Mechanism-based inactivation of 17 beta,20 alpha-hydroxysteroid dehydrogenase by an acetylenic secoestradiol

14,15-Secoestra-1,3,5(10)-trien-15-yne-3,17 beta-diol (1) is a mechanism-based inactivator of human placental 17 beta,20 alpha-hydroxysteroid dehydrogenase (estradiol dehydrogenase, EC 1.1.1.62). Inactivation with alcohol 1 requires NAD-dependent enzymic oxidation and follows approximately pseudo-first-order kinetics with a limiting t1/2 of 82 min and a "Ki" of 2.0 microM at pH 9.2 and 25 degrees C. At saturating concentrations of NAD, the initial rate of inactivation is slower than in the presence of 5 microM NAD, suggesting that cofactor binding to free enzyme impedes the inactivation process. Glutathione completely protects the enzyme from inactivation at both cofactor concentrations. Inactivation with 45 microM tritiated alcohol 1 followed by dialysis and gel filtration demonstrates a covalent interaction and affords an estimated stoichiometry of 1.4 molecules of steroid per subunit (2.8 per dimer). Chemically prepared 3-hydroxy-14,15-secoestra-1,3,5(10)-trien-15-yn-17-one (2) rapidly inactivates estradiol dehydrogenase with biphasic kinetics. From the latter phase, a Ki of 2.8 microM and a limiting t1/2 of 12 min at pH 9.2 were determined. Estradiol, NADH, and NAD all retard this latter inactivation phase. We propose that enzymatically generated ketone 2 inactivates estradiol dehydrogenase after its release from and return to the active site of free enzyme.     

Identification of 7 alpha,12 alpha-dihydroxy-5 beta-cholestan-3-one 3 alpha-hydroxysteroid dehydrogenase

A reductase catalyzing the reduction of the 3-ketone group of 7 alpha,12 alpha-dihydroxy-5 beta-cholestan-3-one and 7 alpha-hydroxy-5 beta-cholestan-3-one, which are the intermediates in the conversion of cholesterol to cholic acid and chenodeoxycholic acid, respectively, into the 3 alpha-hydroxyl group, was purified about 250-fold as judged by the activity from the 100,000 X g supernatant of rat liver homogenate. The purified enzyme was electrophoretically homogeneous, and its molecular weight determined by sodium dodecyl sulfate-polyacrylamide gel electrophoretography was 32,000. The absorption spectrum of the purified enzyme showed only a peak at 280 nm due to aromatic amino acids, precluding the presence of a chromophoric prosthetic group in the molecule. The enzyme showed activity toward a variety of substrates, including 3-oxo-5 beta-cholanoic acid, androsterone, 9,10-phenanthrenquinone, p-nitrobenzaldehyde, but not toward glucuronic acid, DL-glyceraldehyde, and glycolaldehyde. The optimal pH for the reduction of 7 alpha-hydroxy-5 beta-cholestan-3-one was 7.4, and the cofactor required was either NADPH or NADH, though the former gave the higher activity. Judging from the chromatography behavior as well as substrate specificity, the enzyme was identified as 3 alpha-hydroxysteroid dehydrogenase (3 alpha-hydroxysteroid:NAD(P)+ oxidoreductase, EC 1.1.1.50).     

Identification of a human stomach alcohol dehydrogenase with distinctive kinetic properties

A new form of alcohol dehydrogenase, designated mu-alcohol dehydrogenase, was identified in surgical human stomach mucosa by isoelectric focusing and kinetic determinations. This enzyme was anodic to class I (alpha, beta, gamma) and class II (pi) alcohol dehydrogenases on agarose isoelectric focusing gels. The partially purified mu-alcohol dehydrogenase, specifically using NAD+ as cofactor, catalyzed the oxidation of aliphatic and aromatic alcohols with long chain alcohols being better substrates, indicating a barrel-shape hydrophobic binding pocket for substrate. mu-Alcohol dehydrogenase stood out in high Km values for both ethanol (18 mM) and NAD+ (340 microM) as well as in high Ki value (320 microM) for 4-methylpyrazole, a competitive inhibitor for ethanol. mu-Alcohol dehydrogenase may account for up to 50% of total stomach alcohol dehydrogenase activity and appeared to play a significant role in first-pass metabolism of ethanol in human.     

Evidence that enzyme-generated aromatic Michael acceptors covalently modify the nucleotide-binding site of 3 alpha-hydroxysteroid dehydrogenase.

The non-steroidal allylic and acetylenic alcohols 1-(4'-nitrophenyl)prop-2-en-1-ol (I) and 1-(4'-nitrophenyl)prop-2-yn-1-ol (II) are oxidized by homogeneous 3 alpha-hydroxysteroid dehydrogenase to the corresponding alpha beta-unsaturated ketones 1-(4'-nitrophenyl)prop-2-en-1-one (III) and 1-(4'-nitrophenyl)prop-2-yn-1-one (IV), which then inactivate the enzyme selectively with high affinity; low effective partition ratios are observed for the parent alcohols [Ricigliano & Penning (1989) Biochem. J. 262, 139-149]. Inactivation of 3 alpha-hydroxysteroid dehydrogenase by compound (I) displays an NAD+ concentration optimum. Scavenging experiments indicate that the enzyme-generated inactivators (III) and (IV) alkylate the enzyme via a release-and-return mechanism. Several lines of evidence suggest that compounds (III) and (IV) covalently modify the NAD(P)(+)-binding site. First, micromolar concentrations of NAD(P)H offer substantial protection against enzyme inactivation mediated by Michael acceptors (III) and (IV). In these protection studies Kd measurements for NAD(P)H approached those measured by fluorescence titration of free enzyme. Secondly, under initial-velocity conditions compounds (III) and (IV) act essentially as competitive inhibitors of NAD+ binding, and as mixed competitive or non-competitive inhibitors against androsterone binding. Thirdly, enzyme inactivated with either compound (III) or compound (IV) fails to bind to NAD+ affinity columns (e.g. Affi-gel Blue). Under the same conditions of chromatography native enzyme and enzyme affinity-labelled at the steroid-binding site with 17 beta-bromoacetoxy-5 alpha-dihydrotestosterone is retained on the affinity column. A kinetic scheme that represents the inactivation of the homogeneous dehydrogenase by the enzyme-generated alkylators (III) and (IV) is presented.     

Camphoroquinone reduction: another reaction catalyzed by rat liver cytosol 3 alpha-hydroxysteroid dehydrogenase

A new purification scheme is described for the female rat liver cytosolic enzyme dually catalyzing the oxidation of androsterone (3 alpha-hydroxysteroid:NAD(P)+ oxidoreductase, EC 1.1.1.50) and acenaphthenol (trans-1,2-dihydrobenzene-1,2-diol:NADP+ oxidoreductase, EC 1.3.1.20). This purification procedure yielded the most highly purified preparation of this enzyme thus far published as adjudged from its androsterone oxidation activity. In addition, we have demonstrated that this purified enzyme also catalyzes the reduction of camphoroquinone, a natural monoterpene, non-aromatic quinone. The nature of the products of the camphoroquinone reduction has been partially elucidated and agrees with previously published results (Robertson, J.S. and Solomon, E. (1971) Biochem. J. 121, 503-509). Kinetic studies of the metabolism of androsterone, camphoroquinone and acenaphthenol by the enzyme have been performed, yielding respective Km and Vmax values. The results of these studies allow a clarification of the mechanism of action of this enzyme, particularly with respect to its dihydrodiol dehydrogenase activity.     

Fast kinetics analysis of the peroxidatic reaction catalysed by horse liver alcohol dehydrogenase

The kinetics of the enzymatic step of the peroxidatic reaction between NAD and hydrogen peroxide, catalysed by horse liver alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1), has been investigated at pH 7 at high enzyme concentration. Under such conditions no burst phase has been observed, thus indicating that the rate-limiting step in the process, which converts NAD into Compound I, either precedes or coincides with the chemical step responsible for the observed spectroscopic change. Kinetic analysis of the data, performed according to a simplified reaction scheme suggests that the rate-limiting step is coincident with the spectroscopic (i.e., chemical) step itself. Furthermore, the absence of a proton burst phase indicates the proton release step does not precede the chemical step, in contrast with the case of ethanol oxidation. A kinetic effect of different premixing conditions on the reaction rate has been observed and attributed to the presence of NADH formed in the 'blank reaction' between NAD and residual ethanol tightly bound to alcohol dehydrogenase. A molecular mechanism for the enzymatic peroxidation step is finally proposed, exploiting the knowledge of the much better known reaction of ethanol oxidation. Inhibition of this reaction by NADH has been investigated with respect to H2O2 (noncompetitive, Ki about 10 microM) and to NAD (competitive, Ki about 0.7 microM). The effect of temperature on the steady-state reaction state (about 65 kJ/mol activation energy) has also been studied.     

Purification and characterization of 17 beta-hydroxysteroid dehydrogenase from Cylindrocarpon radicicola

An NAD+-linked 17 beta-hydroxysteroid dehydrogenase was purified to homogeneity from a fungus, Cylindrocarpon radicicola ATCC 11011 by ion exchange, gel filtration, and hydrophobic chromatographies. The purified preparation of the dehydrogenase showed an apparent molecular weight of 58,600 by gel filtration and polyacrylamide gel electrophoresis. SDS-gel electrophoresis gave Mr = 26,000 for the identical subunits of the protein. The amino-terminal residue of the enzyme protein was determined to be glycine. The enzyme catalyzed the oxidation of 17 beta-hydroxysteroids to the ketosteroids with the reduction of NAD+, which was a specific hydrogen acceptor, and also catalyzed the reduction of 17-ketosteroids with the consumption of NADH. The optimum pH of the dehydrogenase reaction was 10 and that of the reductase reaction was 7.0. The enzyme had a high specific activity for the oxidation of testosterone (Vmax = 85 mumol/min/mg; Km for the steroid = 9.5 microM; Km for NAD+ = 198 microM at pH 10.0) and for the reduction of androstenedione (Vmax = 1.8 mumol/min/mg; Km for the steroid = 24 microM; Km for NADH = 6.8 microM at pH 7.0). In the purified enzyme preparation, no activity of 3 alpha-hydroxysteroid dehydrogenase, 3 beta-hydroxysteroid dehydrogenase, delta 5-3-ketosteroid-4,5-isomerase, or steroid ring A-delta-dehydrogenase was detected. Among several steroids tested, only 17 beta-hydroxysteroids such as testosterone, estradiol-17 beta, and 11 beta-hydroxytestosterone, were oxidized, indicating that the enzyme has a high specificity for the substrate steroid. The stereospecificity of hydrogen transfer by the enzyme in dehydrogenation was examined with [17 alpha-3H]testosterone.     

Purification and characterization of a novel form of 20 alpha-hydroxysteroid dehydrogenase from Clostridium scindens.

We have purified a steroid-inducible 20 alpha-hydroxysteroid dehydrogenase from Clostridium scindens to apparent homogeneity. The final enzyme preparation was purified 252-fold, with a recovery of 14%. Denaturing and nondenaturing polyacrylamide gradient gel electrophoresis showed that the native enzyme (Mr, 162,000) was a tetramer composed of subunits with a molecular weight of 40,000. The isoelectric point was approximately pH 6.1. The purified enzyme was highly specific for adrenocorticosteroid substrates possessing 17 alpha, 21-dihydroxy groups. The purified enzyme had high specific activity for the reduction of cortisone (Vmax, 280 nmol/min per mg of protein; Km, 22 microM) but was less reactive with cortisol (Vmax, 120 nmol/min per mg of protein; Km, 32 microM) at pH 6.3. The apparent Km for NADH was 8.1 microM with cortisone (50 microM) as the cosubstrate. Substrate inhibition was observed with concentrations of NADH greater than 0.1 mM. The purified enzyme also catalyzed the oxidation of 20 alpha-dihydrocortisol (Vmax, 200 nmol/min per mg of protein; Km, 41 microM) at pH 7.9. The apparent Km for NAD+ was 526 microM. The initial reaction velocities with NADPH were less than 50% of those with NADH. The amino-terminal sequence was determined to be Ala-Val-Lys-Val-Ala-Ile-Asn-Gly-Phe-Gly-Arg. These results indicate that this enzyme is a novel form of 20 alpha-hydroxysteroid dehydrogenase.     

A novel dihydrodiol dehydrogenase in bovine liver cytosol: purification and characterization of multiple forms of dihydrodiol dehydrogenase.

Three enzymes (DD1, DD2, and DD3) having dihydrodiol dehydrogenase activity were purified to homogeneity from bovine cytosol. DD1 and DD2 were identified as 3 alpha-hydroxysteroid dehydrogenase and high-Km aldehyde reductase, respectively, as judged from their molecular weights, substrate specificities and inhibitor sensitivities. DD3 was a unique enzyme which could specifically catalyze the dehydrogenation of trans-benzenedihydrodiol and trans-naphthalenedihydrodiol without any activity toward the other tested alcohols, aldehydes, ketones, and quinones. The Km value of DD3 (0.18 mM) for benzenedihydrodiol was lower than those of other dihydrodiol dehydrogenases so far reported. DD3 immunologically crossreacted with DD1, but showed no crossreactivity with DD2. Additionally, DD3 was inhibited in a competitive manner, with a low Ki value of 1 microM, by androsterone, which was a good substrate for DD1. It was assumed that DD3 is a novel enzyme which is specific to dihydrodiols, exhibiting similarity to DD1 in immunological and structural properties.     

In vitro inhibition by ketoconazole of human testicular steroid oxidoreductases

An oral antimycotic agent, ketoconazole has been demonstrated to be an inhibitor of cytochrome P-450-dependent monooxygenases. To investigate its effect on steroid oxidoreductases, in vitro studies were carried out using subcellular fractions of human testes. Ketoconazole competitively inhibited activities of 3 beta-hydroxy-5-ene-steroid oxidoreductase/isomerase and NADH-linked 20 alpha-hydroxysteroid oxidoreductase for steroid substrate and the Ki values were 2.9 and 0.9 microM, respectively. In contrast, ketoconazole inhibited neither 17 beta-hydroxysteroid oxidoreductase nor NADPH-linked 20 alpha-hydroxysteroid oxidoreductase, indicating that the two 20 alpha-hydroxysteroid oxidoreductases are distinct. Further, ketoconazole inhibited non-competitively the above enzyme activities for the corresponding cofactors of NAD and NADH. From the binding mode of ketoconazole to cytochrome P-450 and the present findings, it appears likely that the agent binds to a site which is different from that of steroids or pyridine nucleotides.     

Steady-state kinetics of formaldehyde dehydrogenase and formate dehydrogenase from a methanol-utilizing yeast, Candida boidinii.

Initial velocity studies and product inhibition studies were conducted for the forward and reverse reactions of formaldehyde dehydrogenase (formaldehyde: NAD oxidoreductase, EC 1.2.1.1) isolated from a methanol-utilizing yeast Candida boidinii. The data were consistent with an ordered Bi-Bi mechanism for this reaction in which NAD+ is bound first to the enzyme and NADH released last. Kinetic studies indicated that the nucleoside phosphates ATP, ADP and AMP are competitive inhibitors with respect to NAD and noncompetitive inhibitors with respect to S-hydroxymethylglutathione. The inhibitions of the enzyme activity by ATP and ADP are greater at pH 6.0 and 6.5 than at neutral or alkaline pH values. The kinetic studies of formate dehydrogenase (formate:NAD oxidoreductase, EC 1.2.1.2) from the methanol grown C. boidinii suggested also an ordered Bi-Bi mechanism with NAD being the first substrate and NADH the last product. Formate dehydrogenase the last enzyme of the dissimilatory pathway of the methanol metabolism is also inhibited by adenosine phosphates. Since the intracellular concentrations of NADH and ATP are in the range of the Ki values for formaldehyde dehydrogenase and formate dehydrogenase the activities of these main enzymes of the dissimilatory pathway of methanol metabolism in this yeast may be regulated by these compounds.     

3alpha-, 7alpha- and 12alpha-hydroxysteroid dehydrogenase activities from Clostridium perfringens.

25 strains of Clostridium perfringens were screened for hydroxysteroid dehydrogenase activity; 19 contained NADP-dependent 3alpha-hydroxysteroid dehydrogenase and eight contained NAD-dependent 12alpha-hydroxysteroid dehydrogenase active against conjugated and unconjugated bile salts. All strains containing 12alpha-hydroxysteroid dehydrogenase also contained 3alpha-hydroxysteroid dehydrogenase although 12alpha-hydroxysteroid dehydrogenase was invariably in lesser quantity than the 3alpha-hydroxysteroid dehydrogenase. In addition, 7alpha-hydroxysteroid dehydrogenase activity was evident only when 3alpha, 7alpha, 12alpha-trihydroxy-5beta-cholanoate was substrate but notably absent when 3alpha, 7alpha-dihydroxy-5beta-cholanoate was substrate. The oxidation product 12alpha-hydroxy-3, 7-diketo-5beta-cholanoate is rapidly further degraded to an unknown compound devoid of either 3alpha- or 7alpha-OH groups. Group specificity of these enzymes was confirmed by thin-layer chromatography studies of the oxidation products. These enzyme systems appear to be constitutive rather than inducible. In contrast to C. perfringens. Clostridium paraputrificum (five strains tested) contained no measurable hydroxysteroid dehydrogenase activity. pH studies of the C. perfringens enzymes revealed a sharp pH optimum at pH 11.3 and 10.5 for the 3alpha-OH- and 12alpha-OH-oriented activities, respectively. Kinetic studies gave Km estimates of approx. 5 X 10(-5) and 8 X 10(-4) M with 3alpha, 7a-dihydroxy-5beta-cholanoate and 3alpha, 12alpha-dihydroxy-5beta-cholanoate as substrates for two respective enzymes. 3alpha-hydroxysteroid dehydrogenase was active against 3alpha-OH-containing steroids such as androsterone regardless of the sterochemistry of the 5H (Both A/B cis and A/B trans steroides were substrates). There was no activity against 3beta-OH-containing steroids. The 3alpha- and 12alpha-hydroxysteroid dehydrogenase activities, although differing in cofactor requirements cannot be distinguished by their appearance in the growth curve, their mobility on disc gel electrophoresis, elution volume on passage through Sephadex G-200 or heat inactivation studies.     

Kinetic characterization of the pyruvate and oxoglutarate dehydrogenase complexes from human heart

The Michaelis constant values for the highly purified pyruvate dehydrogenase complex (PDC) from human heart are 25, 13 and 50 microM for pyruvate, CoA and NAD, respectively. Acetyl-CoA produces a competitive inhibition of PDC (Ki = 35 microM) with respect to CoA, whereas NADH produces the same type of inhibition with respect to NAD (Ki = 36 microM). The oxoglutarate dehydrogenase complex (OGDC) from human heart has active sites with two different affinities for 2-oxoglutarate ([S]0.5 of 30 and 120 microM). ADP (1 mM) decreases the [S]0.5 values by a half. The inhibition of OGDC (Ki = 81 microM) by succinyl-CoA is of a competitive type with respect to CoA (Km = 2.5 microM), whereas that of NADH (Ki = 25 microM) is of a mixed type with respect to NAD (Km = 170 microM).     

Kinetic properties of a sn-glycerol-3-phosphate dehydrogenase purified from the unicellular alga Chlamydomonas reinhardtii

A sn-glycerol-3-phosphate dehydrogenase (sn-glycerol-3-phosphate:NAD+ 2-oxidoreductase, EC 1.1.1.8) has been purified from the unicellular green alga Chlamydomonas reinhardtii 3400-fold to a specific activity of 34 mumol/mg protein per min by a simple procedure involving two chromatographic steps on affinity dyes. The pH optimum for reduction of dihydroxyacetone phosphate was 6.8 and for glycerol 3-phosphate oxidation it was 9.5. In the direction of dihydroxyacetone phosphate reduction, the enzyme showed Michaelis-Menten kinetics. The enzyme reacted specifically with NADH and dihydroxyacetone phosphate as substrates with affinity constants of 16 and 12 microM, respectively. Product inhibition as well as competitive inhibition pattern indicated a random-bi-bi reaction mechanism for sn-glycerol-3-phosphate dehydrogenase from C. reinhardtii. The effective control of dihydroxyacetone reduction catalysed via this enzyme by ATP, Pi and NAD gave evidence for a physiological role of the enzyme in plastidic glycolysis.     

Affinity-labelling of the anti-inflammatory drug and prostaglandin-binding site of 3 alpha-hydroxysteroid dehydrogenase of rat liver cytosol with 17 beta- and 21-bromoacetoxysteroids.

The homogeneous 3 alpha-hydroxysteroid dehydrogenase of rat liver cytosol binds prostaglandins with low micromolar affinity at its active site and is competitively inhibited by the non-steroidal and steroidal anti-inflammatory drugs [Penning, Mukharji, Barrows & Talalay (1984) Biochem. J. 222, 601-611]. To examine the portion of this binding site that accommodates the glucocorticoid side chain, we have synthesized 17 beta-bromoacetoxy-5 alpha-dihydrotestosterone (BrDHT) and 21-bromoacetoxydesoxycorticosterone (BrDOC) as affinity-labelling agents. Both these agents promote rapid inactivation of the purified enzyme in a time- and concentration-dependent manner. Analyses of the inactivation progress curves gave estimates of Ki for the inactivators and half-life (t1/2) for the enzyme at saturation (tau) as follows: Ki = 33 microM and tau = 18 s for BrDHT, and Ki = 10 microM and tau = 203 s for BrDOC. Under initial-velocity conditions BrDHT and BrDOC act as competitive inhibitors, yielding Ki values identical with those measured in the inactivation experiments. Both indomethacin and prostaglandin E2 can protect the enzyme from inactivation, yielding Ki values for these ligands consistent with those measured independently by competitive-inhibition studies. These data confirm that the bromoacetoxysteroids label the active site, which is coincident with the prostaglandin- and anti-inflammatory-drug-binding site. Neither gel filtration nor extensive dialysis restores activity to the enzyme inactivated with either affinity-labelling agent. Use of radioactive BrDHT or BrDOC, in which either the steroid portion is labelled with 3H or the bromoacetate portion is labelled with 14C, indicates that inactivation is accompanied by a stoichiometric incorporation of 0.7-1.0 molecules of inhibitor per enzyme monomer. The linkage that forms between the dehydrogenase with either [14C]BrDHT or [14C]BrDOC is stable to acid and base treatment. Complete acid hydrolysis of the enzyme inactivated with [14C]BrDHT, followed by amino acid analyses, indicates that 87% of the radioactivity is eluted with carboxymethylcysteine. An almost identical result is obtained with [14C]BrDOC, where at least 75% of the radioactivity is eluted with this amino acid. Thus BrDHT and BrDOC alkylate at least one reactive cysteine residue at the active site that may be of functional importance in binding the glucocorticoid side chain.     

Rv1106c from Mycobacterium tuberculosis is a 3beta-hydroxysteroid dehydrogenase

New approaches are required to combat Mycobacterium tuberculosis (Mtb), especially the multi-drug resistant and extremely drug resistant organisms (MDR-TB and XDR-TB). There are many reports that mycobacteria oxidize 3beta-hydroxysterols to 3-ketosteroids, but the enzymes responsible for this activity have not been identified in mycobacterial species. In this work, the Rv1106c gene that is annotated as a 3beta-hydroxysteroid dehydrogenase in Mtb has been cloned and heterologously expressed. The purified enzyme was kinetically characterized and found to have a pH optimum between 8.5 and 9.5. The enzyme, which is a member of the short chain dehydrogenase superfamily, uses NAD+ as a cofactor and oxidizes cholesterol, pregnenolone, and dehydroepiandrosterone to their respective 3-keto-4-ene products. The enzyme forms a ternary complex with NAD+ binding before the sterol. The enzyme shows no substrate preference for dehydroepiandrosterone versus pregnenolone with second-order rate constants (kcat/Km) of 3.2 +/- 0.4 and 3.9 +/- 0.9 microM-1 min-1, respectively, at pH 8.5, 150 mM NaCl, 30 mM MgCl2, and saturating NAD+. Trilostane is a competitive inhibitor of dehydroepiandrosterone with a Ki of 197 +/- 8 microM. The expression of the 3beta-hydroxysteroid dehydrogenase in Mtb is intracellular. Disruption of the 3beta-hydroxysteroid dehydrogenase gene in Mtb abrogates mycobacterial cholesterol oxidation activity. These data are consistent with the Rv1106c gene being the one responsible for 3beta-hydroxysterol oxidation in Mtb.     

Human glutamic-gamma-semialdehyde dehydrogenase. Kinetic mechanism.

The kinetic mechanism of homogeneous human glutamic-gamma-semialdehyde dehydrogenase (EC 1.5.1.12) with glutamic gamma-semialdehyde as substrate was determined by initial-velocity, product-inhibition and dead-end-inhibition studies to be compulsory ordered with rapid interconversion of the ternary complexes (Theorell-Chance). Product-inhibition studies with NADH gave a competitive pattern versus varied NAD+ concentrations and a non-competitive pattern versus varied glutamic gamma-semialdehyde concentrations, whereas those with glutamate gave a competitive pattern versus varied glutamic gamma-semialdehyde concentrations and a non-competitive pattern versus varied NAD+ concentrations. The order of substrate binding and release was determined by dead-end-inhibition studies with ADP-ribose and L-proline as the inhibitors and shown to be: NAD+ binds to the enzyme first, followed by glutamic gamma-semialdehyde, with glutamic acid being released before NADH. The Kia and Kib values were 15 +/- 7 microM and 12.5 microM respectively, and the Ka and Kb values were 374 +/- 40 microM and 316 +/- 36 microM respectively; the maximal velocity V was 70 +/- 5 mumol of NADH/min per mg of enzyme. Both NADH and glutamate were product inhibitors, with Ki values of 63 microM and 15,200 microM respectively. NADH release from the enzyme may be the rate-limiting step for the overall reaction.     

Plant succinic semialdehyde dehydrogenase. Cloning, purification, localization in mitochondria, and regulation by adenine nucleotides.

Succinic semialdehyde dehydrogenase (SSADH) is one of three enzymes constituting the gamma-aminobutyric acid shunt. We have cloned the cDNA for SSADH from Arabidopsis, which we designated SSADH1. SSADH1 cDNA encodes a protein of 528 amino acids (56 kD) with high similarity to SSADH from Escherichia coli and human (>59% identity). A sequence similar to a mitochondrial protease cleavage site is present 33 amino acids from the N terminus, indicating that the mature mitochondrial protein may contain 495 amino acids (53 kD). The native recombinant enzyme and the plant mitochondrial protein have a tetrameric molecular mass of 197 kD. Fractionation of plant mitochondria revealed its localization in the matrix. The purified recombinant enzyme showed maximal activity at pH 9.0 to 9.5, was specific for succinic semialdehyde (K(0.5) = 15 microM), and exclusively used NAD+ as a cofactor (Km = 130 +/- 77 microM). NADH was a competitive inhibitor with respect to NAD+ (Ki = 122 +/- 86 microM). AMP, ADP, and ATP inhibited the activity of SSADH (Ki = 2.5-8 mM). The mechanism of inhibition was competitive for AMP, noncompetitive for ATP, and mixed competitive for ADP with respect to NAD+. Plant SSADH may be responsive to mitochondrial energy charge and reducing potential in controlling metabolism of gamma-aminobutyric acid.     

Non-K-region o-quinones as enzyme-generated inactivators of dihydrodiol dehydrogenase

Homogeneous 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase from rat liver cytosol catalyzes the NAD(P)+-dependent oxidation of non-K-region trans-dihydrodiols of polycyclic aromatic hydrocarbons, many of which are proximate carcinogens. These reactions proceed with Km values in the millimolar range to yield highly reactive o-quinones that can be trapped as thioether adducts [Smithgall, T. E., Harvey, R. G., & Penning, T. M. (1988) J. Biol. Chem. 263, 1814-1820]. The enzymatically generated o-quinones, e.g., naphthalene-1,2-dione and benzo[a]pyrene-7,8-dione are potent inhibitors of the dehydrogenase, yielding IC50 values of 5.0 and 10.0 microM, respectively. Naphthalene-1,2-dione was found to be an efficient irreversible inhibitor of the enzyme and can inactivate equimolar concentrations of the dehydrogenase, yielding a t 1/2 for the enzyme of 10 s or less. By contrast (+/-)-trans-1,2-dihydroxy-1,2-dihydronaphthalene promotes a slower inactivation of the dehydrogenase, yielding a Kd of 70 microM and a limiting rate constant that corresponds to a t 1/2 at saturation of 23.2 min. Inactivation by this dihydrodiol has an obligatory requirement for NADP+. Examination of the kcat for the oxidation of (+/-)-trans-1,2-dihydroxy-1,2-dihydronaphthalene yields a partition ratio for the dihydrodiol of 200,000, suggesting that alkylation from the parent dihydrodiol is a rare occurrence. Benzo[a]pyrene-7,8-dione, which is the product of the enzymatic oxidation of (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene, also promotes a time- and concentration-dependent inactivation of the dehydrogenase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inactivation of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase by koningic acid

Koningic acid, a sesquiterpene antibiotic, is a specific inhibitor of the enzyme glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12). In the presence of 3 mM of NAD+, koningic acid irreversibly inactivated the enzyme in a time-dependent manner. The pseudo-first-order rate constant for inactivation (kapp) was dependent on koningic acid concentration in saturate manner, indicating koningic acid and enzyme formed a reversible complex prior to the formation of an inactive, irreversible complex; the inactivation rate (k 3) was 5.5.10(-2) s-1, with a dissociation constant for inactivation (Kinact) of 1.6 microM. The inhibition was competitive against glyceraldehyde 3-phosphate with a Ki of 1.1 microM, where the Km for glyceraldehyde 3-phosphate was 90 microM. Koningic acid inhibition was uncompetitive with respect to NAD+. The presence of NAD+ accelerated the inactivation. In its absence, the charcoal-treated NAD+-free enzyme showed a 220-fold decrease in apparent rate constant for inactivation, indicating that koningic acid sequentially binds to the enzyme next to NAD+. The enzyme, a tetramer, was inactivated when maximum two sulfhydryl groups, possibly cysteine residues at the active sites of the enzyme, were modified by the binding of koningic acid. These observations demonstrate that koningic acid is an active-site-directed inhibitor which reacts predominantly with the NAD+-enzyme complex.