. The pre-steady-state kinetics of P450 C21-catalyzing reactions were studied using a UNISOKU MX-200. Solution A (100 μl) contained 5 pmol of P450 C21 proteoliposomes with 5 pmol of the reductase and 100 pmol of3H-substrate (1 μCi) in the reaction buffer. Solution B (100 μl) contained 10 nmol of NADPH but did not contain an unlabeled substrate. The contents of solutions C and D are the same as those in the single turnover experiments. In these reaction conditions, multicycle turnover reactions of P450 C21 can occur. The time courses of P450 C21-catalyzing reactions of progesterone and 17α-hydroxyprogesterone were measured in the initial 3 s at 37 °C. Computer simulations for the pre-steady-state kinetics were performed with HopKINSIM version 1.7.2 provided by D. Wachsstock (Johns Hopkins University) using a Macintosh computer (iMac; Apple Computer Inc., Cupertino, CA) equipped with FPU 3.07 (John Neil & Associates, Cupertino, CA) (36Barshop B.A. Wrenn R.F. Frieden C. Anal. Biochem. 1983; 130: 134-145Crossref PubMed Scopus (670) Google Scholar, 37Frieden C. Trends Biochem. Sci. 1993; 18: 58-60Abstract Full Text PDF PubMed Scopus (53) Google Scholar). [1,2,6,7-3H]progesterone and [4-14C]androstenedione were obtained from PerkinElmer Life Sciences. 17α-[3H]Hydroxyprogesterone was produced from 3H-labeled progesterone by an enzymatic reaction using P450 17α-proteolipsomes and purified with HPLC (31Kominami S. Inoue S. Higuchi A. Takemori S. Biochim. Biophys. Acta. 1989; 985: 293-299Crossref PubMed Scopus (38) Google Scholar).l-α-Phosphatidylcholine from egg yolk was obtained from Sigma, and l-α-phosphatidylethanolamine from egg yolk andl-α-phosphatidylserine from bovine spinal cord were from Lipid Products (Surrey, United Kingdom). Other chemicals were of the highest grade commercially available. The reaction system of P450 C21 was reconstituted in liposomal membranes. The incorporation of P450 C21 and NADPH-P450 reductase in the liposome membranes was confirmed by density gradient centrifugation (31Kominami S. Inoue S. Higuchi A. Takemori S. Biochim. Biophys. Acta. 1989; 985: 293-299Crossref PubMed Scopus (38) Google Scholar, 32Kominami S. Ikushiro S. Takemori S. Biochim. Biophys. Acta. 1987; 905: 143-150Crossref PubMed Scopus (9) Google Scholar). Almost all of the P450 C21 in the proteoliposomes is reduced upon the addition of the reductase and NADPH, suggesting that the heme domain of P450 C21 interacts with the functional domain of the reductase on the outer surface of the vesicles. Fig.1 a shows the time courses of 21-hydroxylation of progesterone and 17α-hydroxyprogesterone catalyzed by P450 C21 at 37 °C in the presence of NADPH-P450 reductase at an equimolar amount of P450 C21 (10 pmol) in the liposome membranes (15 μg of phospholipids in 0.5 ml of reaction solution). The amounts of deoxycortisol and deoxycorticosterone, which were the 21-hydroxylated products from 17α-hydroxyprogesterone and progesterone, respectively, increased linearly with the reaction time. The rates of product formation by P450 C21, which were calculated from the slopes of the lines, were 0.33 ± 0.04 and 0.17 ± 0.03 nmol/min for 17α-hydroxyprogesterone and progesterone, respectively. The rates of product formation both from progesterone and 17α-hydroxyprogesterone were not altered by change in the volume of the reaction solution as long as the total amounts of P450 C21, the reductase, and phospholipids were kept constant in the reaction solution, as shown in Fig. 1 b. The rate of product formation in the steady state increased hyperbolically with the amount of the reductase in the membranes as shown in Fig.2, where the rate for 17α-hydroxyprogesterone was almost twice that for progesterone in the concentration range observed. An increase in the rate of product formation by various P450s has been observed with the increase in the amount of the reductase in the reaction solution and has been attributed to formation of an active complex between P450 and the reductase (14Miwa G.T. Lu A.Y.H. Arch. Biochem. Biophys. 1984; 234: 161-166Crossref PubMed Scopus (54) Google Scholar, 15Miwa G.T. West S.B. Huang M. Lu A.Y.H. J. Biol. Chem. 1979; 254: 5695-5700Abstract Full Text PDF PubMed Google Scholar). The dissociation constant of the active P450 C21-reductase complex (1:1, mol/mol) was estimated by the method of Miwa et al. (15Miwa G.T. West S.B. Huang M. Lu A.Y.H. J. Biol. Chem. 1979; 254: 5695-5700Abstract Full Text PDF PubMed Google Scholar). We obtained 16 ± 4 and 15 ± 4 nm for Kd(app), the apparent dissociation constants for the P450 C21-reductase complex, from the dependence of the rates of 21-hydroxylations of 17α-hydroxyprogesterone and progesterone, respectively. The apparent dissociation constants were calculated under the assumption that P450 C21 and the reductase are distributed homogeneously in the reaction solution. These Kd(app) values are a little smaller than those reported for other P450 systems (14Miwa G.T. Lu A.Y.H. Arch. Biochem. Biophys. 1984; 234: 161-166Crossref PubMed Scopus (54) Google Scholar, 38Muller-Enoch D. Churchill P. Fleischer S. Guengerich F.P. J. Biol. Chem. 1984; 259: 8174-8182Abstract Full Text PDF PubMed Google Scholar). Below, we discuss the calculation of dissociation constants of the complex using effective concentrations of the enzymes defined with the volume of lipid phase of the membranes. Vmax, which is the rate in the presence of an excess amount of the reductase with 0.01 nmol of P450 C21, was 0.78 ± 0.06 nmol of deoxycortisol produced per min for the reaction of 17α-hydroxyprogesterone and 0.48 ± 0.06 nmol of deoxycorticosterone produced per min for progesterone. Thelines in Fig. 2 are the theoretical curves drawn with the above values. It was remarkable that Vmax was about 2 times higher for the 17α-hydroxyprogesterone reaction than for the progesterone reaction but that Kd(app) was about the same for both substrates, suggesting that the difference in the substrates had little effect on the interaction between P450 C21 and the reductase. To obtain the rate of conversion of the substrate to the 21-hydroxylated product in the single turnover reaction, a solution containing a P450-reductase-3H-substrate ternary complex was mixed rapidly with excess amounts of NADPH and the unlabeled substrate. Since3H-substrate and3H-products once released from the enzyme do not rebind to P450 C21 in the presence of an excess amount of unlabeled substrate, only the change in the3H-substrate in the P450 C21-reductase complex at the initiation of the reaction is observable under these conditions. Fig.3, a and b, shows the time courses of reactions of progesterone and 17α-hydroxyprogesterone, respectively, under the single turnover conditions at 37 °C. The amount of [3H]progesterone decreased exponentially with the reaction time, and the decrease corresponded exactly to the increase in 21-hydroxylated3H-product, [3H]deoxycorticosterone. The decrease in 17α-[3H]hydroxyprogesterone and the corresponding increase in [3H]deoxycortisol were apparently slower than those for progesterone. The first-order rate constants for reactions of progesterone and 17α-hydroxyprogesterone, which were obtained by fitting the observed data to single exponential curves, were 4.3 and 1.8 s−1, respectively. It is concluded that the higher activity of P450 C21 for the reaction of 17α-hydroxyprogesterone in the steady state is not due to the higher conversion rate of 17α-hydroxyprogesterone than that of progesterone. The time courses for reactions of progesterone in the single turnover conditions were also dependent on the amount of the reductase in the reaction solution as shown in Fig. 4. The amount of [3H]progesterone that was converted to [3H]deoxycorticosterone in the single turnover reaction increased with the amount of the reductase in the membranes, but the rate of the conversion did not vary, remaining 4.0 ± 0.5 s−1. The increase in the amount of product in the single turnover condition could be attributed to the increase in the amount of active complex in the reaction solution.Figure 4Effects of the amount of NADPH-cytochrome P450 reductase on the single turnover experiments for the conversion of progesterone (open symbols) to deoxycorticosterone (closed symbols) catalyzed by P450 C21 proteoliposomes. The experiments were performed in the presence of a constant amount (50 pmol) of P450 C21 and 50, 100, and 300 pmol of the reductase in liposome membranes, corresponding to the data points of circles,squares, and triangles, respectively. Other experimental conditions are the same as those described in the legend to Fig. 3. All of the curves are drawn using the first-order rate constant of 4.0 s−1. The details are described under “Results.”View Large Image Figure ViewerDownload Hi-res image Download (PPT) We measured the time courses of the 21-hydroxylation reactions at 37 °C under the pre-steady-state reaction conditions using a rapid mixing device, UNISOKU MX-200. A solution (100 μl) containing equimolar amounts of P450 C21 and the reductase (5 pmol) and 100 pmol of radioactive substrate was mixed rapidly with an equal volume of NADPH solution, and the conversions of substrates were measured in the range of 0–3 s. Fig. 5, a and b, show the conversions of progesterone and 17α-hydroxyprogesterone to the products, respectively, in which the amount of deoxycorticosterone increased rapidly in the initial 300 ms with a slower linear increase after that, while the amount of deoxycortisol increased almost linearly up to 3 s. The burst increase of deoxycorticosterone in the pre-steady state showed that the rate of conversion from progesterone to deoxycorticosterone must be significantly faster than the rate of dissociation of product deoxycorticosterone from the enzyme (39Bender M.L. Begue-Canton M.L. Blakeley R.L. Brubacher L.J. Feder J. Gunter C.R. Kezdy F.J. Killheffer Jr., J.V. Marshall T.H. Miller C.G. Roeske R.W. Stoops J.K. J. Am. Chem. Soc. 1966; 88: 5890-5913Crossref PubMed Scopus (331) Google Scholar). Thelines in Figs. 5 are the theoretical curves drawn using the computer software HopKINSIM (36Barshop B.A. Wrenn R.F. Frieden C. Anal. Biochem. 1983; 130: 134-145Crossref PubMed Scopus (670) Google Scholar, 37Frieden C. Trends Biochem. Sci. 1993; 18: 58-60Abstract Full Text PDF PubMed Scopus (53) Google Scholar). If P450 C21 and the reductase were distributed homogeneously in the reaction solution, the effective concentrations of P450 C21 and the reductase would be decreased to one-tenth by a 10-fold increase in the volume of the reaction solution. Fig. 1 b shows, however, that there was almost no change in the rate of product formation with an increase in the volume of the reaction solution, suggesting that the formation of the active complex does not depend on the volume of the reaction solution. The concentrations of P450 C21 and the reductase in the lipid phase of the membranes are not affected by change in the volume of the reaction solution as long as the absolute amounts of P450 C21, the reductase, and the phospholipids are kept constant. P450 C21 proteoliposomes contain 1 mol of P450 C21 in 2000 × 770 g of phospholipids, where 770 is the average molecular weight of the phospholipids. The concentration of P450 C21 was 0.65 mm in the lipid phase under the assumption that 1 g of phospholipids occupies 1 ml of the volume in the membranes (34Huang C. Mason J.T. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 308-310Crossref PubMed Scopus (543) Google Scholar). The apparent dissociation constants, Kd(app), for P450 C21-reductase complex were calculated from the reductase dependence of the rate of product formation under the assumption of a homogeneous distribution of enzymes in the reaction solution using the equation Kd(app) = ((P/V)(R/V))/(PR/V), where P, R, and PR represent the absolute amounts (mol) of free P450C21, free reductase, and P450 C21-reductase complex, respectively, and V is the volume of the reaction solution (0.0005 liters in Fig. 2). The amount of P450 C21-reductase complex can be calculated using the equationPR (mol) = (observed rate of product formation/Vmax) × (total amount of P450 C21 (mol)), where Vmax is the rate when all of the P450 C21 is in the form of a complex with the reductase. The concentration of P450 C21 in the lipid phase can be defined asP/Lip, where Lip represents the volume in liters of the lipid phase of the membranes. The dissociation constant of the complex defined in the lipid phase can be written asKd(lip) = ((P/Lip)(R/Lip))/(PR/Lip) = Kd(app) × (V/Lip) (29Kominami S. Itoh Y. Takemori S. J. Biol. Chem. 1986; 261: 2077-2083Abstract Full Text PDF PubMed Google Scholar). In the experiments for Fig. 2 using 10 pmol of P450 C21, the volume of the lipid phase can be calculated as 10 × 10−12 × 2000 × 770 ml, and Vis 0.5 ml. The value for V/Lip is 3.2 × 104, and the value for Kd(lip) becomes 520 ± 120 μm for P450 C21-reductase complex, which must be the real dissociation constant for the P450 C21-reductase complex in the liposome membranes. A similar Kd(lip) value, about 0.5 mm, is calculated for a hepatic P450-reductase complex in egg yolk liposomes from the data of Miwa and Lu (14Miwa G.T. Lu A.Y.H. Arch. Biochem. Biophys. 1984; 234: 161-166Crossref PubMed Scopus (54) Google Scholar). In the experiments for Fig. 4, the amount of P450 C21 was kept constant (50 pmol in 0.077 mg of phospholipids), and the amount of reductase was increased from 50 to 100 and then to 300 pmol. The amount of [3H]progesterone hydroxylated by P450 C21 in the single turnover reaction increased with the amount of the reductase in the liposome membranes, but the rate of conversion of [3H]progesterone did not change. [3H]Deoxycorticosterone must be produced from the active ternary complex of P450 C21-[3H]progesterone-reductase that exists at the initial stage of the reaction. The amounts of active complex between P450 C21 and the reductase in the lipid phase can be calculated using the equation Kd(lip) = ((Pt −PR)/Lip)(Rt −PR)/Lip)/(PR/Lip), wherePt and Rt represent the total amounts of P450 C21 and the reductase (mol) in the reaction solution, respectively, andLip is 7.7 × 10−8 liters for the experiments in Fig. 4. The amounts of complex were calculated usingKd(lip) = 520 μm to be 21, 31, and 43 pmol in the presence of 50, 100, and 300 pmol of the reductase with 50 pmol of P450 C21 in the membranes, respectively. These are almost proportional to the amounts of product formed in the single turnover reactions (0.4, 0.55, and 0.7 pmol, respectively) in Fig. 4. The small amounts of product formation are attributed to the low concentration of 3H-substrate (2.5 pmol) in the reaction solution. On the other hand, in the model of P450 C21 and the reductase being distributed homogeneously in the reaction solution, the amounts of the complex calculated as 39, 47, and 49.4 pmol usingKd(app) do not increase significantly with an increase in the amount of the reductase. Fig. 4 clearly demonstrates that the effective concentrations of the membrane proteins should be defined in the lipid phase. To analyze the reaction mechanism of P450 C21 in the steady state, we divided one cycle of the P450 C21 reaction into three steps, as shown in Fig. 6. It is assumed that the complex between P450 C21 and the reductase is the active species in the reaction. The presence of an excess amount of the substrate prevents reactions in the reverse direction. The rate for the conversion of the substrate to the product obtained in the single turnover experiment corresponds to k2. The steady-state velocity of product formation can be written as follows (40Roberts D.V. Enzyme Kinetics. Cambridge University Press, Cambridge, United Kingdom1977: 35-40Google Scholar), v=k1k2k3[S][E0]/((k3k2+k1[S](k2+k3))Equation 1 where [E0] represents the concentration of the active complex. This equation can be simplified asv/[E0] =k2 k3/(k2+ k3) in the presence of an excess amount of substrate, wherek2 k3/k1[S] is much smaller than k2 + k3. We can calculate the values of k3 for the reactions of progesterone and 17α-hydroxyprogesterone in the steady state using the observed values of Vmax for v, the rates of conversion of the substrate, k2, and the total amount of P450 C21 in the reaction solution. In the presence of an excess amount of the reductase, nearly all P450 C21 in the reaction solution must be in the form of an active complex, and the rate of product formation can be expressed as Vmax. The calculated values for k3 are listed together with the values of Vmax andk2 in TableI. It is quite interesting that the rate of dissociation of the product from the enzyme,k3, was about one-fourth less than the rate of the conversion of the substrate, k2, in the reaction of progesterone, whereas k3 was about 2.5 times larger than k2 for 17α-hydroxyprogesterone. It is clarified that the rate-determining steps for the reactions of progesterone and 17α-hydroxyprogesterone are the dissociation of the product and the conversion of the substrate, respectively.Table IKinetic parameters for P450 C21 reactions in the membrane reconstituted systemSubstrateVmaxk2k3nmol product/mins−1s−1Progesterone0.48 ± 0.064.3 ± 0.71.0 ± 0.217α-Hydroxyprogesterone0.78 ± 0.061.8 ± 0.54.5 ± 0.8Vmax is the rate of product formation by 10 pmol of P450 C21 in the presence of an excess amount of the reductase in the membranes. k2 is the rate constant for the conversion of the substrate to the product, and k3is that for the product dissociation from P450 C21-reductase complex in the scheme shown in Fig. 6. Values are means ± S.D. of at least triplicate determinations. The details of the calculation are described under “Discussion.” Open table in a new tab Vmax is the rate of product formation by 10 pmol of P450 C21 in the presence of an excess amount of the reductase in the membranes. k2 is the rate constant for the conversion of the substrate to the product, and k3is that for the product dissociation from P450 C21-reductase complex in the scheme shown in Fig. 6. Values are means ± S.D. of at least triplicate determinations. The details of the calculation are described under “Discussion.” The difference in the rate-determining steps in P450 C21 reactions must affect the pre-steady-state kinetics of the reactions of progesterone and 17α-hydroxyprogesterone (1Bell L.C. Guengerich F.P. J. Biol. Chem. 1997; 272: 29643-29651Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). The time courses of product formation from progesterone and 17α-hydroxyprogesterone in the range of 0–3 s are shown in Fig. 5, a and b, respectively. As discussed above, the rate of conversion of progesterone to the product in the active complex is about 4 times faster than the dissociation of deoxycorticosterone from the active complex, which is reflected by the burst and the subsequent slow linear increase of the product. On the other hand, the conversion of 17α-hydroxyprogesterone to deoxycortisol is the slowest step in the reaction cycle, and we did not observe any burst formation of deoxycortisol. The lines in Fig. 5 are the simulated curves obtained using the simulation program HopKINSIM version 1.7.2 with the rate constants in Table I, where the value of k1was selected to be 0.02 (nm·s)−1for the binding of both progesterone and 17α-hydroxyprogesterone (29Kominami S. Itoh Y. Takemori S. J. Biol. Chem. 1986; 261: 2077-2083Abstract Full Text PDF PubMed Google Scholar,36Barshop B.A. Wrenn R.F. Frieden C. Anal. Biochem. 1983; 130: 134-145Crossref PubMed Scopus (670) Google Scholar, 37Frieden C. Trends Biochem. Sci. 1993; 18: 58-60Abstract Full Text PDF PubMed Scopus (53) Google Scholar). The simulation study shows that the burst increase in the product formation is due to the fast conversion of the substrate to the product with subsequent slow dissociation of the product from the enzyme. The successive reactions catalyzed by P450 2E1, P450 11β, and P450 17α had as rate-determining steps the dissociation of the products, with slow dissociation of intermediate products facilitating the further monooxygenation of intermediates without dissociation from the enzyme active sites (2Bell-Parikh L.C. Guengerich F.P. J. Biol. Chem. 1999; 274: 23833-23840Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 3Imai T. Yamazaki T. Kominami S. Biochemistry. 1998; 37: 8097-8104Crossref PubMed Scopus (30) Google Scholar, 4Yamazaki T. Ohno T. Sakaki T. Akiyoshi-Shibata M. Yabusaki Y. Imai T. Kominami S. Biochemistry. 1998; 37: 2800-2806Crossref PubMed Scopus (43) Google Scholar, 5Tagashira H. Kominami S. Takemori S. Biochemistry. 1995; 34: 10939-10945Crossref PubMed Scopus (25) Google Scholar). The rate-determining step in the hydroxylation reaction of progesterone catalyzed by P450 C21 is the product dissociation, and P450 C21 does not catalyze a multistep reaction. This means that the rate-determining product release from the enzymes was not restricted to the successive reactions. The rate-determining product release might not be a special phenomenon in P450 reactions. White and Coon (41White R.E. Coon M.J. Annu. Rev. Biochem. 1980; 49: 315-356Crossref PubMed Scopus (930) Google Scholar) had speculated some 20 years ago that the difference in the activity of one species of P450 for different substrates might be explained by the difference in the product dissociation rate. The difference in the rates of product release might be due to the difference in the hydrophobicities of the products (42Al-Gailany K.A.S. Houston J.B. Bridges J.W. Biochem. Pharmacol. 1978; 27: 783-788Crossref PubMed Scopus (54) Google Scholar, 43Backes W.I. Canady W.J. J. Biol. Chem. 1981; 256: 7213-7227Abstract Full Text PDF PubMed Google Scholar). Deoxycortisol has two hydroxyl groups, but deoxycorticosterone has only one. It is not surprising that deoxycortisol dissociates faster from P450 C21 than the more hydrophobic deoxycorticosterone (29Kominami S. Itoh Y. Takemori S. J. Biol. Chem. 1986; 261: 2077-2083Abstract Full Text PDF PubMed Google Scholar). Since it could be the rate-determining step in the P450 reaction, much more attention should be paid to the product dissociation from P450s.