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- W1980377002 abstract "Thromboxane synthase (TXAS) is a “non-classical” cytochrome P450. Without any need for an external electron donor, or for a reductase or molecular oxygen, it uses prostaglandin H2 (PGH2) to catalyze either an isomerization reaction to form thromboxane A2(TXA2) or a fragmentation reaction to form 12-l-hydroxy-5,8,10-heptadecatrienoic acid and malondialdehyde (MDA) at a ratio of 1:1:1 (TXA2:heptadecatrienoic acid:MDA). We report here kinetics of TXAS with heme ligands in binding study and with PGH2 in enzymatic study. We determined that 1) binding of U44069, an oxygen-based ligand, is a two-step process; U44069 first binds TXAS, then ligates the heme-iron with a maximal rate constant of 105–130 s−1; 2) binding of cyanide, a carbon-based ligand, is a one-step process with kon of 2.4m−1 s−1 andkoff of 0.112 s−1; and 3) both imidazole and clotrimazole (nitrogen-based ligands) bind TXAS in a two-step process; an initial binding to the heme-iron with on-rate constants of 8.4 × 104m−1s−1 and 1.5 × 105m−1 s−1 for imidazole and clotrimazole, respectively, followed by a slow conformational change with off-rate constants of 8.8 s−1 and 0.53 s−1, respectively. The results of our binding study indicate that the TXAS active site is hydrophobic and spacious. In addition, steady-state kinetic study revealed that TXAS consumed PGH2 at a rate of 3,800 min−1 and that thekcat/Km for PGH2 consumption was 3 × 106m−1 s−1. Based on these data, TXAS appears to be a very efficient catalyst. Surprisingly, rapid-scan stopped-flow experiments revealed marginal absorbance changes upon mixing TXAS with PGH2, indicating minimal accumulation of any heme-derived intermediates. Freeze-quench EPR measurements for the same reaction showed minimal change of heme redox state. Further kinetic analysis using a combination of rapid-mixing chemical quench and computer simulation showed that the kinetic parameters of TXAS-catalyzed reaction are: PGH2 bound TXAS at a rate of 1.2–2.0 × 107m−1s−1; the rate of catalytic conversion of PGH2to TXA2 or MDA was at least 15,000 s−1 and the lower limit of the rates for products release was 4,000–6,000 s−1. Given that the cellular PGH2concentration is quite low, we concluded that under physiological conditions, the substrate-binding step is the rate-limiting step of the TXAS-catalyzed reaction, in sharp contrast with “classical” P450 enzymes. Thromboxane synthase (TXAS) is a “non-classical” cytochrome P450. Without any need for an external electron donor, or for a reductase or molecular oxygen, it uses prostaglandin H2 (PGH2) to catalyze either an isomerization reaction to form thromboxane A2(TXA2) or a fragmentation reaction to form 12-l-hydroxy-5,8,10-heptadecatrienoic acid and malondialdehyde (MDA) at a ratio of 1:1:1 (TXA2:heptadecatrienoic acid:MDA). We report here kinetics of TXAS with heme ligands in binding study and with PGH2 in enzymatic study. We determined that 1) binding of U44069, an oxygen-based ligand, is a two-step process; U44069 first binds TXAS, then ligates the heme-iron with a maximal rate constant of 105–130 s−1; 2) binding of cyanide, a carbon-based ligand, is a one-step process with kon of 2.4m−1 s−1 andkoff of 0.112 s−1; and 3) both imidazole and clotrimazole (nitrogen-based ligands) bind TXAS in a two-step process; an initial binding to the heme-iron with on-rate constants of 8.4 × 104m−1s−1 and 1.5 × 105m−1 s−1 for imidazole and clotrimazole, respectively, followed by a slow conformational change with off-rate constants of 8.8 s−1 and 0.53 s−1, respectively. The results of our binding study indicate that the TXAS active site is hydrophobic and spacious. In addition, steady-state kinetic study revealed that TXAS consumed PGH2 at a rate of 3,800 min−1 and that thekcat/Km for PGH2 consumption was 3 × 106m−1 s−1. Based on these data, TXAS appears to be a very efficient catalyst. Surprisingly, rapid-scan stopped-flow experiments revealed marginal absorbance changes upon mixing TXAS with PGH2, indicating minimal accumulation of any heme-derived intermediates. Freeze-quench EPR measurements for the same reaction showed minimal change of heme redox state. Further kinetic analysis using a combination of rapid-mixing chemical quench and computer simulation showed that the kinetic parameters of TXAS-catalyzed reaction are: PGH2 bound TXAS at a rate of 1.2–2.0 × 107m−1s−1; the rate of catalytic conversion of PGH2to TXA2 or MDA was at least 15,000 s−1 and the lower limit of the rates for products release was 4,000–6,000 s−1. Given that the cellular PGH2concentration is quite low, we concluded that under physiological conditions, the substrate-binding step is the rate-limiting step of the TXAS-catalyzed reaction, in sharp contrast with “classical” P450 enzymes. thromboxane A2 thromboxane A2 synthase thromboxane B2 prostaglandin H2 12-l-hydroxy-5,8,10-heptadecatrienoic acid malondialdehyde thiobarbituric acid high performance liquid chromatography Thromboxane A2(TXA2)1 is a potent inducer of vasoconstriction and platelet aggregation. It is believed to be a crucial factor contributing to a variety of cardiovascular and pulmonary diseases such as atherosclerosis, myocardial infarction, and primary pulmonary hypertension (1Samuelsson B. Goldyne M. Granstrom E. Hamberg M. Hammarstrom S. Malmsten C. Annu. Rev. Biochem. 1978; 47: 997-1029Crossref PubMed Scopus (972) Google Scholar, 2Vane J.R. Botting R. FASEB J. 1987; 1: 89-96Crossref PubMed Scopus (451) Google Scholar). TXA2 is rather labile, being hydrolyzed to the biologically inactive thromboxane B2 (TXB2) with a half-life of about 30 s in aqueous solution at 37 °C (3Hamberg M. Svensson J. Samuelsson B. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 2994-2998Crossref PubMed Scopus (2790) Google Scholar). Its biosynthesis is accomplished by thromboxane synthase (TXAS) using prostaglandin H2 (PGH2) as the substrate. Notably, the formation of TXA2 is accompanied by those of 12-l-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and malondialdehyde (MDA) at a ratio of 1:1:1 (4Haurand M. Ullrich V. J. Biol. Chem. 1985; 260: 15059-15067Abstract Full Text PDF PubMed Google Scholar) (SchemeFS1). The biological functions of MDA and HHT are unclear. However, MDA can form adducts with lysine residues of proteins or with amine head groups of phospholipids and its adducts have been detected in atherosclerotic lesions of human aorta (5Uchida K. Trends Cardiovasc. Med. 1999; 9: 109-113Crossref PubMed Scopus (268) Google Scholar). It was also shown to participate in the formation of an important endogenous DNA adduct which may contribute to the etiology of human genetic disease and cancer (6Chaudhary A.K. Nokubo M. Reddy G.R. Yeola S.N. Morrow J.D. Blair I.A. Marnett L.J. Science. 1994; 265: 1580-1582Crossref PubMed Scopus (413) Google Scholar).TXAS is a member of the cytochrome P450 superfamily and is associated with endoplasmic reticulum (4Haurand M. Ullrich V. J. Biol. Chem. 1985; 260: 15059-15067Abstract Full Text PDF PubMed Google Scholar, 7Ruan K.-H. Wang L.-H. Wu K.K. Kulmacz R.J. J. Biol. Chem. 1993; 268: 19483-19490Abstract Full Text PDF PubMed Google Scholar). Human TXAS was assigned as CYP 5A1. Unlike other microsomal P450s that require the ubiquitous P450 reductase to shuttle electrons for the mono-oxygenation reaction, TXAS undergoes an isomerization reaction without reductase or molecular oxygen. A “classical” P450 accepts an electron from the reductase and converts heme iron to the Fe(II) state, followed by binding of molecular oxygen and another one-electron reduction. The subsequent steps, including oxygen bond cleavage and oxidation of the substrate, occur rapidly and have been difficult to resolve (8Mueller E.J. Loida P.J. Sligar S.G. Ortiz de Montellano P.R. Cytochrome P450; Structure, Mechanism and Biochemistry. Plenum Press, New York1995: 83-124Crossref Google Scholar). However, for P450cam, the rate constants of oxygen binding and substrate hydroxylation were determined to be 4.1 × 105m−1 s−1 and 34 s−1, respectively (9Gerber N.C. Sligar S.G. J. Biol. Chem. 1994; 269: 4260-4266Abstract Full Text PDF PubMed Google Scholar). The intermediates of the later steps in the P450cam reaction were well characterized in a recent time-resolved x-ray crystallographic study in which the presence of an oxyferryl intermediate after breakdown of molecular oxygen was identified (10Schlichting I. Berendzen J. Chu K. Stock A.M. Maves S.A. Benson D.E. Sweet R.M. Ringe D. Petsko G.A. Sligar S.G. Science. 2000; 287: 1615-1622Crossref PubMed Scopus (1197) Google Scholar).TXAS, which uses an endoperoxide as the substrate, apparently undergoes a different mechanism. Hecker and Ullrich (11Hecker M. Ullrich V. J. Biol. Chem. 1989; 264: 141-150Abstract Full Text PDF PubMed Google Scholar) proposed a mechanistic model for TXAS involving a homolytic scission of PGH2 that led to the formation of heme-Fe(IV) and alkoxy radical intermediates for enzyme and substrate species, respectively. In the present study, we attempted to characterize the intermediates of the TXAS reaction employing optical and EPR spectroscopies. EPR studies failed to identify any radical species. Stopped-flow spectroscopy revealed no significant absorbance changes at the heme center. Further kinetic analysis using the rapid-mixing chemical quench and computer simulation showed that the substrate-binding step is the rate-limiting step of TXAS-catalyzed reaction, and that the catalytic conversion of PGH2 to TXA2 or MDA by TXAS was remarkably fast.DISCUSSIONTXAS, as a resting enzyme, has a typical low spin P450 heme with an oxygen-based distal ligand (4Haurand M. Ullrich V. J. Biol. Chem. 1985; 260: 15059-15067Abstract Full Text PDF PubMed Google Scholar, 13Hsu P.-Y. Tsai A.-L. Kulmacz R.J. Wang L.-H. J. Biol. Chem. 1999; 274: 762-769Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Several types of heme ligands including U44069 (oxygen-based ligand), imidazole, clotrimazole (both are nitrogen-based ligands), and cyanide (carbon-based ligand) were studied to determine their kinetic binding characteristics with TXAS using stopped-flow spectroscopy. All these ligands caused Type II spectral changes, indicating that the exogenous ligands formed a low-spin coordinate heme complex. However, their binding kinetics to TXAS were quite different. U44069 and cyanide binding to TXAS exhibited monophasic kinetics, in contrast to the biphasic kinetics found for imidazole and clotrimazole. Analysis ofkobs versus ligand concentrations further indicated that cyanide underwent a one-step binding process, whereas U44069, imidazole, and clotrimazole underwent two-step binding processes.Interestingly, the binding mechanisms of those ligands involved in the two-step binding processes are distinctly different. U44069 first binds TXAS, but not heme iron, and then replaces the original heme ligand. The two nitrogen-based ligands, nonetheless, bind heme iron first and then undergo a conformational change before they reach equilibrium. Cyanide, which is charged but is the smallest molecule tested, had the lowest on-rate constant (2.4 m−1s−1). This low rate constant compared with other hemoproteins is likely due to the strong electron-donating thiolate ligand. On the other hand, clotrimazole, although much bulkier than imidazole, had a higher on-rate constant (8.4 × 104m−1 s−1 and 1.5 × 105m−1 s−1 for imidazole and clotrimazole, respectively). These results indicated that the TXAS active site is both hydrophobic and spacious, consistent with our previously reported findings (13Hsu P.-Y. Tsai A.-L. Kulmacz R.J. Wang L.-H. J. Biol. Chem. 1999; 274: 762-769Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar).Classical eukaryotic P450s catalyze hydroxylation reactions at a very slow rate with kcat of 1–10 mol of product formed/min/mol of P450 (21Guengerich F.P. J. Biol. Chem. 1991; 266: 10019-10022Abstract Full Text PDF PubMed Google Scholar). Many prokaryotic P450s, however, have somewhat higher catalytic activities. For example, P450camcatalyzes the reaction at a rate of ∼1900 mol/min/mol of protein (22Peterson J.A. Mock D.M. Cooper D.Y. Rosenthal O. Synder R. Witmer C. Cytochrome P450 and b5. Plenum Press, New York1975: 311-324Google Scholar), and so does P450BM-3 (23Narhi L.O. Fulco A.J. J. Biol. Chem. 1986; 261: 7160-7169Abstract Full Text PDF PubMed Google Scholar), a soluble protein in which the reductase domain is naturally fused to the oxygenase domain. For the “non-classical” P450s such as allene-oxide synthase and nitric-oxide reductase that do not need reductase for catalysis, the turnover numbers seems to be much higher. Allene-oxide synthase, a P450 acting as a dehydrase which converts lipid hydroperoxide to allene oxide, appears to form product at a rate of >60,000 mol/min/mol of protein (24Song W.C. Brash A.R. Science. 1991; 253: 781-784Crossref PubMed Scopus (230) Google Scholar). Nitric-oxide reductase, i.e.P450nor, which catalyzes NO to N2O has a turnover number of 72,000 mol/min/mol of protein (25Shiro Y. Fujii M. Iizuka T. Adachi S. Tsukamoto K. Nakahara K. Shoun H. J. Biol. Chem. 1993; 270: 1617-1623Abstract Full Text Full Text PDF Scopus (184) Google Scholar). However, prostacyclin synthase, another non-classical P450 that uses PGH2 as the natural substrate, has a lower turnover number (∼150 mol/min/mol of protein) (11Hecker M. Ullrich V. J. Biol. Chem. 1989; 264: 141-150Abstract Full Text PDF PubMed Google Scholar, 26Hara S. Miyata A. Yokoyama C. Inoue H. Brugger R. Lottspeich F. Ullrich V. Tanabe T. J. Biol. Chem. 1994; 269: 19897-19903Abstract Full Text PDF PubMed Google Scholar). TXAS, as observed in this study, formed MDA or TXA2 at a rate of 1,900 min−1. In other words, TXAS consumed PGH2 at a rate of 3,800 min−1. Furthermore, the ratio ofkcat/Km (an index of catalytic efficiency) of TXAS for PGH2 consumption is thus 3 × 106m−1s−1. Compared with carbonic anhydrase, an extremely efficient enzyme that has a kcat/Km of 107-108m−1s−1 (27Lindskog S. Engberg P. Forsman C. Ibrahim S.A. Johnson B.H. Simonsson I. Tibell L. Ann. N. Y. Acad. Sci. 1984; 429: 61-75Crossref PubMed Scopus (67) Google Scholar), TXAS should be considered as a very efficient catalyst. It should also be noted that many P450s including P450cam can act as non-classical P450s and convert PGH2 to MDA and HHT, but not TXA2, without reductase, molecular oxygen, or any electron donor (11Hecker M. Ullrich V. J. Biol. Chem. 1989; 264: 141-150Abstract Full Text PDF PubMed Google Scholar). Their “HHT/MDA synthase” activities, nonetheless, are much lower than TXAS. A recent report showed that four microsomal P450s (P4501A2, 2B1, 2E1, and 3A4) had Kd for U44069 of ∼200 μm and when assayed at 50 μmPGH2, their catalytic activities were 1–10 min−1 (28Plastaras J.P. Guengerich P. Nebert D.W. Marnett L.J. J. Biol. Chem. 2000; 275: 11784-11790Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). It would appear that PGH2 was readily converted to MDA and HHT by heme in the context of P450 where a hydrophobic environment is present. A study of chemical models for heme-catalyzed PGH2 reactions showed that 9% of HHT was formed in a phosphate buffer, whereas 33% of HHT was formed in acetonitrile, a less polar solvent (11Hecker M. Ullrich V. J. Biol. Chem. 1989; 264: 141-150Abstract Full Text PDF PubMed Google Scholar). TXAS is more efficient in HHT/MDA synthesis not only because its active site is hydrophobic but it also has a greater affinity for PGH2. Furthermore, several TXAS active site amino acid residues, as we have previously shown (29Wang L.-H. Matijevic-Aleksic N. Hsu P.-Y. Ruan K.-H. Wu K.K. Kulmacz R.J. J. Biol. Chem. 1996; 271: 19970-19975Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar), may be involved in the reaction. It is intriguing to note that prostacyclin synthase, although it has a high affinity for PGH2 (Kd is ∼10 μm), does not catalyze HHT/MDA formation (11Hecker M. Ullrich V. J. Biol. Chem. 1989; 264: 141-150Abstract Full Text PDF PubMed Google Scholar). What structural elements determine the HHT/MDA activity of P450 remains a challenging topic.In this study, the rate constant of each catalytic step,i.e. PGH2 to TXA2 or MDA/HHT, is estimated to be ∼15,000 s−1. The half-life of the intermediate(s) at 23 °C is therefore much less than the dead-time of the stopped-flow apparatus (∼1.5 ms). Furthermore, the rate constant of substrate binding is 12–20 × 106m−1 s−1 and the in vivo binding constant could be calculated if the cellular PGH2 concentration was known. To the best of our knowledge, cellular PGH2 concentration has not been reported due in part to the instability of PGH2 in aqueous solution. However, concentrations of arachidonic acid, the substrate for prostaglandin H synthase, in many inflammatory blood cells and lung tissues were less than 10 μm (30Triggiani M. Oriente A. Seeds M.C. Bass D.A. Marone G. Chilton F.H. J. Exp. Med. 1995; 182: 1181-1190Crossref PubMed Scopus (77) Google Scholar, 31Chilton F.H. Fonteh A.N. Surette M.E. Triggiani M. Winkler J.D. Biochim. Biophys. Acta. 1996; 1299: 1-15Crossref PubMed Scopus (209) Google Scholar). In pancreatic islets, cellular un-esterified arachidonate concentrations of 38–75 μm were reported under glucose-induced conditions. But the maximally effective concentration, including exogenous arachidonic acid, was 30–40 μm (32Wolf B.A. Pasquale S.M. Turk J. Biochemistry. 1991; 30: 6372-6379Crossref PubMed Scopus (110) Google Scholar). Since arachidonic acid is a substrate for many other pathways in addition to prostaglandin H synthase, it is probably safe to assume that the cellular PGH2 concentration is generally less than 40 μm. If that is the case, the substrate-binding rate constant for TXAS in the physiological conditions would be less than 800 s−1, and this value is much smaller than the other forward rate constants. We therefore conclude that the substrate-binding step is the rate-limiting step in the TXAS-catalyzed reaction.P450s were also classified according to the identity of their electron donors. Class I P450s require both ferredoxin and ferredoxin reductase for electron transfer, whereas Class II P450s require only a P450 reductase (33Graham-Lorence S. Peterson J.A. FASEB. J. 1996; 10: 206-214Crossref PubMed Scopus (124) Google Scholar). In both Class I and Class II P450s, the rate-limiting step of the overall reaction is at the stage of the second electron transfer to the heme center. Class III P450s that do not require any reductase, are not well characterized regarding their kinetic mechanisms. An elegant study on P450nor, a Class III P450, revealed that binding of NO and NADH substrates were fast to form the intermediate I, an NO-bound two-electron reduced species (25Shiro Y. Fujii M. Iizuka T. Adachi S. Tsukamoto K. Nakahara K. Shoun H. J. Biol. Chem. 1993; 270: 1617-1623Abstract Full Text Full Text PDF Scopus (184) Google Scholar). The intermediate I was slowly converted to the products. The rate-limiting step of P450nor is thus in the chemical steps. In contrast to other P450s, the rate-limiting step of TXAS is at the step of substrate binding. To the best of our knowledge, this kinetic mechanism is the first example of Class III P450s in which substrate binding is the rate-limiting step. Thromboxane A2(TXA2)1 is a potent inducer of vasoconstriction and platelet aggregation. It is believed to be a crucial factor contributing to a variety of cardiovascular and pulmonary diseases such as atherosclerosis, myocardial infarction, and primary pulmonary hypertension (1Samuelsson B. Goldyne M. Granstrom E. Hamberg M. Hammarstrom S. Malmsten C. Annu. Rev. Biochem. 1978; 47: 997-1029Crossref PubMed Scopus (972) Google Scholar, 2Vane J.R. Botting R. FASEB J. 1987; 1: 89-96Crossref PubMed Scopus (451) Google Scholar). TXA2 is rather labile, being hydrolyzed to the biologically inactive thromboxane B2 (TXB2) with a half-life of about 30 s in aqueous solution at 37 °C (3Hamberg M. Svensson J. Samuelsson B. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 2994-2998Crossref PubMed Scopus (2790) Google Scholar). Its biosynthesis is accomplished by thromboxane synthase (TXAS) using prostaglandin H2 (PGH2) as the substrate. Notably, the formation of TXA2 is accompanied by those of 12-l-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and malondialdehyde (MDA) at a ratio of 1:1:1 (4Haurand M. Ullrich V. J. Biol. Chem. 1985; 260: 15059-15067Abstract Full Text PDF PubMed Google Scholar) (SchemeFS1). The biological functions of MDA and HHT are unclear. However, MDA can form adducts with lysine residues of proteins or with amine head groups of phospholipids and its adducts have been detected in atherosclerotic lesions of human aorta (5Uchida K. Trends Cardiovasc. Med. 1999; 9: 109-113Crossref PubMed Scopus (268) Google Scholar). It was also shown to participate in the formation of an important endogenous DNA adduct which may contribute to the etiology of human genetic disease and cancer (6Chaudhary A.K. Nokubo M. Reddy G.R. Yeola S.N. Morrow J.D. Blair I.A. Marnett L.J. Science. 1994; 265: 1580-1582Crossref PubMed Scopus (413) Google Scholar). TXAS is a member of the cytochrome P450 superfamily and is associated with endoplasmic reticulum (4Haurand M. Ullrich V. J. Biol. Chem. 1985; 260: 15059-15067Abstract Full Text PDF PubMed Google Scholar, 7Ruan K.-H. Wang L.-H. Wu K.K. Kulmacz R.J. J. Biol. Chem. 1993; 268: 19483-19490Abstract Full Text PDF PubMed Google Scholar). Human TXAS was assigned as CYP 5A1. Unlike other microsomal P450s that require the ubiquitous P450 reductase to shuttle electrons for the mono-oxygenation reaction, TXAS undergoes an isomerization reaction without reductase or molecular oxygen. A “classical” P450 accepts an electron from the reductase and converts heme iron to the Fe(II) state, followed by binding of molecular oxygen and another one-electron reduction. The subsequent steps, including oxygen bond cleavage and oxidation of the substrate, occur rapidly and have been difficult to resolve (8Mueller E.J. Loida P.J. Sligar S.G. Ortiz de Montellano P.R. Cytochrome P450; Structure, Mechanism and Biochemistry. Plenum Press, New York1995: 83-124Crossref Google Scholar). However, for P450cam, the rate constants of oxygen binding and substrate hydroxylation were determined to be 4.1 × 105m−1 s−1 and 34 s−1, respectively (9Gerber N.C. Sligar S.G. J. Biol. Chem. 1994; 269: 4260-4266Abstract Full Text PDF PubMed Google Scholar). The intermediates of the later steps in the P450cam reaction were well characterized in a recent time-resolved x-ray crystallographic study in which the presence of an oxyferryl intermediate after breakdown of molecular oxygen was identified (10Schlichting I. Berendzen J. Chu K. Stock A.M. Maves S.A. Benson D.E. Sweet R.M. Ringe D. Petsko G.A. Sligar S.G. Science. 2000; 287: 1615-1622Crossref PubMed Scopus (1197) Google Scholar). TXAS, which uses an endoperoxide as the substrate, apparently undergoes a different mechanism. Hecker and Ullrich (11Hecker M. Ullrich V. J. Biol. Chem. 1989; 264: 141-150Abstract Full Text PDF PubMed Google Scholar) proposed a mechanistic model for TXAS involving a homolytic scission of PGH2 that led to the formation of heme-Fe(IV) and alkoxy radical intermediates for enzyme and substrate species, respectively. In the present study, we attempted to characterize the intermediates of the TXAS reaction employing optical and EPR spectroscopies. EPR studies failed to identify any radical species. Stopped-flow spectroscopy revealed no significant absorbance changes at the heme center. Further kinetic analysis using the rapid-mixing chemical quench and computer simulation showed that the substrate-binding step is the rate-limiting step of TXAS-catalyzed reaction, and that the catalytic conversion of PGH2 to TXA2 or MDA by TXAS was remarkably fast. DISCUSSIONTXAS, as a resting enzyme, has a typical low spin P450 heme with an oxygen-based distal ligand (4Haurand M. Ullrich V. J. Biol. Chem. 1985; 260: 15059-15067Abstract Full Text PDF PubMed Google Scholar, 13Hsu P.-Y. Tsai A.-L. Kulmacz R.J. Wang L.-H. J. Biol. Chem. 1999; 274: 762-769Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Several types of heme ligands including U44069 (oxygen-based ligand), imidazole, clotrimazole (both are nitrogen-based ligands), and cyanide (carbon-based ligand) were studied to determine their kinetic binding characteristics with TXAS using stopped-flow spectroscopy. All these ligands caused Type II spectral changes, indicating that the exogenous ligands formed a low-spin coordinate heme complex. However, their binding kinetics to TXAS were quite different. U44069 and cyanide binding to TXAS exhibited monophasic kinetics, in contrast to the biphasic kinetics found for imidazole and clotrimazole. Analysis ofkobs versus ligand concentrations further indicated that cyanide underwent a one-step binding process, whereas U44069, imidazole, and clotrimazole underwent two-step binding processes.Interestingly, the binding mechanisms of those ligands involved in the two-step binding processes are distinctly different. U44069 first binds TXAS, but not heme iron, and then replaces the original heme ligand. The two nitrogen-based ligands, nonetheless, bind heme iron first and then undergo a conformational change before they reach equilibrium. Cyanide, which is charged but is the smallest molecule tested, had the lowest on-rate constant (2.4 m−1s−1). This low rate constant compared with other hemoproteins is likely due to the strong electron-donating thiolate ligand. On the other hand, clotrimazole, although much bulkier than imidazole, had a higher on-rate constant (8.4 × 104m−1 s−1 and 1.5 × 105m−1 s−1 for imidazole and clotrimazole, respectively). These results indicated that the TXAS active site is both hydrophobic and spacious, consistent with our previously reported findings (13Hsu P.-Y. Tsai A.-L. Kulmacz R.J. Wang L.-H. J. Biol. Chem. 1999; 274: 762-769Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar).Classical eukaryotic P450s catalyze hydroxylation reactions at a very slow rate with kcat of 1–10 mol of product formed/min/mol of P450 (21Guengerich F.P. J. Biol. Chem. 1991; 266: 10019-10022Abstract Full Text PDF PubMed Google Scholar). Many prokaryotic P450s, however, have somewhat higher catalytic activities. For example, P450camcatalyzes the reaction at a rate of ∼1900 mol/min/mol of protein (22Peterson J.A. Mock D.M. Cooper D.Y. Rosenthal O. Synder R. Witmer C. Cytochrome P450 and b5. Plenum Press, New York1975: 311-324Google Scholar), and so does P450BM-3 (23Narhi L.O. Fulco A.J. J. Biol. Chem. 1986; 261: 7160-7169Abstract Full Text PDF PubMed Google Scholar), a soluble protein in which the reductase domain is naturally fused to the oxygenase domain. For the “non-classical” P450s such as allene-oxide synthase and nitric-oxide reductase that do not need reductase for catalysis, the turnover numbers seems to be much higher. Allene-oxide synthase, a P450 acting as a dehydrase which converts lipid hydroperoxide to allene oxide, appears to form product at a rate of >60,000 mol/min/mol of protein (24Song W.C. Brash A.R. Science. 1991; 253: 781-784Crossref PubMed Scopus (230) Google Scholar). Nitric-oxide reductase, i.e.P450nor, which catalyzes NO to N2O has a turnover number of 72,000 mol/min/mol of protein (25Shiro Y. Fujii M. Iizuka T. Adachi S. Tsukamoto K. Nakahara K. Shoun H. J. Biol. Chem. 1993; 270: 1617-1623Abstract Full Text Full Text PDF Scopus (184) Google Scholar). However, prostacyclin synthase, another non-classical P450 that uses PGH2 as the natural substrate, has a lower turnover number (∼150 mol/min/mol of protein) (11Hecker M. Ullrich V. J. Biol. Chem. 1989; 264: 141-150Abstract Full Text PDF PubMed Google Scholar, 26Hara S. Miyata A. Yokoyama C. Inoue H. Brugger R. Lottspeich F. Ullrich V. Tanabe T. J. Biol. Chem. 1994; 269: 19897-19903Abstract Full Text PDF PubMed Google Scholar). TXAS, as observed in this study, formed MDA or TXA2 at a rate of 1,900 min−1. In other words, TXAS consumed PGH2 at a rate of 3,800 min−1. Furthermore, the ratio ofkcat/Km (an index of catalytic efficiency) of TXAS for PGH2 consumption is thus 3 × 106m−1s−1. Compared with carbonic anhydrase, an extremely efficient enzyme that has a kcat/Km of 107-108m−1s−1 (27Lindskog S. Engberg P. Forsman C. Ibrahim S.A. Johnson B.H. Simonsson I. Tibell L. Ann. N. Y. Acad. Sci. 1984; 429: 61-75Crossref PubMed Scopus (67) Google Scholar), TXAS should be considered as a very efficient catalyst. It should also be noted that many P450s including P450cam can act as non-classical P450s and convert PGH2 to MDA and HHT, but not TXA2, without reductase, molecular oxygen, or any electron donor (11Hecker M. Ullrich V. J. Biol. Chem. 1989; 264: 141-150Abstract Full Text PDF PubMed Google Scholar). Their “HHT/MDA synthase” activities, nonetheless, are much lower than TXAS. A recent report showed that four microsomal P450s (P4501A2, 2B1, 2E1, and 3A4) had Kd for U44069 of ∼200 μm and when assayed at 50 μmPGH2, their catalytic activities were 1–10 min−1 (28Plastaras J.P. Guengerich P. Nebert D.W. Marnett L.J. J. Biol. Chem. 2000; 275: 11784-11790Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). It would appear that PGH2 was readily converted to MDA and HHT by heme in the context of P450 where a hydrophobic environment is present. A study of chemical models for heme-catalyzed PGH2 reactions showed that 9% of HHT was formed in a phosphate buffer, whereas 33% of HHT was formed in acetonitrile, a less polar solvent (11Hecker M. Ullrich V. J. Biol. Chem. 1989; 264: 141-150Abstract Full Text PDF PubMed Google Scholar). TXAS is more efficient in HHT/MDA synthesis not only because its active site is hydrophobic but it also has a greater affinity for PGH2. Furthermore, several TXAS active site amino acid residues, as we have previously shown (29Wang L.-H. Matijevic-Aleksic N. Hsu P.-Y. Ruan K.-H. Wu K.K. Kulmacz R.J. J. Biol. Chem. 1996; 271: 19970-19975Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar), may be involved in the reaction. It is intriguing to note that prostacyclin synthase, although it has a high affinity for PGH2 (Kd is ∼10 μm), does not catalyze HHT/MDA formation (11Hecker M. Ullrich V. J. Biol. Chem. 1989; 264: 141-150Abstract Full Text PDF PubMed Google Scholar). What structural elements determine the HHT/MDA activity of P450 remains a challenging topic.In this study, the rate constant of each catalytic step,i.e. PGH2 to TXA2 or MDA/HHT, is estimated to be ∼15,000 s−1. The half-life of the intermediate(s) at 23 °C is therefore much less than the dead-time of the stopped-flow apparatus (∼1.5 ms). Furthermore, the rate constant of substrate binding is 12–20 × 106m−1 s−1 and the in vivo binding constant could be calculated if the cellular PGH2 concentration was known. To the best of our knowledge, cellular PGH2 concentration has not been reported due in part to the instability of PGH2 in aqueous solution. However, concentrations of arachidonic acid, the substrate for prostaglandin H synthase, in many inflammatory blood cells and lung tissues were less than 10 μm (30Triggiani M. Oriente A. Seeds M.C. Bass D.A. Marone G. Chilton F.H. J. Exp. Med. 1995; 182: 1181-1190Crossref PubMed Scopus (77) Google Scholar, 31Chilton F.H. Fonteh A.N. Surette M.E. Triggiani M. Winkler J.D. Biochim. Biophys. Acta. 1996; 1299: 1-15Crossref PubMed Scopus (209) Google Scholar). In pancreatic islets, cellular un-esterified arachidonate concentrations of 38–75 μm were reported under glucose-induced conditions. But the maximally effective concentration, including exogenous arachidonic acid, was 30–40 μm (32Wolf B.A. Pasquale S.M. Turk J. Biochemistry. 1991; 30: 6372-6379Crossref PubMed Scopus (110) Google Scholar). Since arachidonic acid is a substrate for many other pathways in addition to prostaglandin H synthase, it is probably safe to assume that the cellular PGH2 concentration is generally less than 40 μm. If that is the case, the substrate-binding rate constant for TXAS in the physiological conditions would be less than 800 s−1, and this value is much smaller than the other forward rate constants. We therefore conclude that the substrate-binding step is the rate-limiting step in the TXAS-catalyzed reaction.P450s were also classified according to the identity of their electron donors. Class I P450s require both ferredoxin and ferredoxin reductase for electron transfer, whereas Class II P450s require only a P450 reductase (33Graham-Lorence S. Peterson J.A. FASEB. J. 1996; 10: 206-214Crossref PubMed Scopus (124) Google Scholar). In both Class I and Class II P450s, the rate-limiting step of the overall reaction is at the stage of the second electron transfer to the heme center. Class III P450s that do not require any reductase, are not well characterized regarding their kinetic mechanisms. An elegant study on P450nor, a Class III P450, revealed that binding of NO and NADH substrates were fast to form the intermediate I, an NO-bound two-electron reduced species (25Shiro Y. Fujii M. Iizuka T. Adachi S. Tsukamoto K. Nakahara K. Shoun H. J. Biol. Chem. 1993; 270: 1617-1623Abstract Full Text Full Text PDF Scopus (184) Google Scholar). The intermediate I was slowly converted to the products. The rate-limiting step of P450nor is thus in the chemical steps. In contrast to other P450s, the rate-limiting step of TXAS is at the step of substrate binding. To the best of our knowledge, this kinetic mechanism is the first example of Class III P450s in which substrate binding is the rate-limiting step. TXAS, as a resting enzyme, has a typical low spin P450 heme with an oxygen-based distal ligand (4Haurand M. Ullrich V. J. Biol. Chem. 1985; 260: 15059-15067Abstract Full Text PDF PubMed Google Scholar, 13Hsu P.-Y. Tsai A.-L. Kulmacz R.J. Wang L.-H. J. Biol. Chem. 1999; 274: 762-769Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Several types of heme ligands including U44069 (oxygen-based ligand), imidazole, clotrimazole (both are nitrogen-based ligands), and cyanide (carbon-based ligand) were studied to determine their kinetic binding characteristics with TXAS using stopped-flow spectroscopy. All these ligands caused Type II spectral changes, indicating that the exogenous ligands formed a low-spin coordinate heme complex. However, their binding kinetics to TXAS were quite different. U44069 and cyanide binding to TXAS exhibited monophasic kinetics, in contrast to the biphasic kinetics found for imidazole and clotrimazole. Analysis ofkobs versus ligand concentrations further indicated that cyanide underwent a one-step binding process, whereas U44069, imidazole, and clotrimazole underwent two-step binding processes. Interestingly, the binding mechanisms of those ligands involved in the two-step binding processes are distinctly different. U44069 first binds TXAS, but not heme iron, and then replaces the original heme ligand. The two nitrogen-based ligands, nonetheless, bind heme iron first and then undergo a conformational change before they reach equilibrium. Cyanide, which is charged but is the smallest molecule tested, had the lowest on-rate constant (2.4 m−1s−1). This low rate constant compared with other hemoproteins is likely due to the strong electron-donating thiolate ligand. On the other hand, clotrimazole, although much bulkier than imidazole, had a higher on-rate constant (8.4 × 104m−1 s−1 and 1.5 × 105m−1 s−1 for imidazole and clotrimazole, respectively). These results indicated that the TXAS active site is both hydrophobic and spacious, consistent with our previously reported findings (13Hsu P.-Y. Tsai A.-L. Kulmacz R.J. Wang L.-H. J. Biol. Chem. 1999; 274: 762-769Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Classical eukaryotic P450s catalyze hydroxylation reactions at a very slow rate with kcat of 1–10 mol of product formed/min/mol of P450 (21Guengerich F.P. J. Biol. Chem. 1991; 266: 10019-10022Abstract Full Text PDF PubMed Google Scholar). Many prokaryotic P450s, however, have somewhat higher catalytic activities. For example, P450camcatalyzes the reaction at a rate of ∼1900 mol/min/mol of protein (22Peterson J.A. Mock D.M. Cooper D.Y. Rosenthal O. Synder R. Witmer C. Cytochrome P450 and b5. Plenum Press, New York1975: 311-324Google Scholar), and so does P450BM-3 (23Narhi L.O. Fulco A.J. J. Biol. Chem. 1986; 261: 7160-7169Abstract Full Text PDF PubMed Google Scholar), a soluble protein in which the reductase domain is naturally fused to the oxygenase domain. For the “non-classical” P450s such as allene-oxide synthase and nitric-oxide reductase that do not need reductase for catalysis, the turnover numbers seems to be much higher. Allene-oxide synthase, a P450 acting as a dehydrase which converts lipid hydroperoxide to allene oxide, appears to form product at a rate of >60,000 mol/min/mol of protein (24Song W.C. Brash A.R. Science. 1991; 253: 781-784Crossref PubMed Scopus (230) Google Scholar). Nitric-oxide reductase, i.e.P450nor, which catalyzes NO to N2O has a turnover number of 72,000 mol/min/mol of protein (25Shiro Y. Fujii M. Iizuka T. Adachi S. Tsukamoto K. Nakahara K. Shoun H. J. Biol. Chem. 1993; 270: 1617-1623Abstract Full Text Full Text PDF Scopus (184) Google Scholar). However, prostacyclin synthase, another non-classical P450 that uses PGH2 as the natural substrate, has a lower turnover number (∼150 mol/min/mol of protein) (11Hecker M. Ullrich V. J. Biol. Chem. 1989; 264: 141-150Abstract Full Text PDF PubMed Google Scholar, 26Hara S. Miyata A. Yokoyama C. Inoue H. Brugger R. Lottspeich F. Ullrich V. Tanabe T. J. Biol. Chem. 1994; 269: 19897-19903Abstract Full Text PDF PubMed Google Scholar). TXAS, as observed in this study, formed MDA or TXA2 at a rate of 1,900 min−1. In other words, TXAS consumed PGH2 at a rate of 3,800 min−1. Furthermore, the ratio ofkcat/Km (an index of catalytic efficiency) of TXAS for PGH2 consumption is thus 3 × 106m−1s−1. Compared with carbonic anhydrase, an extremely efficient enzyme that has a kcat/Km of 107-108m−1s−1 (27Lindskog S. Engberg P. Forsman C. Ibrahim S.A. Johnson B.H. Simonsson I. Tibell L. Ann. N. Y. Acad. Sci. 1984; 429: 61-75Crossref PubMed Scopus (67) Google Scholar), TXAS should be considered as a very efficient catalyst. It should also be noted that many P450s including P450cam can act as non-classical P450s and convert PGH2 to MDA and HHT, but not TXA2, without reductase, molecular oxygen, or any electron donor (11Hecker M. Ullrich V. J. Biol. Chem. 1989; 264: 141-150Abstract Full Text PDF PubMed Google Scholar). Their “HHT/MDA synthase” activities, nonetheless, are much lower than TXAS. A recent report showed that four microsomal P450s (P4501A2, 2B1, 2E1, and 3A4) had Kd for U44069 of ∼200 μm and when assayed at 50 μmPGH2, their catalytic activities were 1–10 min−1 (28Plastaras J.P. Guengerich P. Nebert D.W. Marnett L.J. J. Biol. Chem. 2000; 275: 11784-11790Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). It would appear that PGH2 was readily converted to MDA and HHT by heme in the context of P450 where a hydrophobic environment is present. A study of chemical models for heme-catalyzed PGH2 reactions showed that 9% of HHT was formed in a phosphate buffer, whereas 33% of HHT was formed in acetonitrile, a less polar solvent (11Hecker M. Ullrich V. J. Biol. Chem. 1989; 264: 141-150Abstract Full Text PDF PubMed Google Scholar). TXAS is more efficient in HHT/MDA synthesis not only because its active site is hydrophobic but it also has a greater affinity for PGH2. Furthermore, several TXAS active site amino acid residues, as we have previously shown (29Wang L.-H. Matijevic-Aleksic N. Hsu P.-Y. Ruan K.-H. Wu K.K. Kulmacz R.J. J. Biol. Chem. 1996; 271: 19970-19975Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar), may be involved in the reaction. It is intriguing to note that prostacyclin synthase, although it has a high affinity for PGH2 (Kd is ∼10 μm), does not catalyze HHT/MDA formation (11Hecker M. Ullrich V. J. Biol. Chem. 1989; 264: 141-150Abstract Full Text PDF PubMed Google Scholar). What structural elements determine the HHT/MDA activity of P450 remains a challenging topic. In this study, the rate constant of each catalytic step,i.e. PGH2 to TXA2 or MDA/HHT, is estimated to be ∼15,000 s−1. The half-life of the intermediate(s) at 23 °C is therefore much less than the dead-time of the stopped-flow apparatus (∼1.5 ms). Furthermore, the rate constant of substrate binding is 12–20 × 106m−1 s−1 and the in vivo binding constant could be calculated if the cellular PGH2 concentration was known. To the best of our knowledge, cellular PGH2 concentration has not been reported due in part to the instability of PGH2 in aqueous solution. However, concentrations of arachidonic acid, the substrate for prostaglandin H synthase, in many inflammatory blood cells and lung tissues were less than 10 μm (30Triggiani M. Oriente A. Seeds M.C. Bass D.A. Marone G. Chilton F.H. J. Exp. Med. 1995; 182: 1181-1190Crossref PubMed Scopus (77) Google Scholar, 31Chilton F.H. Fonteh A.N. Surette M.E. Triggiani M. Winkler J.D. Biochim. Biophys. Acta. 1996; 1299: 1-15Crossref PubMed Scopus (209) Google Scholar). In pancreatic islets, cellular un-esterified arachidonate concentrations of 38–75 μm were reported under glucose-induced conditions. But the maximally effective concentration, including exogenous arachidonic acid, was 30–40 μm (32Wolf B.A. Pasquale S.M. Turk J. Biochemistry. 1991; 30: 6372-6379Crossref PubMed Scopus (110) Google Scholar). Since arachidonic acid is a substrate for many other pathways in addition to prostaglandin H synthase, it is probably safe to assume that the cellular PGH2 concentration is generally less than 40 μm. If that is the case, the substrate-binding rate constant for TXAS in the physiological conditions would be less than 800 s−1, and this value is much smaller than the other forward rate constants. We therefore conclude that the substrate-binding step is the rate-limiting step in the TXAS-catalyzed reaction. P450s were also classified according to the identity of their electron donors. Class I P450s require both ferredoxin and ferredoxin reductase for electron transfer, whereas Class II P450s require only a P450 reductase (33Graham-Lorence S. Peterson J.A. FASEB. J. 1996; 10: 206-214Crossref PubMed Scopus (124) Google Scholar). In both Class I and Class II P450s, the rate-limiting step of the overall reaction is at the stage of the second electron transfer to the heme center. Class III P450s that do not require any reductase, are not well characterized regarding their kinetic mechanisms. An elegant study on P450nor, a Class III P450, revealed that binding of NO and NADH substrates were fast to form the intermediate I, an NO-bound two-electron reduced species (25Shiro Y. Fujii M. Iizuka T. Adachi S. Tsukamoto K. Nakahara K. Shoun H. J. Biol. Chem. 1993; 270: 1617-1623Abstract Full Text Full Text PDF Scopus (184) Google Scholar). The intermediate I was slowly converted to the products. The rate-limiting step of P450nor is thus in the chemical steps. In contrast to other P450s, the rate-limiting step of TXAS is at the step of substrate binding. To the best of our knowledge, this kinetic mechanism is the first example of Class III P450s in which substrate binding is the rate-limiting step. We thank Dr. Graham Palmer at Rice University for generous access to the EPR facility and Dr. Richard J. Kulmacz for valuable suggestions and helpful assistance in preparation and purification of PGH2." @default.
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- W1980377002 title "Substrate Binding Is the Rate-limiting Step in Thromboxane Synthase Catalysis" @default.
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