Enzyme kinetics in reversed micelles. 3. Behaviour of 20 beta-hydroxysteroid dehydrogenase.
(17/27)The kinetic parameters of 20 beta-hydroxysteroid dehydrogenase were determined in aqueous solutions and in reversed micellar media composed with either an anionic, a cationic or a nonionic surfactant, at low and at high ionic strength. The velocity data were analysed in two ways: first by extrapolation to infinite concentrations of both substrates to determine 'apparent' Michaelis constants and V values, and secondly by comparison to reaction rates calculated using the model presented (see first of this series of papers in this issue of the journal). Data analysis according to the first method reveals some differences in the kinetic parameters in reversed micelles as compared to those in aqueous solution, though the kinetic parameters of the enzyme seem not to be much affected by enclosure in reversed micelles. It is shown that the changes that do occur are not caused by a shift of the intramicellar pH or by electrostatic interactions between the enzyme and the surfactant head groups. Interpretation of the data using the second method assumes that the enzyme is not affected by the enclosure in reversed micelles, and that deviations with respect to the aqueous parameters are caused by exchange phenomena between distinct aqueous droplets in the organic phase and by a high effective intramicellar substrate concentration. This model is able to predict reaction rates that agree rather well with experimentally determined rates and explains why the enzyme mechanism in reversed micelles is, at all progesterone concentrations used, the same as observed at high progesterone concentrations in aqueous solution. Furthermore it clarifies the occurrence of substrate inhibition in sodium-di(ethylhexyl)sulphosuccinate-reversed micelles and the observed low activity in Triton-reversed micelles, as arising from the high partition coefficient of progesterone and the slow rate of diffusion of progesterone into the reversed micelles. From these results, and those reported for enoate reductase (see preceding paper in this issue of the journal) it can be concluded that the theory presented before (see first of this series of papers in this issue of the journal) offers a good explanation for the observed kinetic behaviour in reversed micelles, and emphasizes the importance of exchange processes between micelles. (+info)
Zebrafish 20beta-hydroxysteroid dehydrogenase type 2 is important for glucocorticoid catabolism in stress response.
Crystals of active tetramers of 3 alpha, 20 beta-hydroxysteroid dehydrogenase.
(19/27)The NADH-dependent steroid metabolizing enzyme 3 alpha, 20 beta-hydroxysteroid dehydrogenase (EC 220.127.116.11), from Streptomyces hydrogenans, has been crystallized in the active tetrameric form. Single crystals (approximately 0.75 X 0.40 X 0.40 mm) of square bipyramid shape have been grown reproducibly at room temperature in the presence of excess NADH. Diffraction experiments have been performed at the Cornell High Energy Synchrotron Source. The space group is P43212 or its enantiomorph, and the cell dimensions are a = 106.0(5) A and c = 204(1) A. The asymmetric unit is a tetramer of identical subunits of approximately 25,000 daltons each. The specific volume is 2.8 A3/dalton. A native data set at 2.5-A resolution has been collected. Two potential heavy atom derivatives, with K2Pt(CN)4 and KAu(CN)2, have been identified from the diffraction photographs. (+info)
Steroid desmolase synthesis by Eubacterium desmolans and Clostridium cadavaris.
(20/27)The synthesis of a steroid desmolase was demonstrated in two obligate anaerobes: a new bacterial species, Eubacterium desmolans, isolated from cat fecal flora, and Clostridium cadavaris, recovered from sewage of New York City. The enzyme cleaves the C-17-C-20 bond of corticoids possessing hydroxyl functions at C-17 and C-21. The conversion is quantitative, provided the substrate concentration is less than 100 micrograms/ml and the organisms are in the log phase. The velocity of transformation parallels the bacterial growth curve and in the log phase is higher for E. desmolans than for C. cadavaris. In addition, both organisms synthesize a 20 beta-hydroxysteroid dehydrogenase. (+info)
"Affinity" chromatography of steroid-transforming enzymes with a non-steroidal ligand.
(21/27)The chromatographic behaviour of an avian oestradiol-17 beta dehydrogenase, the 3(17) beta-hydroxy steroid dehydrogenase from Pseudomonas testosteroni and cortisone reductase from Streptomyces dehydrogenans was studied on columns of p-(phenoxypropoxy)aniline attached to CNBr-activated Sepharose. The ligand was effective in adsorbing the oestradiol dehydrogenase from a partially purified extract of chicken liver, and the cortisone reductase was perferentially retained when mixtures of the three dehydrogenases were applied to columns in 10mM-buffer. Under these conditions the 3(17)beta-hydroxy steroid dehydrogenase was eluted in the front, but was adsorbed in the presence of 3 M-KCl. beta-N-Acetylglucosaminidase present in the liver preparation was not retained by the ligand, whereas lactate dehydrogenase from rabbit muscle was adsorbed in a manner similar to the retention pattern found on affinity chromatography with 2',5'-ADP--Sepharose. The mean overall purification of the oestradiol dehydrogenase was 13-fold, with a mean recovery of 53%. p-(Phenoxypropoxy)aniline offers promise for the purification of steroid-transforming enzymes where elution with substrate or cofactor is not wanted. It is also suggested that the ligand may be of service in the purification of receptors of hormonal steroids. (+info)
Rules for the regulation of enzyme activity in reserved micelles as illustrated by the conversion of apolar steroids by 20 beta-hydroxysteroid dehydrogenase.
(22/27)20 beta-Hydroxysteroid dehydrogenase was enclosed in reversed micellar media consisting of cetyltrimethyl-ammonium bromide, hexanol, organic solvent and Hepes buffer. The influence of the composition of these media on the enzymatic reduction of the apolar steroids progesterone and prednisone was investigated by varying the water content, concentration of hexanol and type of organic solvent. By changing the water content and the type of organic solvent, the hexanol to cetyltrimethylammonium bromide ratio in the interphase can be varied. This ratio was determined by phase boundary titrations. It was found that the higher this ratio, the higher the rate of steroid conversion. From variations of the hexanol content it was concluded that the rate of steroid conversion is determined by the hydrophobicity of the steroid relative to the hydrophobicity of the continuous phase and the hydrophobicity of the interphase. The hydrophobicity of the phases was expressed in log P-values. Log P is defined as the logarithm of the partition coefficient in an octanol-water two-phase system. This enabled us to derive the following relations between the hydrophobicity values for the substrate (log Ps), for the interphase (log Pi) and for the continuous phase (log Peph): [log Pi-log Ps] must be minimal to ensure a high steroid concentration in the interphase and [log Pcph-log Ps] must be large to keep the steroid concentration in the continuous phase low. With these considerations, for any given apolar compound, a medium can be composed that gives optimal enzymatic conversion. (+info)
5'-bromoacetamido-5'-deoxyadenosine. A novel reagent for labeling adenine nucleotide sites in proteins.
(23/27)We have synthesized and characterized 5'-bromoacetamido-5'-deoxyadenosine (5'-BADA), a new reagent for labeling adenine nucleotide binding sites in enzymatic and regulatory proteins. 5'-BADA possessed exceptionally high solubility and stability in aqueous buffers between pH 5.0 and 8.6 at 25 degrees C. A Dixon plot of data from enzyme kinetic measurements showed that 5'-BADA is a competitive inhibitor of NADH oxidation by 3 alpha,20 beta-hydroxysteroid dehydrogenase with a Ki value of 11.8 mM. This compares with a Ki value of 10 mM for adenosine under similar experimental conditions. Incubating 5'-BADA with a 3 alpha,20 beta-hydroxysteroid dehydrogenase at pH 7.0 and 25 degrees C caused simultaneous loss of both 3 alpha and 20 beta activity. The enzyme inactivation reaction proceeded by a first order kinetic process. The rates of enzyme inactivation as a function of 5'-BADA concentration obeyed saturation kinetics. 2-Bromoacetamide, at ten times the maximum concentration of 5'-BADA, had no measurable effect on enzyme activity during 25 h of incubation. NADH and AMP protected 3 alpha,20 beta-hydroxysteroid dehydrogenase against inactivation by 5'-BADA. The results suggest that 5'-BADA inactivates the enzyme by irreversibly binding to the adenine domain of the NADH cofactor binding region at the catalytic site of 3 alpha,20 beta-hydroxysteroid dehydrogenase. Irreversible binding follows from an alkylation reaction between the bromoacetamido side chain of 5'-BADA and an amino acid at or near the enzyme catalytic site. 5'-BADA is presented as a new reagent for selectively labeling amino acid residues at the adenine nucleotide binding sites of enzymatic and regulatory proteins. (+info)
20beta-Hydroxysteroid oxidoreductase. Kinetics and binding of corticosteroids and corticosteroid-21-aldehydes.
(24/27)Corticosteroid-21-aldehydes were reduced only at C-20 by 20 beta-hydroxysteroid dehydrogenase (EC 18.104.22.168) of Streptomyces hydrogenans, and the reduction occurred by transfer of hydrogen from the B-side of NADH. A kinetic investigation of cortisol, cortisone, cortexolone, and the 21-aldehydes of each indicated: (a) the magnitude of the Michaelis constant for any substrate was independent of the second substrate concentration; (b) the 21-aldehydes had larger Michaelis constants (5- to 8-fold) and larger maximum velocities (16- to 40-fold) than the steroids from which they were synthesized; (c) the Michaelis constant for NADH, 29 muM, was independent of the steroid substrate. With cortisol and cortisol-21-aldehyde, product inhibition patterns showed only slope effects with steroid product and NAD+, suggesting a "random" mechanism. Inhibition studies with the "poor" substrate cortisol indicated that cortisol and cortisol-21-aldehyde were reduced at the same site. The inhibition constant (180 muM) agreed with the Michaelis constant of cortisol (140 muM). The steroid product, 20beta-hydroxyprogesterone, gives noncompetitive inhibition patterns with respect to NADH and cortisol-21-aldehyde, indicating a separate binding site exists on the enzyme for this inhibitor. The intrinsic protein fluorescence of 20beta-hydroxysteroid dehydrogenase was quenched by NADH (56%) with a dissociation constant of 16 muM. NAD" quenched the protein fluorescence somewhat less (31%) with a dissociation constant of 104 muM. The fluorescence of 2-p-toluidine-6-naphthalene sulfonate is enhanced in the presence of enzyme, and there is a blue shift in the emission wavelength maximum. The enzyme-enhanced 2-p-toluidine-6-naphthalene sulfonate fluorescence is quenched by NAD+ (32%) with a dissociation constant of 128 muM. Corticosteroids and their corresponding 21-aldehydes completely quench the enhanced 2-p-toluidine-6-naphthalene sulfonate fluorescence and this feature can be used to determine enzyme-steroid dissociation constants. Corticosteroid-21-aldehydes and NAD+ dissociation constants determined in this manner agree with values obtained in kinetic measurements. The dissociation constants determined for cortisol, cortisone, cortexolone, progesterone, and 20beta-hydroxyprogesterone were at least 1 order of magnitude greater than the corresponding kinetic constants, and these findings suggest the presence of a kinetically insignificant binding site. (+info)