Low-temperature reactions of trypsin with p-nitroanilide substrates: tetrahedral intermediate formation or enzyme isomerization

The reactions of trypsin with the p-nitroanilides of N alpha-carbobenzoxy-L-lysine, N alpha-carbobenzoxy-L-arginine, and N alpha-benzoyl-L-arginine have been studied in the 0 to -30 degrees C temperature region, over a range of pH values, using 65% (v/v) aqueous dimethyl sulfoxide cryosolvent. At alkaline pH, -30 degrees C, the catalytic reaction appears as a slow "burst" of product (or intermediate) followed by turnover. For all three substrates, the rate of the burst phase is identical. Preincubation of the enzyme at -30 degrees C abolishes the burst. On addition of trypsin to the cryosolvent at -30 degrees C, a time-dependent decrease in fluorescence emission is observed with the same rate as that of the burst with the anilides. The burst phase is thus interpreted as reflecting a temperature/solvent-induced isomerization of trypsin to a less catalytically efficient form, rather than the previously suggested formation of a tetrahedral intermediate [Compton, P. D., & Fink, A. L. (1980) Biochem. Biophys. Res. Commun. 93, 427-431]. The isomerization is not observed at high temperature (greater than or equal to 0 degree C) or at neutral pH. The burst phase was not observed with aqueous methanol cryosolvent, indicating that it is sensitive to both cosolvent and temperature.     

Subzero-temperature stabilization and spectroscopic characterization of homogeneous oxyferrous complexes of the cytochrome P450 BM3 (CYP102) oxygenase domain and holoenzyme

We describe herein for the first time the formation and spectroscopic characterization of homogeneous oxyferrous complexes of the cytochrome P450 BM3 (CYP102) holoenzyme and heme domain (BMP) at -55 degrees C using a 70/30 (v/v) glycerol/buffer cryosolvent. The choice of buffer is a crucial factor with Tris [tris(hydroxymethyl)aminomethane] buffer being significantly more effective than phosphate. The oxyferrous complexes have been characterized with magnetic circular dichroism spectroscopy and the resulting spectra compared to those of the more well-characterized oxyferrous cytochrome P450-CAM. The formation of a stable substrate-bound oxyferrous CYP BM3 holoenzyme, despite the fact that it has the necessary reducing equivalents for turnover, indicates that electron transfer from the flavin domain to the oxyferrous center is very slow at this temperature. The ability to prepare stable homogeneous oxyferrous derivatives of both BMP and the CYP BM3 holoenzyme will enable these species to be used as starting materials for mechanistic investigation of dioxygen activation.     

Direct observation of a novel perturbed oxyferrous catalytic intermediate during reduced putidaredoxin-initiated turnover of cytochrome P-450-CAM: probing the effector role of putidaredoxin in catalysis

The single turnover of (1R)(+)-camphor-bound oxyferrous cytochrome P450-CAM with one equivalent of dithionite-reduced putidaredoxin (Pdx) was monitored for the appearance of transient intermediates at 3 degrees C by double mixing rapid scanning stopped-flow spectroscopy. With excess camphor, three successive species were observed after generating oxyferrous P450-CAM and reacting versus reduced Pdx: a perturbed oxyferrous derivative, a species that was a mixture of high and low spin Fe(III), and high spin ferric camphor-bound enzyme. The rates of the first two steps, approximately 140 and approximately 85 s(-1), were assigned to formation of the perturbed oxyferrous intermediate and to electron transfer from reduced Pdx, respectively. In the presence of stoichiometric substrate, three phases with similar rates were seen even though the final state is low spin ferric P450-CAM. This is consistent with substrate being hydroxylated during the reaction. The single turnover reaction initiated by adding dioxygen to a preformed reduced P450-CAM.Pdx complex with excess camphor also led to phases with similar rates. It is proposed that formation of the perturbed oxyferrous intermediate reflects alteration of H-bonding to the proximal Cys, increasing the reduction potential of the oxyferrous state and triggering electron transfer from reduced Pdx. This species may be a direct spectral signature of the effector role of Pdx on P450-CAM reactivity (i.e. during catalysis). The substrate-free oxyferrous enzyme also reacted readily with reduced Pdx, showing that the inability of substrate-free P450-CAM to accept electrons from reduced Pdx and function as an NADH oxidase is completely due to the incapacity of reduced Pdx to deliver the first but not the second electron.     

High-pressure stopped-flow spectrometry at low temperatures

A stopped-flow instrument operating over temperature and pressure ranges of +30 to -20 degrees C and 10(-3) to 2 kbar , respectively, is described. The system has been designed so that it can be easily interfaced with many commercially available spectrophotometers of fast response time, with the aid of quartz fiber optics. The materials used for the construction are inert, metal free and the apparatus has proven to be leak free at temperatures as low as -20 degrees C under a pressure of 2 kbar . The performance of the instrument was tested by measuring the rate of reduction of cytochrome c with sodium dithionite and the 2,6-dichloroindophenol/ascorbate reaction. The dead time of the system has been evaluated to be 20, 50, and congruent to 100 ms in water at 20 degrees C, in 40% ethylene glycol/water, and at 20 degrees C and -15 degrees C, respectively. These values are rather pressure independent up to 2 kbar . Application of the bomb was demonstrated using the cytochrome c peroxidase/ethyl peroxide reaction. This process occurred in two phases and an increase in pressure decreased the rates of reactions indicating two positive volumes of activation (delta V not equal to app (fast) = 9.2 +/- 1.5 ml X mol-1; delta V not equal to app (slow) = 14 +/- 1.5 ml X mol-1, temperature 2 degrees C). The data suggest that the fast reaction could involve a hydrophobic bond, whereas the slow process could be associated with a stereochemical change of the protein. The problem of temperature equilibrium for high-pressure experiments is also discussed.     

Stabilized isoporphyrin intermediates in the inactivation of horseradish peroxidase by alkylhydrazines

The reaction of horseradish peroxidase with alkylhydrazines results in delta-meso-alkylation of the prosthetic heme group and enzyme inactivation (Ator, M. A., David, S. K., and Ortiz de Montellano, P. R. (1987) J. Biol. Chem. 262, 14954-14960). As reported here, enzyme inactivation is associated with the accumulation of intermediates that absorb at approximately 835 nm. The properties of these intermediates, including their collapse to give meso-alkylhemes, identify them as isoporphyrins. The t1/2 values for inactivation and formation of the isoporphyrin intermediate at 25 degrees C are, respectively, 11.6 and 12.5 min for methylhydrazine (2.0 mM), 8.7 and 7.2 min for ethylhydrazine (1.0 mM), and 30 and 25 s for phenylethylhydrazine (50 microM). The isoporphyrin intermediates are surprisingly long-lived, with half-lives (35 degrees C, pH 7.0) of 9, 28, 96, and 450 min for, respectively, the phenylethyl, methyl, n-butyl, and ethyl analogues. pH studies show that protonation of a group with pKa = 5.0-6.5 accelerates isoporphyrin decay and decreases steady state isoporphyrin accumulation. Horseradish peroxidase reconstituted with delta-meso-methylheme, unlike horseradish peroxidase with a heme that has a larger meso-substituent, is catalytically active but is more sensitive to H2O2-mediated degradation of the prosthetic group than is the native enzyme. The delta-meso-methylheme prosthetic group is converted in the reaction with H2O2 to a biliverdin-like product. The results implicate highly stabilized isoporphyrin intermediates in the inactivation of horseradish peroxidase by alkylhydrazines and indicate that inactivation by the meso-alkyl groups is due to steric interference with electron delivery to the heme edge rather than to intrinsic electronic consequences of meso-alkylation. The structural features that stabilize the cationic isoporphyrins may also be involved in stabilization of the Compound I porphyrin radical cation.     

The dioxygen cycle. Spectral, kinetic, and thermodynamic characteristics of ferryl and peroxy intermediates observed by reversal of the cytochrome oxidase reaction.

The catalytic mechanism of O2 reduction by cytochrome oxidase was studied in isolated mitochondria and mitoplasts by partial reversal of the reaction. At a high redox potential (Eh) of cytochrome c, high pH, and a high electrochemical proton gradient (delta mu H+) across the inner mitochondrial membrane, the initial ferriccupric state (O) of the oxidized enzyme's bimetallic oxygen reaction center is converted to ferryl (F) and peroxy (P) intermediates, the optical spectroscopic properties of which are reported in detail. This is associated with reversed electron transfer from the bimetallic center to ferricytochrome c. The kinetics of reduction of ferricytochrome c by the reversed electron transfer process are compared with the kinetics of formation of F and P. The results are consistent with transfer of one electron from the ferric-cupric bimetallic center (O) to cytochrome c, yielding the F intermediate, followed by transfer of one electron from the latter to cytochrome c, yielding the P state. In the absence of an effective redox buffer, poising cytochrome c highly oxidized, these primary events are immediately followed by reoxidation of cytochrome c, which is ascribed to forward electron transfer to enzyme molecules still in the O state. This forward reaction also results in accumulation of the P intermediate. Kinetic stimulations of the data predict equilibrium constants for the reversed electron transfer steps, and Em,7 values of approximately 1.1 and 1.2 V may be calculated for the F/O and P/F redox couples, respectively, at delta mu H+ and delta psi equal to zero. Taken together with previously measured Em,7 values, these data indicate that it is the two-electron reduction of bound dioxygen to bound peroxide that is responsible for the irreversibility of the catalytic dioxygen cycle of cell respiration.     

The expression of a catalytically active cholesterol 7 alpha-hydroxylase cytochrome P450 in Escherichia coli

We have recently cloned a full-length cDNA encoding the rat hepatic cholesterol 7 alpha-hydroxylase cytochrome P450 (P450c7) (Li, Y. C., Wang, D. P., and Chiang, J. Y. L. (1990) J. Biol. Chem. 265, 12012-12019), which catalyzes the rate-limiting reaction of bile acid synthesis in the liver. By using the polymerase chain reaction, we have designed two P450c7 cDNAs. One has the second Met codon deleted and the third Thr codon replaced with an Ala. The other lacks codons for the NH2-terminal hydrophobic sequence of amino acids 2-24 (P450c7 delta 2-24). The cDNAs were separately cloned into the expression vector pKK233-2 and transformed into Escherichia coli. After induction with isopropyl-beta-D-thiogalactopyranoside, bacteria harboring recombinant plasmids expressed a polypeptide which reacted with the antibody against cholesterol 7 alpha-hydroxylase in immunoblots. The slightly modified full-length enzyme was expressed to 0.2% of the total bacterial lysate and was located in the membrane fraction, whereas P450c7 delta 2-24 was expressed at a 10-fold higher level (2%), of which 85% was in the cytosol and the remaining associated with the membranes. We have purified P450c7 delta 2-24 which showed a typical reduced-CO difference spectrum of cytochrome P450 and reconstituted cholesterol 7 alpha-hydroxylase activity in the presence of NADPH-cytochrome P450 reductase. P450c7 delta 2-24 has a similar Km for cholesterol (24.6 microM) but a lower Vmax (0.10 nmol/min) and a lower turnover number (1.93 min-1) as compared with the enzyme isolated from rat liver microsomes. The purified P450c7 delta 2-24 has an unique hydrophilic NH2 terminus and contains monomers and dimers in equal amounts. This is the first report demonstrating that a genetically engineered cytochrome P450 enzyme lacking a typical NH2-terminal hydrophobic sequence is mainly cytosolic and catalytically active.     

Thermodynamics of the two step formation of horseradish peroxidase compound I

The effects of temperature (20 to -38 degrees C), pressure (normal pressures to 1.2 kbar) and solvent (water, 60% DMSO and 50% methanol) on the reaction of hydrogen peroxide or ethyl peroxide with horseradish peroxidase were studied. The formation of compound I was followed at 403 nm in a stopped flow apparatus adapted for high pressure and low temperature work. As with the alkaline form (Job and Dunford 1978), the neutral form of the peroxidase binds peroxide substrates in two steps. It was the combined use of organic solvents and low temperatures which revealed saturation kinetics: (Formula: see text) compound I, where E = horseradish peroxidase and S peroxide substrate. In water and organic solvents at temperatures above -10 degrees C, K1 was too small and k2 too large to be measured, here K1 X k2 was obtained. k-2 was too small for measurement under all conditions. Whereas K1 was insensitive to the peroxide substrate and solvent composition, k2 was very sensitive. The thermodynamic parameters delta H, delta S and delta V for K1 and k2 were obtained under different experimental conditions and the data are interpreted within the available thermodynamic theories.     

Biodehalogenation: reactions of cytochrome P-450 with polyhalomethanes

The products, stoichiometry, and kinetics of the oxidation of the enzyme cytochrome P-450 cam by five polyhalomethanes and chloronitromethane are described. The reactivity of the enzyme is compared with that of deuteroheme and with the enzyme in its native cell, Pseudomonas putida (PpG-786). In all cases, the reaction entails hydrogenolysis of the carbon-halogen bond: 2FeIIP + RCXn----2FeIIIP + RCHXn-1 (P = porphyrin or P-450 cam in vitro and in vivo). Trichloronitromethane was the fastest reacting substrate, and chloroform was the slowest. The results establish that P. putida is a valid whole cell model for the reductase activity of the P-450 complement in these reactions. The reactions of cytochrome P-450 with polyhaloalkanes proceed in a manner quite analogous to other iron(II) proteins in the G conformation. The chemistry observed for the enzyme parallels that of its iron(II) porphyrin active site. Iron-bonded carbenes are not intermediates, and hydrolytically stable iron alkyls are not products of these reactions.     

The ferrous-dioxygen intermediate in human cytochrome P450 3A4. Substrate dependence of formation and decay kinetics

The oxy-ferrous complex is the first of three branching intermediates in the catalytic cycle of cytochrome P450, in which the total efficiency of substrate turnover is curtailed by the side reaction of autoxidation. For human membrane-bound cytochromes P450, the oxy complex is believed to be the primary source of cytotoxic superoxide and peroxide, although information on the properties and stability of this intermediate is lacking. Here we document stopped-flow spectroscopic studies of the formation and decay of the oxy-ferrous complex in the most abundant human cytochrome P450 (CYP3A4) as a function of temperature in the substrate-free and substrate-bound form. CYP3A4 solubilized in purified monomeric form in nanoscale POPC bilayers is functionally and kinetically homogeneous. In substrate-free CYP3A4, the oxy complex is extremely unstable with a half-life of approximately 30 ms at 5 degrees C. Saturation with testosterone or bromocriptine stabilizes the oxy-ferrous intermediate. Comparison of the autoxidation rates with the available data on CYP3A4 turnover kinetics suggests that the oxy complex may be an important route for uncoupling.     

High pressure, a tool for exploring heme protein active sites

High pressure is an interesting and suitable parameter in the study of the dynamics and stability of proteins. The effects of pressure on proteins delineates its volumic (deltaV degrees ) and energetic (deltaG degrees ) parameters. An enormous amount of effort has been invested by several laboratories in developing basic theory and high pressure techniques that allow the determination of barotropic parameters. Cytochrome P450s, one of the largest super families of heme proteins, are good models for high pressure studies. Two distinct pressure-induced spin transitions of the heme iron in the active site and a P450 to P420 inactivation process have been characterized. The obtained reaction volumes of these two processes for a series of analog-bound cytochrome P450s are compared. We have shown that both the spin volume and the inactivation volume are dependent on the substrate analogs which are known to modulate the polarity and hydration of the heme pocket. Several linear correlations were found between these reaction volumes and the physico-chemical properties of the heme protein such as the polarity-induced exposure of tyrosines, the hydration of the cytochrome CYP101 heme pocket, and the mobility and binding of the substrates indicate that they constitute the main contribution to the complex thermodynamic reaction volume parameters. This interpretation allows us to conclude that cytochrome CYP101, CYP2B4 and CYP102 possess a similar mechanism of substrate binding. Interestingly the barotropic behaviors of monomeric cytochrome P450s are quite different from those of oligomeric and hetorooligomeric cytochrome P450s. The interactions of heterooligomeric subunits influence the stability of individual cytochrome P450s and the asymmetric organization of subunits which can control and modulate the activity and the recognition with NADPH-cytochrome P450 reductase.     

My passion for extreme conditions to solve the cytochrome P450 and NO synthase reaction mechanisms

Cytochrome P450, and especially its reaction mechanism, has always been a major research interest of Professor I.C. Gunsalus. The reaction cycle of this enzyme is complex, containing many elementary steps and intermediates, and constitutes a challenge for the scientific community. During his repeated stays in our laboratories in Paris and in Montpellier, he contributed decisively to our study of the P450 reaction mechanism under extreme conditions, i.e., at subzero temperatures and at high pressure. From this initial impulse, we continued the work with different forms of cytochrome P450 and later on with nitric oxide synthase. This paper gives an overview of the insights into these enzymes gained by the use of extreme conditions. These exciting achievements were initiated by numerous discussions with Professor Gunsalus and also reflect a long-lasting collaboration and friendship.     

Caffeine-inducible enzyme activity in Pseudomonas putida ATCC 700097

Caffeine (1,3,7-trimethylxanthine), a ubiquitous component of human diet has been suggested as a chemical indicator of ecosystem impacts of sewage spills and treated effluent discharges because it is not sufficiently metabolized by wastewater microorganisms. This study identified enzymes responsible for caffeine metabolism in sewage bacteria. Pseudomonas putida biotype A (ATCC 700097) originally isolated as a rare caffeine-degrading organism in domestic wastewater exhibited diauxic growth on caffeine, concomitant with the expression of a P450-type cytochrome and peroxidase enzyme activities. Initial growth phase lasted 13.8 ± 1.4 h with a growth rate that was five times slower than the secondary growth phase that lasted 5.5 ± 1.2 h. Molecular and enzymatic characteristics of the cytochrome P450-type enzyme differ from the previously described cytochrome P450 (P450_cam) of P. putida (ATCC 17453) involved in camphor metabolism. The caffeine-inducible cytochrome P450-type enzyme exhibited a carbon monoxide difference spectrum peak at 450 nm, but does not allow growth on camphor. Caffeine induced production of haem-associated peroxidase activity was confirmed with 3,3, 5,5-tetramethylbenzidine–H₂O₂ reaction in polyacrylamide gels. Polymerase chain reaction (PCR) primers derived from the gene for cytochrome P450_cam (camC) of P. putida (ATCC 17453) did not yield an amplification product when DNA extracted from P. putida strain ATCC 700097 was used as template. The data demonstrate that caffeine is metabolized through a specific biphasic pathway driven by oxygen-demanding enzymes.     

Effect of solvent, pressure and temperature on reaction rates of the multiheme hydroxylamine oxidoreductase. Evidence for conformational change

Hydroxylamine oxidoreductase (HAO) of the ammonia-oxidizing bacterium Nitrosomonas catalyzes the oxidation: NH2OH + H2O----HNO2 + 2e- + 2 H+. The heme-like chromophore P460 is part of a site which binds substrate, extracts electrons and then passes them to the many c hemes of the enzyme. Reduction of the c hemes by hydroxylamine is biphasic with apparent first-order rate constants k1 and k2. CO binds to ferrous P460 with apparent first-order rate constants, k1,CO. In this work we have measured the binding of CO to ferrous P460 of hydroxylamine oxidoreductase and the reduction by substrate of some of the 24 c hemes of the ferric enzyme. These reactions have been studied in water and 40% ethylene glycol, at temperatures ranging from -15 degrees C to 20.7 degrees C and at hydrostatic pressures ranging over 0.1-80 MPa. From the measurements, thermodynamic parameters delta V+ (activation volume), delta G+, delta H+, and delta S+ have been calculated. CO binding. Binding of CO to ferrous P460 was similar to the binding of CO to ferrous horseradish peroxidase. The change of solvent had only a limited effect on delta V+ (-30 ml.mol-1), delta G+, delta H+ or delta S+ and did not cause an inflection in the Arrhenius plot or downward displacement of the linear relationship between ln k1,CO and P at a critical temperature. Binding was exothermic at high temperatures. The response of the binding of CO to solvent, temperature and pressure suggested that the CO binding site had little access to solvent and was not susceptible to change in protein conformation. Fast phase of reduction of c hemes. Changing the solvent from water to 40% ethylene glycol resulted in a decrease from 90% to 50% in the relative number of c hemes reduced during the fast phase, an increase in activation volume from -3.6 ml.mol-1 to 57 ml.mol-1 and changes in other thermodynamic parameters. The activation volume increased with decreasing temperature. The Arrhenius plot had a downward inflection at about 0 degrees C and, in water or ethylene glycol, the linear dependence of ln k1 on P was displaced downwards as the temperature changed from 3.5 degrees C to -15 degrees C. Slow phase of reduction of c hemes. Changing the solvent from water to 40% ethylene glycol resulted in an increase in the relative number of c hemes reduced during the slow phase from 10% to 50%. The activation volume, which was not measurable in water because of the low absorbance change, was -30 ml.mol-1 in ethylene glycol. The activation volume increased with increasing temperature.(ABSTRACT TRUNCATED AT 400 WORDS)     

A molecular model for the enzyme cytochrome P450(17 alpha), a major target for the chemotherapy of prostatic cancer

The enzyme cytochrome P450(17 alpha) catalyses two key steps in the biosynthesis of the androgens from pregnanes: the 17 alpha hydroxylation step and the subsequent 17-20 lyase reaction. Using a variety of techniques, including sequence alignment, secondary structure prediction, molecular mechanics and molecular dynamics, we have constructed a model for the three-dimensional structure of P450(17 alpha) based on that of P450cam, the only cytochrome P450 enzyme for which the crystal structure is known. The model suggests the possibility of two modes of binding of steroid substrates at the active site, perhaps reflecting the dual functionality of the enzyme.     

The catalytic mechanism of Pseudomonas cytochrome c peroxidase

The catalytic mechanism of Pseudomonas cytochrome c peroxidase has been studied using rapid-scan spectrometry and stopped-flow measurements. The reaction of the totally ferric form of the enzyme with H₂O₂ was slow and the complex formed was inactive in the peroxidatic cycle, whereas partially reduced enzyme formed highly reactive intermediates with hydrogen peroxide. Rapid-scan spectrometry revealed two different spectral forms, one assignable to Compound I and the other to Compound II as found in the reaction cycle of other peroxidases. The formation of Compound I was rapid approaching that of diffusion control. The stoichiometry of the peroxidation reaction, deduced from the formation of oxidized electron donor, indicates that both the reduction of Compound I to Compound II and the conversion of Compound II to resting (partially reduced) enzyme are one-electron steps. It is concluded that the reaction mechanism generally accepted for peroxidases is applicable also to Pseudomonas cytochrome c peroxidase, the intramolecular source of one electron in Compound I formation, however, being reduced heme c.     

Enzyme systems for biodegradation of polychlorinated dibenzo-p-dioxins

The angular dioxygenase, cytochrome P450, lignin peroxidase, and dehalogenase are known as dioxin-metabolizing enzymes. All of these enzymes have metal ions in their active centers, and the enzyme systems except for peroxidase have each distinct electron transport chain. Although the enzymatic properties of the angular dioxygenase, lignin peroxidase, and cytochrome P450 have been studied well, the information about dehalogenase is much less than other enzyme systems due to its instability under the aerobic conditions. However, this enzyme system appears to be quite promising from the viewpoint of practical use for bioremediation, because dehalogenases are capable of degradation of polychlorinated dibenzo-p-dioxins (PCDDs) with more than four chlorine substituents, whereas the other three enzyme systems prefer low-chlorinated PCDDs. On the other hand, protein engineering of angular dioxygenase, lignin peroxidase, and cytochrome P450 based on their tertiary structures has great potential to generate highly efficient dioxin-metabolizing enzymes. Actually, we successfully generated 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-metabolizing enzyme by site-directed mutagenesis of cytochrome P450. We hope that recombinant microorganisms harboring genetically engineered dioxin-metabolizing enzymes will be used for bioremediation of soil contaminated with PCDDs and polychlorinated dibenzofurans in the near future.     

Deoxysarpagine hydroxylase--a novel enzyme closing a short side pathway of alkaloid biosynthesis in Rauvolfia

Microsomal preparations from cell suspension cultures of the Indian plant Rauvolfia serpentina catalyze the hydroxylation of deoxysarpagine under formation of sarpagine. The newly discovered enzyme is dependent on NADPH and oxygen. It can be inhibited by typical cytochrome P450 inhibitors such as cytochrome c, ketoconazole, metyrapone, tetcyclacis and carbon monoxide. The CO-effect is reversible with light (450 nm). The data indicate that deoxysarpagine hydroxylase is a novel cytochrome P450-dependent monooxygenase. A pH optimum of 8.0 and a temperature optimum of 35 degrees C were determined. K(m) values were 25 microM for NADPH and 7.4 microM for deoxysarpagine. Deoxysarpagine hydroxylase activity was stable in presence of 20% sucrose at -25 degrees C for >3 months. The analysis of presence of the hydroxylase in nine cell cultures of seven different families indicates a very limited taxonomic distribution of this enzyme.     

Human placental estrogen synthetase (aromatase). Effect of environment on the kinetics of protein-protein and substrate-protein interactions and the production of 19-oxygenated androgen intermediates in the purified reconstituted cytochrome P450 enzyme system

Estrogen synthetase (aromatase) catalyzes the conversion of androgen into estrogen via two hydroxylations at C19 and a subsequent C19-10 lyase reaction. We report here the results of a reconstitution study using a highly purified aromatase cytochrome P450 monooxygenase enzyme system, with both protein components (cytochrome P450 and NADPH-cytochrome P450 reductase) obtained from human term placental microsomes. By varying one of the components (amounts of cytochrome P450, NADPH-cytochrome P450 reductase, or androgen substrate) as the other two were held constant in four different environments (phospholipid, non-ionic detergent, mixture of phospholipid and non-ionic detergent and buffer alone), we obtained evidence supporting the following conclusions. The reconstituted enzyme is more active and the protein components exhibit much lower apparent Km values in the detergent and/or lipid environment compared with buffer alone. Although the apparent Km and Vmax values for each aromatase protein component differ significantly in most cases with the particular limiting component and environment, the catalytic efficiency (Kcat/Km) was independent of the limiting protein component and varied with the environment only (highest in the lipid-detergent mixture and lowest in lipid alone). When the concentration of androgen substrate (androstenedione or testosterone) was varied at constant amounts of the aromatase protein components (NADPH-cytochrome P450 reductase saturating), the Km was lower and the Vmax was higher for adrostenedione. The specificity constant (Vmax/Km) was a function of the reconstitution environment (highest in lipid alone and lowest in detergent alone) and was, on average, about 4-fold higher for androstenedione in a particular environment. The extent of production of 19-oxygenated androgen intermediates (19-hydroxy and 19-oxo androstenedione) was examined at three different levels of aromatase cytochrome P450 (subsaturating, saturating, super-saturating) relative to the NADPH-cytochrome P450 reductase component in the three different hydrophobic environments using androstenedione as substrate. Both 19-oxygenated androgens, each made in comparable amounts relative to control, were isolatable in greatest amounts under cytochrome P450 super-saturating conditions in the detergent-lipid mixed environment, and in least amounts under cytochrome P450 subsaturating conditions in the lipid-only environment. Based on these data, we propose that 19-oxygenated androgen intermediates are biosynthesized sequentially in a step-wise fashion as the cytochrome P450 and NADPH-cytochrome P450 reductase form transient complexes, and that the amount of isolatable 19-oxygenated androgen is proportional to the amount of excess cytochrome P450 component.     

Activation of the microsomal glutathione S-transferase by metabolites of alpha-methyldopa

Rat liver microsomes contain a membrane-bound GSH S-transferase (GSH-tr), an enzyme that is involved in the detoxication of xenobiotics. Also located on rat liver microsomes is the cytochrome P450 system, an enzyme complex that catalyzes the conversion of several xenobiotics into reactive intermediates. In this study, it was demonstrated that reactive products from alpha-methyldopa formed by the cytochrome P450 system are able to stimulate microsomal GSH-tr. Also, products formed from alpha-methyldopa that are generated by H2O2-horseradish peroxidase and tyrosinase are able to stimulate the activity of microsomal GSH-tr. GSH was able to prevent the activation of microsomal GSH-tr. Our results indicate that the ortho-quinone or semi-ortho-quinone radical of alpha-methyldopa is responsible for the stimulation of microsomal GSH-tr, probably via arylation of the free sulfhydryl group of microsomal GSH-tr. This conclusion was supported by the observation that 4-methyl-ortho-quinone itself was able to stimulate microsomal GSH-tr via sulfhydryl arylation. Our results are in conformity with the hypothesis that reactive products formed by the cytochrome P450 complex are able to stimulate microsomal GSH-tr and possibly in this way enhance their detoxication.