Positioning of a spin-labeled substrate analogue into the structure of delta 5-3-ketosteroid isomerase by combined kinetic, magnetic resonance, and X-ray diffraction methods

We have shown by kinetic and magnetic resonance measurements that a spin-labeled substrate analogue, spiro[doxyl-2,3'-5' alpha-androstan]-17'beta-ol, binds at the substrate site of crystalline delta 5-3-ketosteroid isomerase (steroid delta-isomerase; EC 5.3.3.1) of Pseudomonas testosteroni. The spin-labeled steroid is a linear competitive inhibitor with a Ki value (25 +/- 5 microM) that is consistent with dissociation constants obtained by direct binding measurements based on changes in the electron paramagnetic resonance spectrum of the nitroxide, longitudinal relaxation rates of water protons, and longitudinal and transverse relaxation rates of carbon-bound protons of the isomerase. These binding studies yield a stoichiometry for the nitroxide of 1 per subunit of the enzyme. Measurements of the longitudinal relaxation rates of water protons indicate that the 3-doxyl portion of the spin-label is highly immobilized yet is exposed to solvent. Paramagnetic effects of the nitroxide on T1 defined distances to several previously assigned [Benisek, W. F., & Ogez, J. R. (1982) Biochemistry 21, 5816-5825] and newly assigned protons of the enzyme. These distances were then used to locate (with an accuracy of +/- 2 A) the nitroxide moiety at a unique position in a partially refined 2.5-A resolution X-ray structure of native isomerase. Three of five additional proton resonance peaks, attributed to ring-shielded methyl groups, could be assigned to specific residues on the basis of distances from the spin-label in the X-ray structure. The remaining portion of the spin-labeled steroid was then docked into the X-ray structure in a hydrophobic cavity of the enzyme. This position of the steroid is consistent with the steroid binding site previously proposed [Westbrook, E. M., Piro, O. E., & Sigler, P. B. (1984) J. Biol. Chem. 259, 9096-9103]. However, the rotational orientation of this steroid about its long axis could not be unambiguously established. If we assume that steroid substrates and the spin-labeled inhibitor bind to the same site, but with reversal of the 3- and 17-positions, then the phenolic hydroxyl of Tyr-55 is optimally positioned to function as the general acid that protonates the 3-keto group of the substrate, facilitated by the negative end of the dipole of a 10-residue alpha-helix, the only helix in the molecule.(ABSTRACT TRUNCATED AT 400 WORDS)     

Structure-based inhibitor design for an enzyme that binds different steroids: a potent inhibitor for human type 5 17beta-hydroxysteroid dehydrogenase

Human type 5 17beta-hydroxysteroid dehydrogenase plays a crucial role in local androgen formation in prostate tissue. Several chemicals were synthesized and tested for their ability to inhibit this enzyme, and a series of estradiol derivatives bearing a lactone on the D-ring were found to inhibit its activity efficiently. The crystal structure of the type 5 enzyme in complex with NADP and such a novel inhibitor, EM1404, was determined to a resolution of 1.30 A. Significantly more hydrogen bonding and hydrophobic interactions were defined between EM1404 and the enzyme than in the substrate ternary complex. The lactone ring of EM1404 accounts for important interactions with the enzyme, whereas the amide group at the opposite end of the inhibitor contributes to the stability of three protein loops involved in the construction of the substrate binding site. EM1404 has a strong competitive inhibition, with a Ki of 6.9+/-1.4 nM, demonstrating 40 times higher affinity than that of the best inhibitor previously reported. This is observed despite the fact that the inhibitor occupies only part of the binding cavity. Attempts to soak the inhibitor into crystals of the binary complex with NADP were unsuccessful, yielding a structure with a polyethylene glycol fragment occupying the substrate binding site. The relative crystal packing is discussed. Combined studies of small molecule inhibitor synthesis, x-ray crystallography, enzyme inhibition, and molecular modeling make it possible to analyze the plasticity of the substrate binding site of the enzyme, which is essential for developing more potent and specific inhibitors for hormone-dependent cancer therapy.     

Crystallographic studies of the mechanism of xylose isomerase

The mechanism of xylose isomerase (EC 5.3.1.5) has been studied with X-ray crystallography. Four refined crystal structures are reported at 3-A resolution: native enzyme, enzyme + glucose, enzyme + glucose + Mg2+, and enzyme + glucose + Mn2+. One of these structures (E.G.Mg) was determined in a crystal mounted in a flow cell. The other structures were equilibrium experiments carried out by soaking crystals in substrate containing solution. These structures and other studies suggest that, contrary to expectation, xylose isomerase may not use the generally expected base-catalyzed enolization mechanism. A mechanism involving a hydride shift is consistent with the structures presented here and warrants further investigation. Additional evidence in support of a hydride shift comes from comparing xylose isomerase with triosephosphate isomerase which is known to catalyze an analogous reaction via an enediol intermediate. Evidence is presented that suggests that aldose-ketose isomerases can be divided into two groups. Phospho sugar isomerases generally do not require a metal ion for activity and show exchange of substrate protons with solvent. In contrast, simple sugar isomerases all require a metal ion and show very low solvent exchange. These observations are rationalized on the basis of the need for stereospecific sugar binding.     

Kinetic and ultraviolet spectroscopic studies of active-site mutants of delta 5-3-ketosteroid isomerase

delta 5-3-Ketosteroid isomerase (EC 5.3.3.1) of Pseudomonas testosteroni promotes the highly efficient isomerization of delta 5-3-ketosteroids to delta 4-3-ketosteroids by means of a direct and stereospecific transfer of the 4 beta-proton to the 6 beta-position, via an enolic intermediate. An acidic residue responsible for the protonation of the 3-carbonyl function of the steroid and a basic group concerned with the proton transfer have been implicated in the catalytic mechanism. Recent NMR studies with a nitroxide spin-labeled substrate analogue have allowed positioning of the steroid into the 2.5-A X-ray crystal structure of the enzyme [Kuliopulos, A., Westbrook, E.M., Talalay, P., & Mildvan, A.S. (1987) Biochemistry 26, 3927-3937], thereby corroborating the approximate location of the steroid binding site deduced from a difference Fourier X-ray diffraction map of the 4-(acetoxymercuri)estradiol-isomerase complex [Westbrook, E.M., Piro, O.E., & Sigler, P.B. (1984) J. Biol. Chem. 259, 9096-9103]. The steroid lies in a hydrophobic cavity near Asp-38, Tyr-14, and Tyr-55. In order to assess the role of these amino acid residues in catalysis, the gene for isomerase was cloned, sequenced, and overexpressed in Escherichia coli [Kuliopulos, A., Shortle, D., & Talalay, P. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8893-8897], and the following mutants were prepared: Asp-38 to asparagine (D38N) and Tyr-14 and Tyr-55 to phenylalanine (Y14F and Y55F, respectively). The kcat value of the D38N mutant enzyme is 10(5.6)-fold lower than that of the wild-type enzyme, suggesting that Asp-38 functions as the base which abstracts the 4 beta-proton of the steroid in the rate-limiting step. Threefold lower Km values in all mutants indicate tighter binding of the substrate to the more hydrophobic sites. In comparison with the wild-type enzyme, the Y55F mutant shows only a 4-fold decrease in kcat while the Y14F mutant shows a 10(4.7)-fold decrease in kcat, suggesting that Tyr-14 is the general acid. The red shift of the ultraviolet absorption maximum of the competitive inhibitor 19-nortestosterone from 248 to 258-260 nm, which occurs upon binding to the wild-type enzyme [Wang, S.F., Kawahara, F.S., & Talalay, P. (1963) J. Biol. Chem. 238, 576-585], is mimicked in strong acid. This spectral shift was also observed with the D38N and Y55F mutants, but not on binding of the steroid to the Y14F mutant.(ABSTRACT TRUNCATED AT 400 WORDS)     

Chemical modification of chalcone isomerase by diethyl pyrocarbonate: histidine residues are not essential for catalysis

Chalcone isomerase form soybean is inactivated by treatment with diethyl pyrocarbonate (DEP). The competitive inhibitor 4',4-dihydroxychalcone provides kinetic protection against inactivation by DEP with a binding constant at the site of protection in agreement with its binding constant at the active site. Very high concentrations of the competitive inhibitors 4',4-dihydroxychalcone or morin hydrate offer a 10- to 40-fold maximal protection, suggesting a second slower mechanism for inactivation which cannot be prevented by blockage of the active site. Blockage of the only cysteine residue in chalcone isomerase with p-mercuribenzoate does not affect the rate constant for DEP-dependent inactivation and indicates that the modification of the cysteine residue is not responsible for the activity loss observed in the presence of DEP. Treatment of inactivated enzyme with hydroxylamine does not restore catalytic activity, indicating that the modification of histidine or tyrosine residues is not responsible for the activity loss. All five histidines of chalcone isomerase are modified by DEP at pH 5.7 and ionic strength 1.0 M. The rate constant for the modification of the histidine residues of chalcone isomerase is close to that for the reaction of N-acetyl histidine with DEP, indicating that the histidine residues are quite accessible to the modifying reagent. The rate of histidine modification is the same in native enzyme, in urea-denatured enzyme, and in the presence of a competitive inhibitor. In the presence of the competitive inhibitor morin hydrate, all of the histidine residues of chalcone isomerase can be modified without significant loss in catalytic activity. These results demonstrate that the histidine residues of chalcone isomerase are not essential for catalysis and therefore cannot function as nucleophilic catalysts as previously proposed.     

Glucosephosphate isomerase from Trypanosoma brucei. Cloning and characterization of the gene and analysis of the enzyme

In Trypanosoma brucei the enzyme glucose-6-phosphate isomerase, like most other enzymes of the glycolytic pathway, resides in a microbody-like organelle, the glycosome. Here we report a detailed study of this enzyme, involving a determination of its kinetic properties and the cloning and sequence analysis of its gene. The gene codes for a polypeptide of 606 amino acids, with a calculated Mr of 67280. The protein predicted from the gene sequence has 54-58% positional identity with its yeast and mammalian counterparts. Compared to those other glucose-6-phosphate isomerases the trypanosomal enzyme contains an additional 38-49 amino acids in its N-terminal domain, as well as a number of small insertions and deletions. The additional amino acids are responsible for the 5-kDa-larger subunit mass of the T. brucei enzyme, as measured by gel electrophoresis. The glucose-6-phosphate isomerase of the trypanosome has no excess of positive residues and, consequently, no high isoelectric point, in contrast to the other glycolytic enzymes that are present in the glycosome. However, similar to other glycosomal proteins analyzed so far, specific clusters of positive residues can be recognized in the primary structure. Comparison of the kinetic properties of the T. brucei glucose-6-phosphate isomerase with those of the yeast and rabbit muscle enzymes did not reveal major differences. The three enzymes have very similar pH profiles. The affinity for the substrate fructose 6-phosphate (Km = 0.122 mM) and the inhibition constant for the competitive inhibitor gluconate 6-phosphate (Ki = 0.14 mM) are in the same range as those of the similar enzymes. The Km shows the same strong dependence on salt as the rabbit muscle enzyme, although somewhat less than the yeast glucose-6-phosphate isomerase. The trypanocidal drug suramin inhibits the T. brucei and yeast enzymes to the same extent (Ki = 0.29 and 0.36 mM, respectively), but it had no effect on the rabbit muscle enzyme. Agaricic acid, a potent inhibitor of various glycosomal enzymes of T. brucei, has also a strong, irreversible effect on glucose-6-phosphate isomerase, while leaving the yeast and mammalian enzymes relatively unaffected.     

Crystallization and preliminary X-ray diffraction studies of mitochondrial short-chain delta 3,delta 2-enoyl-CoA isomerase from rat liver.

Crystals of short-chain delta 3,delta 2-enoyl-CoA isomerase (EC 5.3.3.8) from rat liver mitochondria have been grown using the hanging-drop vapour diffusion technique. The enoyl-CoA isomerase is an auxiliary enzyme in the beta-oxidation pathway of fatty acid metabolism, and catalyzes the isomerization of unsaturated fatty acids to produce the metabolizable delta 2-trans isomer. The crystals belong to the orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions a = 47.9, b = 118.4 and c = 164.8 A, and diffract to 3 A.     

The kinetic mechanism of the anterior pituitary progesterone 5 alpha-reductase

An analysis of the kinetic mechanism of the microsomal NADPH-linked progesterone 5 alpha-reductase obtained from female rat anterior pituitaries was performed. Initial velocity, product inhibition and dead-end inhibition studies indicate that the kinetic mechanism for the progesterone 5 alpha-reductase is equilibrium ordered sequential. Analysis of the initial velocity data resulted in intersecting double reciprocal plots suggesting a sequential mechanism [apparent Km(progesterone) = 88.2 +/- 8.2 nM; apparent Kia(NADPH) = 7.7 +/- 1.1 microM]. Furthermore, the plot of 1/v vs 1/progesterone intersected on the ordinate which is indicative of an equilibrium ordered mechanism. Additional support for ordered substrate binding was provided by the product inhibition studies with NADPH versus NADP and progesterone versus NADP. NADP is a competitive inhibitor versus NADPH (apparent Kis = 7.8 +/- 1.0 microM) and a noncompetitive inhibitor versus progesterone (apparent Kis = 9.85 +/- 2.1 microM and apparent Kii = 63.2 +/- 12.5 microM). These inhibition patterns suggest that NADPH binds prior to progesterone. In sum, these kinetic studies indicate that NADPH binds to the microsomal enzyme in rapid equilibrium and preferentially precedes the binding of progesterone.     

Human placental 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase: purification from microsomes, substrate kinetics, and inhibition by product steroids

In human pregnancy, placental 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase produce progesterone from pregnenolone and metabolize fetal dehydroepiandrosterone sulfate to androstenedione, an estrogen precursor. The enzyme complex was solubilized from human placental microsomes using the anionic detergent, sodium cholate. Purification (500-fold, 3.9% yield) was achieved by ion exchange chromatography (Fractogel-TSK DEAE 650-S) followed by hydroxylapatite chromatography (Bio-Gel HT). The purified enzyme was detected as a single protein band in sodium dodecylsulfate-polyacrylamide gel electrophoresis (monomeric Mr = 19,000). Fractionation by gel filtration chromatography at constant specific enzyme activity supported enzyme homogeneity and determined the molecular mass (Mr = 76,000). The dehydrogenase and isomerase activities copurified. Kinetic constants were determined at pH 7.4, 37 degrees C for the oxidation of pregnenolone (Km = 1.9 microM, Vmax = 32.6 nmol/min/mg) and dehydroepiandrosterone (Km = 2.8 microM, Vmax = 32.0 nmol/min/mg) and for the isomerization of 5-pregnene-3,20-dione (Km = 9.7 microM, Vmax = 618.3 nmol/min/mg) and 5-androstene-3,17-dione (Km = 23.7 microM, Vmax = 625.7 nmol/min/mg). Mixed substrate analyses showed that the dehydrogenase and isomerase reactions use the appropriate pregnene and androstene steroids as alternative, competitive substrates. Dixon analyses demonstrated competitive inhibition of the oxidation of pregnenolone and dehydroepiandrosterone by both product steroids, progesterone and androstenedione. The enzyme has a 3-fold higher affinity for androstenedione than for progesterone as an inhibitor of dehydrogenase activity. Based on these competitive patterns of substrate utilization and product inhibition, the pregnene and androstene activities of 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase may be expressed at a single catalytic site on one protein in human placenta.     

Structure of the complex between trypanosomal triosephosphate isomerase and N-hydroxy-4-phosphono-butanamide: binding at the active site despite an "open" flexible loop conformation.

The structure of triosephosphate isomerase from Trypanosoma brucei complexed with the competitive inhibitor N-hydroxy-4-phosphono-butanamide was determined by X-ray crystallography to a resolution of 2.84 A. Full occupancy binding of the inhibitor is observed only at one of the active sites of the homodimeric enzyme where the flexible loop is locked in a completely open conformation by crystal contacts. There is evidence that the inhibitor also binds to the second active site of the enzyme, but with low occupancy. The hydroxamyl group of the inhibitor forms hydrogen bonds to the side chains of Asn 11, Lys 13, and His 95, whereas each of its three methylene units is involved in nonpolar interactions with the side chain of the flexible loop residue Ile 172. Interactions between the hydroxamyl and the catalytic base Glu 167 are absent. The binding of this phosphonate inhibitor exhibits three unusual features: (1) the flexible loop is open, in contrast with the binding mode observed in eight other complexes between triosephosphate isomerase and various phosphate and phosphonate compounds; (2) compared with these complexes the present structure reveals a 1.5-A shift of the anion-binding site; (3) this is the first phosphonate inhibitor that is not forced by the enzyme into an eclipsed conformation about the P-CH2 bond. The results are discussed with respect to an ongoing drug design project aimed at the selective inhibition of glycolytic enzymes of T. brucei.     

The structure of rhamnose isomerase from Escherichia coli and its relation with xylose isomerase illustrates a change between inter and intra-subunit complementation during evolution

Using a new expression construct, rhamnose isomerase from Escherichia coli was purified and crystallized. The crystal structure was solved by multiple isomorphous replacement and refined to a crystallographic residual of 17.4 % at 1.6 A resolution. Rhamnose isomerase is a tight tetramer of four (beta/alpha)(8)-barrels. A comparison with other known structures reveals that rhamnose isomerase is most similar to xylose isomerase. Alignment of the sequences of the two enzymes based on their structures reveals a hitherto undetected sequence identity of 13 %, suggesting that the two enzymes evolved from a common precursor. The structure and arrangement of the (beta/alpha)(8)-barrels of rhamnose isomerase are very similar to xylose isomerase. Each enzyme does, however, have additional alpha-helical domains, which are involved in tetramer association, and largely differ in structure. The structures of complexes of rhamnose isomerase with the inhibitor l-rhamnitol and the natural substrate l-rhamnose were determined and suggest that an extended loop, which is disordered in the native enzyme, becomes ordered on substrate binding, and may exclude bulk solvent during catalysis. Unlike xylose isomerase, this loop does not extend across a subunit interface but contributes to the active site of its own subunit. It illustrates how an interconversion between inter and intra-subunit complementation can occur during evolution. In the crystal structure (although not necessarily in vivo) rhamnose isomerase appears to bind Zn(2+) at a "structural" site. In the presence of substrate the enzyme also binds Mn(2+) at a nearby "catalytic" site. An array of hydrophobic residues, not present in xylose isomerase, is likely to be responsible for the recognition of l-rhamnose as a substrate. The available structural data suggest that a metal-mediated hydride-shift mechanism, which is generally favored for xylose isomerase, is also feasible for rhamnose isomerase.     

Subcellular distribution and properties of progesterone (delta4-steroid) 5alpha-reductase in rat medial basal hypothalamus.

The subcellular distribution and properties of rat hypothalamic progesterone 5 alpha-reductase, which accelerates the conversion of progesterone to 5 alpha-pregnane-3,20-dione, have been investigated by utilizing 3H-labeled substrate and a reverse isotopic dilution assay system. The enxymic activity was associated primarily with a cell debris-membranes fraction deribed from the 100 x g pellet. This fraction contained mainly membrane-like particulates and was free of nuclei. Little or no activity was associated with the purified nuclei. The hypothalamic 5 alpha-reductase was stimulated by NADPH but not by NADH. The reaction proceeded optimally over a pH range of 6.0 to 7.2 and at a temperaturhe substrate specificity of the enzyme for other delta 4-3-ketosteroids and the ability of these steroids to inhibit the 5 alpha reduction of [1,2-3H]progesterone as well as the effect of 17 beta-estradiol were also studied. 20 alpha-hydroxypregn-4-en-3-one was more reactive that progesterone, while testosterone was the least reactive. The estimated Km for 20 alpha-hydroxypregn-4-en-3-one was 8.6 +/- 1.9 x 10(-7) M, and for testosterone, 1.6 +/- 1.4 x 10(-5) M. The inhibition studies indicate that 20 alpha-hydroxypregn-4-en-3-one and 17 beta-estradiol are competitive and noncompetitive inhibitors, respectively, of the 5 alpha reduction of progesterone with Ki of 6.0 +/- 3.0 x 10(-8) M for 20 alpha-hydroxypregn-4-en-3-one and Kii (intercept inhibition constant) of 2.6 +/- 0.7 x 10(-5) M and Kis (slope inhibition constant) of 3.6 +/- 0.6 x 10(-5) M for 17 beta-estradiol. Testosterone is a poor competitive inhibitor of the reaction.     

Interaction of mouse submaxillary gland renin with a statine-containing, subnanomolar, competitive inhibitor

The interaction between mouse submaxillary gland renin and a statine-containing, iodinated substrate analog inhibitor was studied. The compound, 1 (Boc-His-Pro-Phe-(4-iodo)-Phe-Sta-Leu-Phe-NH2, Sta = (3S,4S)-4-amino-3-hydroxy-6-methyl-heptanoic acid), a statine-containing analog of the renin substrate octapeptide, was a competitive inhibitor of cleavage of synthetic tetradecapeptide renin substrate by mouse submaxillary gland renin, with a Ki of 6.2 x 10(-10) M (pH 7.2, 37 degrees C). Titration of the partial quenching of the tryptophan fluorescence of the enzyme by 1 revealed tight binding with a dissociation constant less than 3 nM and a binding stoichiometry of one mole 1 per mole enzyme. The time course of tight binding of 1 to mouse renin appeared to be fast, with kON greater than or equal to 1.3 x 10(6) s-1 M-1. The UV difference spectrum generated upon binding of 1 to mouse renin had two prominent features: a strong, broad band that had a minimum at 242 nm with delta epsilon (242) = -19,500 cm-1 M-1, and a triplet of enhanced bands centered at 286 nm with delta epsilon (286) about +1100 cm-1 M-1. The strong, broad, negative band was similar to the difference between the UV absorbance of 1 in methanol and in 0.1 M citrate phosphate pH 7.2. A structure-activity correlation for analogs of 1 showed some moieties of 1 that are important for potent inhibition of mouse renin. The inhibition data for these compounds versus human kidney renin suggested that the solution of the crystal structure of 1 bound to mouse renin will provide useful information for the design of inhibitors of human kidney renin.     

Conformational changes in phosphoglucose isomerase induced by ligand binding

Phosphoglucose isomerase (PGI; EC 5.3.1.9) is the second enzyme in glycolysis, where it catalyzes the isomerization of D-glucose-6-phosphate to D-fructose-6-phosphate. It is the same protein as autocrine motility factor, differentiation and maturation mediator, and neuroleukin. Here, we report a new X-ray crystal structure of rabbit PGI (rPGI) without ligands bound in its active site. The structure was solved at 1.8A resolution by isomorphous phasing with a previously solved X-ray crystal structure of the rPGI dimer containing 6-phosphogluconate in its active site. Comparison of the new structure to previously reported structures enables identification of conformational changes that occur during binding of substrate or inhibitor molecules. Ligand binding causes an induced fit of regions containing amino acid residues 209-215, 245-259 and 385-389. This conformational change differs from the change previously reported to occur between the ring-opening and isomerization steps, in which the helix containing residues 513-521 moves toward the bound substrate. Differences between the liganded and unliganded structures are limited to the region within and close to the active-site pocket.     

Energetics of thrombin-fibrinogen interaction.

The kinetic mechanism of thrombin-fibrinogen interaction has been elucidated by steady-state measurements of synthetic substrate hydrolysis by human alpha-thrombin in the presence of human fibrinogen used as a competitive inhibitor and sucrose used as a viscogenic agent. Sucrose greatly affects the FKm for thrombin-fibrinogen interaction, without altering the intrinsic properties of the system. Under conditions of pH 7.5 and 0.1 M NaCl, fibrinogen behaves like a sticky substrate for thrombin, with acylation being comparable to dissociation in the temperature range 20-37 degrees C. In the same temperature range, deacylation is much faster than acylation. The van't Hoff enthalpy of binding for thrombin-fibrinogen interaction is -24 +/- 3 kcal/mol and the entropy is -55 +/- 11 cal mol-1 deg-1. A chemical compensation effect is present in the binding of fibrinogen and synthetic amide substrates to thrombin, with the delta H and delta G values being linked through a linear relationship.     

Affinity alkylation of human placental 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase by 2 alpha-bromoacetoxyprogesterone: evidence for separate dehydrogenase and isomerase sites on one protein

We have copurified human placental 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase, which synthesize progesterone from pregnenolone and androstenedione from fetal dehydroepiandrosterone sulfate, from microsomes as a homogeneous protein based on electrophoretic and NH2-terminal sequencing data. The affinity alkylator, 2 alpha-bromoacetoxyprogesterone, simultaneously inactivates the pregnene and androstene dehydrogenase activities as well as the C21 and C19 isomerase activities in a time-dependent, irreversible manner following first order kinetics. At four concentrations (50/1-20/1 steroid/enzyme M ratios), the alkylator inactivates the dehydrogenase activity (t1/2 = 1.5-3.7 min) 2-fold faster than the isomerase activity. Pregnenolone and dehydroepiandrosterone protect the dehydrogenase activity, while 5-pregnene-3,20-dione, progesterone, and androstenedione protect isomerase activity from inactivation. The protection studies and competitive kinetics of inhibition demonstrate that the affinity alkylator is active site-directed. Kitz and Wilson analyses show that 2 alpha-bromoacetoxyprogesterone inactivates the dehydrogenase activity by a bimolecular mechanism (k3' = 160.9 l/mol.s), while the alkylator inactivates isomerase by a unimolecular mechanism (Ki = 0.14 mM, k3 = 0.013 s-1). Pregnenolone completely protects the dehydrogenase activity but does not slow the rate of isomerase inactivation by 2 alpha-bromoacetoxyprogesterone at all. NADH completely protects both activities from inactivation by the alkylator, while NAD+ protects neither. From Dixon analysis, NADH competitively inhibits NAD+ reduction by dehydrogenase activity. Mixed cofactor studies show that isomerase binds NAD+ and NADH at a common site. Therefore, NADH must not protect either activity by simply binding at the cofactor site. We postulate that NADH binding as an allosteric activator of isomerase protects both the dehydrogenase and isomerase activities from affinity alkylation by inducing a conformational change in the enzyme protein. The human placental enzyme appears to express the pregnene and androstene dehydrogenase activities at one site and the C21 and C19 isomerase activities at a second site on the same protein.     

Microsomal enzymes of cholesterol biosynthesis from lanosterol. Solubilization and purification of steroid 8-isomerase

Steroid-8-ene isomerase that catalyzes isomerization of delta 8- to delta 7-sterols has been solubilized from rat liver microsomes with a mixture of two detergents, octylglucoside and sodium taurodeoxycholic acid. During a 40-fold enrichment of the solubilized enzyme, other enzymes of cholesterol biosynthesis, endogenous lipids, and electron carriers are removed. A comparison of properties of the solubilized and partially purified isomerase with the membrane-bound enzyme shows they are essentially identical with respect to pH profile, effect of inhibitors and cofactors, substrate specificity, and Km values. Addition of phospholipid to the partially purified enzyme stimulates activity as much as 1.8-fold over control rates. Although the relative rate of isomerization of cholesta-8,24-dien-3 beta-ol is six times that observed with cholest-8-en-3 beta-ol, the delta 8 to delta 7 ratio at equilibrium is approximately equal. The reversibility of the reaction has been demonstrated by the direct conversion of cholest-7-en-3 beta-ol to cholest-8-en-3 beta-ol; at equilibrium the delta 7-isomer is predominant (19/1). The purified enzyme does not catalyze isomerization of cholesta-8,14-dien-3 beta-ol and cholest-8(14)-en-3 beta-ol under conditions that result in equilibrium mixtures of isomers from cholest-8(9)-en-3 beta-ol. These results are consistent with the earlier suggestion that delta 8(14)-sterols are neither formed nor metabolized by the same microsomal enzymes that catalyze transformation of lanosterol to cholesterol.     

Human placental 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase: purification from mitochondria and kinetic profiles, biophysical characterization of the purified mitochondrial and microsomal enzymes

In human placenta, 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase, an enzyme complex found in microsomes and mitochondria, synthesizes progesterone from pregnenolone and androstenedione from fetal dehydroepiandrosterone sulfate. The dehydrogenase and isomerase activities of the mitochondrial enzyme were copurified (733-fold) using sequential cholate solubilization, ion exchange chromatography (DEAE-Toyopearl 650S), and hydroxylapatite chromatography (Bio-Gel HT). Enzyme homogeneity was demonstrated by a single protein band in SDS-polyacrylamide gel electrophoresis (monomeric Mr = 41,000), gel filtration at constant specific enzyme activity (Mr = 77,000), and a single NH2-terminal sequence. Kinetic constants were determined for the oxidation of pregnenolone (Km = 1.6 microM, Vmax = 48.6 nmol/min/mg) and dehydroepiandrosterone (Km = 2.4 microM, Vmax = 48.5 nmol/min/mg) and for the isomerization of 5-pregnene-3,20-dione (Km = 9.3 microM, Vmax = 914.2 nmol/min/mg) and 5-androstene-3,17-dione (Km = 27.6 microM, Vmax = 888.4 nmol/min/mg. Mixed substrate studies showed that the dehydrogenase and isomerase activities utilize their respective pregnene and androstene substrates competitively. Dixon analysis demonstrated that the product steroids, progesterone and androstenedione, are competitive inhibitors of the C-21 and C-19 dehydrogenase activities. Enzyme purified from mitochondria and microsomes had similar kinetic profiles with respect to substrate utilization, product inhibition, and cofactor (NAD+) reduction (mean Km +/- SD using C-19 and C-21 dehydrogenase substrates = 26.4 +/- 0.8 microM, mean Vmax = 73.2 +/- 1.3 nmol/min/mg). Pure enzyme from both organelles exhibited identical biophysical properties in terms of molecular weight and subunit composition, pH optima (pH 9.8, dehydrogenase; pH 7.5, isomerase), temperature optimum (37 degrees C), stability in storage and solution, effects of divalent cations, and the single NH2-terminal sequence of 27 amino acids. These results suggest that the mitochondrial and microsomal enzymes are the same protein localized in different organelles.     

Human placental 3beta-hydroxysteroid dehydrogenase: delta5-isomerase. Demonstration of an intermediate in the conversion of 3beta-hydroxypregn-5-en-20-one to pregn-4-ene-3,20-dione.

3beta-Hydroxypregn-5-en-20-one (pregnenolone) and NAD+ were incubated with a solubilized preparation of the coupled enzyme 3beta-hydroxysteroid:NAD(P) oxidoreductase-3-ketosteroid delta4,delta5-isomerase (3beta-hydroxysteroid dehydrogenase: delta5-isomerase) from the mitochondrial fraction of human placenta. Unconverted pregnenolone, pregn-4-ene-3,20-dione (rogesterone), and a small but detectable amount of pregn-5-ene-3,20-dione were isolated from the medium by Sephadex LH-20 chromomatography. The identification of pregn-5-ene-3,20-dione, confirmed by mass fragmentography, has provided the first direct evidence for the formation of the hypothetical delta5,3-ketone intermediate in the conversion of pregnenolone to progesterone. When tritium-labeled pregnenolone and [4-14C]pregnenolone were incubated simultaneously the 3H:14C ratio in isolated pregn-5-ene-3,20-dione was 4.6 times greater than in isolated progesterone and pregnenolone, indicating a kinetic isotope effect in the enzymatic isomerization of tritium-labeled pregn-5-ene-3,20-dione. Exposure of the enzyme to two steroids which inhibit the overall enzyme reaction, 2alpha-cyano-17beta-hydroxy-4,4,17alpha-trimethylandrost-5-en-3-one (cyanoketone) and 3-hydroxyestra-1,3,5(10),6,8-pentaen-17-one (equilenin), increased the relative yield of labeled pregn-5-ene-3,20-dione as well as the recovery of radioactivity remaining as unconverted pregnenolone, suggesting that both the dehydrogenase and isomerase activities were inhibited. Exposure of the enzyme to equilenin increased the ratio of isolated pregn-5-ene-3,20-dione radioactivity to progesterone radioactivity as progesterone synthesis was inhibited. Equilenin also diminished the tritium isotope effect on the isomerase reaction. Both findings suggest that it is possible to inhibit the isomerase to a greater extent than the dehydrogenase. In order to measure the rate of progesterone produced by the coupled enzymes, we have modified a radiochemical method which involves precipitation of pregnenolone by digitonin. Digitonin precipitation proved to be effective in separating unconverted pregnenolone from the steroid products of both enzyme reactions, progesterone and pregn-5-ene-3,20-dione. Neither the steroidal inhibitors nor the kinetic isotope effect altered the accuracy of the method for routine measurement of the overall rate of conversion of delta5,3beta-hydroxysteroid to delta4,3-ketosteroid.     

Anomeric specificity of D-xylose isomerase.

Crystal structures of complexes of D-xylose isomerase with deoxysugars have been determined. Deoxynojirimycin is a structural analogue of alpha-pyranose and mimics the binding of these aldose substrates. The structure of this complex supports the hypothesis that an imidazole group catalyzes ring opening of the pyranose. The steric restrictions in the active site of the enzyme prevent a beta-pyranose from binding in the same way. For the reverse reaction with ketoses, the anomeric specificity is less certain. Dideoxyimino-D-glucitol is a structural analogue of the ketose alpha-D-furanose. The binding of the inhibitor dideoxyimino-D-glucitol to the crystals of the enzyme does not mimic the binding of the reactive alpha-D-fructofuranose. Superposition of the nonphysiological substrate alpha-D-fructofuranose onto the atomic positions of dideoxyimino-D-glucitol is not possible due to the steric restrictions of the active site. However, by utilizing the approximate 2-fold symmetry of the sugar, a stereochemically sensible model is produced which is consistent with other data. In addition to reaction with alpha-D-furanose, the enzyme probably reacts with open ring keto sugars which are present at significant concentrations. Other sugars which resemble furanoses either do not inhibit significantly or are not observed in the crystals bound in a single conformation.