The iron-sulfur-cluster-containing L-serine dehydratase from Peptostreptococcus asaccharolyticus. Stereochemistry of the deamination of L-threonine.

The stereochemistry of the deamination of L-threonine to 2-oxobutyrate, catalyzed by purified L-serine dehydratase of Peptostreptococcus asaccharolyticus, was elucidated. For this purpose the enzyme reaction was carried out with unlabelled L-threonine in 2H2O and in 3HOH, as well as with L-[3-3H]threonine in unlabelled water. Isotopically labelled 2-oxobutyrate thus formed was directly reduced in a coupled reaction with L- or D-lactate dehydrogenase and NADH. The (2R)- or (2S)-2-hydroxybutyrate species obtained were then subjected to configurational analyses of their labelled methylene group. The results from 1H-NMR spectroscopy and, after degradation of 2-hydroxybutyrate to propionate, the transcarboxylase assay consistently indicated that the deamination of L-threonine catalyzed by L-serine dehydratase of P. asaccharolyticus proceeds with inversion and retention in a 2:1 ratio. This partial racemization is the first ever to be observed for a reaction catalyzed by serine dehydratase, therefore confirming the distinction of the L-serine dehydratase of P. asaccharolyticus as an iron-sulfur protein from those dehydratases dependent on pyridoxal phosphate. For the latter enzymes exclusively, retention has been reported.     

Effects of deoxycholic acid and its epimers on lipid peroxidation in isolated rat hepatocytes

We studied the effects of deoxycholic acid and its three epimers with beta-hydroxyl groups (3alpha,12beta-, 3beta,12alpha-, and 3beta,12beta-dihydroxy-5beta-cholan-24-oic acids), which were hydrophilic and less cytotoxic, on lipid peroxidation to elucidate the relationship between structural features of bile acids and their effect on lipid peroxidation. Taurodeoxycholate markedly increased the production of thiobarbituric acid-reactive substances, end products of lipid peroxidation, in isolated rat hepatocytes, whereas epimers of taurodeoxycholate did not. Deoxycholic acid inhibited mitochondrial NADH dehydrogenase and NADH:ferricytochrome c oxidoreductase activities, leading to free radical generation, whereas epimers of deoxycholic acid had no effect on mitochondrial enzymes. These findings suggested that hydrophobic bile acids cause lipid peroxidation by impairment of mitochondrial function, leading to the generation of free radicals; and epimerization of alpha-hydroxyl groups in the steroid nucleus to beta-hydroxyl groups results in a decrease of the toxic effects of deoxycholic acid on lipid peroxidation.     

The stereospecificity of the enzymic reduction of 21-dehydrocortisol by NADH.

(21R)-[21-3H]cortisol and (21S)-[21-3H]cortisol were synthesized by reduction of 21-dehydrocortisol by NADH in the presence of 21-hydroxysteroid dehydrogenase. The stereochemistry at carbon 21 was established after cleaving the side chain and oxidizing the resulting two epimers of tritiated glycolate with glycolate oxidase of known (2-pro-S) stereospecificity. From the distribution of radioactivity in the water and glyoxylate produced in this reaction, it was concluded that the reaction of 21-dehydrocortisol with (4S)-[4-3H]NADH catalyzed by 21-hydroxysteroid dehydrogenase results in a transfer of tritium from the 4S position of the nucleotide to form (21S)-[21-3H]cortisol, and that (21R)-[21-3H]cortisol resulted from the enzyme-catalyzed reduction of 21-dehydro[21-3H]cortisol with NADH. Nuclear magnetic resonance studies on both epimers at position 21 of [21-2H]cortisol and of [21-2H]cortisone prepared enzymically identify the transferring 21-pro-S hydrogen as the relatively downfield of the two 21-hydrogen atoms.     

Characterization of enzymes of the branched-chain amino acid biosynthetic pathway in Methanococcus spp.

Methanococcus aeolicus, Methanococcus maripaludis, and Methanococcus voltae contain similar levels of four enzymes of branched-chain amino acid biosynthesis: acetohydroxy acid synthase, acetohydroxy acid isomeroreductase, dihydroxy acid dehydratase, and transaminase B. Following growth at low partial pressures of H2-CO2, the levels of these enzymes in extracts of M. voltae are reduced three- to fivefold, which suggests that their synthesis is regulated. The enzymes from M. aeolicus were found to be similar to the eubacterial and eucaryotic enzymes with respect to molecular weights, pH optima, kinetic properties, and sensitivities to O2. The acetohydroxy acid isomeroreductase has a specific requirement for Mg2+, and other divalent cations were inhibitory. It was stimulated threefold by K+ and NH4+ ions and was able to utilize NADH as well as NADPH. The partially purified enzyme was not sensitive to O2. The dihydroxy acid dehydratase is extremely sensitive to O2, and it has a half-life under 5% O2 of 6 min at 25 degrees C. Divalent cations were required for activity, and Mg2+, Mn2+, Ni2+, Co2+, and Fe2+ were nearly equally effective. In conclusion, the archaebacterial enzymes are functionally homologous to the eubacterial and eucaryotic enzymes, which implies that this pathway is very ancient.     

Purification of 2-hydroxyglutaryl-CoA dehydratase from Acidaminococcus fermentans. An iron-sulfur protein

1. The (R)-2-hydroxyglutaryl-CoA dehydratase system from Acidaminococcus fermentans was separated by chromatography of cell-free extracts on Q-Sepharose into two components, an activator and the actual dehydratase. The latter enzyme was further purified to homogeneity by chromatography on blue-Sepharose. It is an iron-sulfur protein (Mr 210,000) consisting of two different polypeptides (alpha, Mr 55,000, and beta, Mr 42,000) in an alpha 2 beta 2 structure with probably two [4Fe-4S] centers. After activation this purified enzyme catalysed the dehydration of (R)-2-hydroxyglutarate only in the presence of acetyl-CoA and glutaconate CoA-transferase, demonstrating that the thiol ester and not the free acid is the substrate of the dehydration. The result led to a modification of the hydroxyglutarate pathway of glutamate fermentation. 2. The activation of the dehydratase by the flow-through from Q-Sepharose concentrated by ultrafiltration required NADH, MgCl2, ATP and strict anaerobic conditions. This fraction was designated as Ao. Later when the concentration was performed by chromatography on phenyl-Sepharose, an NADH-independent form of the activator, designated as A*, was obtained. This enzyme, which required only ATP for activation of the dehydratase, was purified further by affinity chromatography on ATP-agarose. It contains neither iron nor inorganic sulfur. A*, as well as the activated dehydratase, were irreversibly inactivated by exposure to air within less than 15 min. The activated dehydratase but not A* was also inactivated by 1 mM hydroxylamine or by 0.1 mM 2,4-dinitrophenol. 3. The (R)-2-hydroxyglutaryl-CoA dehydratase system is closely related the that of (R)-lactoyl-CoA dehydratase from Clostridium propionicum as described by R. D. Kuchta and R. H. Abeles [(1985) J. Biol. Chem. 260, 13,181-13,189].     

Sugar-glycerol cofermentations in lactobacilli: the fate of lactate.

The simultaneous fermentation of glycerol and sugar by lactobacillus brevis B22 and Lactobacillus buchneri B190 increases both the growth rate and total growth. The reduction of glycerol to 1,3-propanediol by the lactobacilli was found to influence the metabolism of the sugar cofermented by channelling some of the intermediate metabolites (e.g., pyruvate) towards NADH-producing (rather than NADH-consuming) reactions. Ultimately, the absolute requirement for NADH to prevent the accumulation of 3-hydroxypropionaldehyde leads to a novel lactate-glycerol cofermentation. As a result, additional ATP can be made not only by (i) converting pyruvate to acetate via acetyl phosphate rather than to the ethanol usually found and (ii) oxidizing part of the intermediate pyruvate to acetate instead of the usual reduction to lactate but also by (iii) reoxidation of accumulated lactate to acetate via pyruvate. The conversion of lactate to pyruvate is probably catalyzed by NAD-independent lactate dehydrogenases that are found only in the cultures oxidizing lactate and producing 1,3-propanediol, suggesting a correlation between the expression of these enzymes and a raised intracellular NAD/NADH ratio. The enzymes metabolizing glycerol (glycerol dehydratase and 1,3-propanediol dehydrogenase) were expressed in concert without necessary induction by added glycerol, although their expression may also be influenced by the intracellular NAD/NADH ratio set by the different carbohydrates fermented.     

Nutrition of Threonine

By administering 2 mg/day of cortisone acetate to adrenalectomized rats, the hepatic threonine dehydratase activity of these rats increased 5 times as much as that of the control. By administering 5 IU/day of ACTH to hypophysectomized rats, both the hepatic threonine dehydratase activity and the adrenal glucose-6-phosphate dehydrogenase activity increased 3 times and 7 times as much as that of the control group, respectively. The effects of excess feeding of lysine or threonine on the increase of the dehydratase activity by the adminitration of cortisone to the adrenalectomized rats and the administration of ACTH to the hypophysetomized rats were negative. When the intact rats were fed on lysine and/or threonine excess diet, the amount of glucocorticoid secretion as measured by the adrenal glucose-6-phosphate dehydrogenase activity increased and the hepatic threonine dehydratase activity increased accordingly. A linear relationship was found between these two activities and no significant deviation from the relationship due to the difference in diet composition was observed. A mechanism was proposed, based on these results, explaining the fact that the hepatic threonine dehydratase activity increased when rats were fed on lysine or threonine excess diet.     

Photoinduced enzymatic epimerization of glucose

The possibility was examined whether weak external electromagnetic radiation can affect the parameters of enzymic reactions. It was found that, in a system involving an aqueous solution of concanavalin A, glucose, and erbium salt exposed to helium-neon laser light of a wavelength of 633 nm, epimers of glucose: allosa, mannose, and galactose are formed. The effect observed was found to represent a photoinduced enzymic reaction in which the protein dissolved in water converts the glucose associated with it into epimers by the action of electromagnetic radiation. The photoepimerase activity of concanavalin A was established for the first time.     

Bull semen nicotinamide adenine dinucleotide nucleosidase. VI. Fluorescence studies of the enzyme with reduced nicotinamide adenine dinucleotide

The binding of NADH to bull semen NAD nucleosidase was observed to be accompanied by a considerable enhancement of the fluorescence of NADH. The fluorescence enhancement observed in the binding of NADH to the enzyme was utilized to study the stoichiometry of binding of this compound to the enzyme. Results obtained from the fluorescence titration of the enzyme with NADH indicated the binding of one mole of NADH per mole of enzyme (36,000 g). The dissociation constant for the enzyme-NADH complex was determined to be 2.52 × 10^?6m. NADH was also found to be a very effective competitive inhibitor of the NADase-catalyzed hydrolysis of NAD, and the inhibitor dissociation constant (K_I) for the enzyme-NADH complex was determined to be 2.99 × 10^?6m which was in good agreement with the value obtained from the fluorescence titration experiments.     

Stereoselective binding of carbenicillin epimers to human serum albumin

Binding of carbenicillin (CBPC) epimers to human serum albumin (HSA) was found to be stereoselective. Epimer-epimer interaction was also observed in the binding to HSA. There were at least three binding sites on HSA for CBPC epimers, one of which (stereoselective site) was more in favor of S-CBPC than R-CBPC. At the stereoselective site, the binding constant of S-CBPC was approximately 4-fold greater than that of R-CBPC. The affinities to other binding sites (non-stereoselective sites) were similar between the epimers, and the affinity of S-CBPC of the non-stereoselective sites was much smaller than that for the stereoselective site. R-CBPC and S-CBPC appeared to displace each other at all the binding sites, i.e., the binding of the epimers was competitive at the non-stereoselective sites as well as at the stereoselective site. By using site marker ligands, it was revealed that CBPC epimers may bind to Site I (warfarin binding site), but not to Site II (diazepam binding site). A binding model with an assumption of competitive interactions at all the binding sites simulated the binding characteristics of CBPC epimers fairly well. © 1996 Wiley-Liss, Inc.     

Kinetics of p-nitrophenyl pivalate hydrolysis catalysed by cytoplasmic aldehyde dehydrogenase.

The effects of modifiers (NAD+, NADH, propionaldehyde, chloral hydrate, diethylstilboestrol and p-nitrobenzaldehyde) on the hydrolysis of p-nitrophenyl (PNP) pivalate (PNP trimethylacetate) catalysed by cytoplasmic aldehyde dehydrogenase are reported. In each case a different inhibition pattern is obtained to that observed when the substrate is PNP acetate; for example, propionaldehyde and chloral hydrate competitively inhibit the hydrolysis of PNP acetate, but are mixed inhibitors with PNP pivalate. The kinetic results can be rationalized in terms of different rate-determining steps: acylation of the enzyme in the case of the pivalate but acyl-enzyme hydrolysis for the acetate. This is confirmed by stopped-flow studies, in which a burst of p-nitrophenoxide is observed when the substrate is PNP acetate, but not when it is the pivalate. PNP pivalate inhibits the dehydrogenase activity of the enzyme competitively with the aldehyde substrate; this is most simply explained if the esterase and dehydrogenase reactions occur at a common enzymic site.     

L-serine dehydratase and L-serine-pyruvate aminotransferase activities in different animal species.

1. A rough inverse correlation between liver serine dehydratase activity and species body size was observed with mammals. This was not found for glutamate, malate or lactate dehydrogenase, fumarase or aspartate-2-oxoglutarate aminotransferase. Serine dehydratase (and certain liver aminotransferases showing a similar species-size dependence) may have a role in the stimulation of heat production. 2. Cold-exposed rats showed a 3-4 fold increase in serine dehydratase activity. 3. No liver serine dehydratase was measurable in amphibia. 4. In mammals high liver serine-pyruvate aminotransferase is associated with a flesh-eating dietary habit. High activity was found in amphibia and goldfish. A gluconeogenic role is suggested.     

Physiologic Mechanisms Involved in Accumulation of 3-Hydroxypropionaldehyde during Fermentation of Glycerol by Enterobacter agglomerans

When grown in 700 mM glycerol within the pH range 6.0 to 7.5, anaerobic pH-regulated cultures of Enterobacter agglomerans exhibited an extracellular accumulation of 3-hydroxypropionaldehyde (3-HPA). This phenomenon, which causes fermentation cessation, occurred earlier when pH was low. In contrast, substrate consumption was complete at pH 8. Levels of glycerol-catabolizing enzymes, i.e., glycerol dehydrogenase and dihydroxyacetone kinase for the oxidative route and glycerol dehydratase and 1,3-propanediol dehydrogenase for the reductive route, as well as the nucleotide pools were determined periodically in the pH 7- and pH 8-regulated cultures. A NAD/NADH ratio of 1.7 was correlated with the beginning of the production of the inhibitory metabolite. Further accumulation was dependent on the ratio of glycerol dehydratase activity to 1,3-propanediol dehydrogenase activity. For a ratio higher than 1, 3-HPA was produced until fermentation ceased, which occurred for the pH 7-regulated culture. At pH 8, a value below 1 was noticed and 3-HPA accumulation was transient, while the NAD/NADH ratio decreased. The low rate of glycerol dissimilation following the appearance of 3-HPA in the culture medium was attributed to the strong inhibitory effect exerted by 3-HPA on glycerol dehydrogenase activity.     

Identification of two GDP-6-deoxy-D-lyxo-4-hexulose reductases synthesizing GDP-D-rhamnose in Aneurinibacillus thermoaerophilus L420-91T

The glycan repeats of the surface layer glycoprotein of Aneurinibacillus thermoaerophilus L420-91T contain d-rhamnose and 3-acetamido-3,6-dideoxy-d-galactose, both of which are also constituents of lipopolysaccharides of Gram-negative plant and human pathogenic bacteria. The two genes required for biosynthesis of the nucleotide-activated precursor GDP-d-rhamnose, gmd and rmd, were cloned, sequenced, and overexpressed in Escherichia coli. The corresponding enzymes Gmd and Rmd were purified to homogeneity, and functional studies were performed. GDP-d-mannose dehydratase (Gmd) converted GDP-d-mannose to GDP-6-deoxy-d-lyxo-4-hexulose, with NADP+ as cofactor. The reductase Rmd catalyzed the second step in the pathway, namely the reduction of the keto-intermediate to the final product GDP-d-rhamnose using both NADH and NADPH as hydride donor. The elution behavior of the intermediate and end product was analyzed by high performance liquid chromatography. Nuclear magnetic resonance spectroscopy was used to identify the structure of the final product of the reaction sequence as GDP-alpha-d-rhamnose. This is the first characterization of a GDP-6-deoxy-d-lyxo-4-hexulose reductase. In addition, Gmd has been shown to be a bifunctional enzyme with both dehydratase and reductase activities. So far, no enzyme catalyzing these two types of reactions has been identified. Both Gmd and Rmd are members of the SDR (short chain dehydrogenase/reductase) protein family.     

Nicotinamide-adenine dinucleotide inhibition of pig kidney alkaline phosphatase.

1. The interaction of NAD+, NADH and various nucleotide analogues with pig kidney alkaline phosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum) EC 3.1.3.1) has been investigated by kinetic means. Some inhibitors act uncompetitively whereas others markedly increase the slopes of double reciprocal plots suggesting they have some affinity for the free enzyme. 2. The compounds seem to bind to alkaline phosphatase through interactions of their bases with a relatively non-specific region of the enzyme, although it is likely that for those nucleotides having some affinity for the free enzyme there is some attraction between the pyrophosphate backbone and the active site. 3. From studies of the effect of NAD+ and NADH on ATPase activity it was concluded that the substrate inhibition that is characteristic of the ATPase activity of alkaline phosphatase originates from binding of ATP to the site assumed to exist for NAD+ and NADH. The potentiation of NAD+-inhibition of ATPase activity by Mg-2+ is probably a result of the depletion of [ATP-4-] the true substrate. The depletion allows NAD+ to complete more effectively for the active site. 4. Binding of NADH is favoured by protonation of an enzymic group with a pK of approx. 9.0 belonging possibly to a tyrosine residue or a zinc hydrate. 5. A large entropy decrease was found to accompany the binding of NAD+ and NADH to alkaline phosphatase. This may be further evidence of an "induced-fit" mechanism previously suspected because of the synergistic inhibitory effects of adenosine and nicotinamide.     

Synthesis of D-ring-substituted (5'R)- and (5'S)-17β-pyrazolinylandrostene epimers and comparison of their potential anticancer activities

Various steroidal benzylidenes were synthetized from pregnenolone with benzaldehyde and p-substituted benzaldehydes. The resulting 17β-chalconyl derivatives of pregnenolone were reacted with hydrazine hydrate in acetic acid solution. Regardless of the starting material, the ring-closure reaction afforded (in contrast with the literature data) a mixture of two steroidal pyrazoline epimers. The epimers were critical isomer pairs, which could be separated only in their acetylated form; their structures were investigated by NMR techniques. The in vitro inhibition of rat testicular C(17,20)-lyase activity and the antiproliferative effects on four human cancer cell lines were measured, and the results obtained from the two epimer series were compared.     

The influence of actinomycin D on the activity of delta-aminolaevulinic acid synthetase and dehydratase in the liver of mice and rats treated by griseofulvin.

The changes of delta-aminolaevulinic acid dehydratase--the second enzyme of porphyrin synthesis--were studied and compared in the liver of mice and rats treated with griseofulvin. The results showed that griseofulvin increased the activity of delta-aminolaevulinic acid dehydratase in the liver of mice and rats treated by griseofulvin. No differences were found between mice and rats as to the effect of griseofulvin on the activity of delta-aminolaevulinic acid dehydratase. The influence of the administration of actinomycin D on the activity of delta-aminolaevulinic acid synthetase and dehydratase in the liver of mice and rats was studied at an early period of experimental porphyria induced by griseofulvin. It was found that the administration of actinomycin D blocked the observed effect of griseofulvin on the activity of both enzymes in the liver of mice and rats.     

Kinetic studies on the esterase activity of cytoplasmic sheep liver aldehyde dehydrogenase.

The hydrolysis of 4-nitrophenyl acetate catalysed by cytoplasmic aldehyde dehydrogenase (EC 1.2.1.3) from sheep liver was studied by steady-state and transient kinetic techniques. NAD+ and NADH stimulated the steady-state rate of ester hydrolysis at concentrations expected on the basis of their Michaelis constants from the dehydrogenase reaction. At higher concentrations of the coenzymes, both NAD+ and NADH inhibited the reaction competitively with respect to 4-nitrophenyl acetate, with inhibition constants of 104 and 197 micron respectively. Propionaldehyde and chloral hydrate are competitive inhibitors of the esterase reaction. A burst in the production of 4-nitrophenoxide ion was observed, with a rate constant of 12 +/- 2s-1 and a burst amplitude that was 30% of that expected on the basis of the known NADH-binding site concentration. The rate-limiting step for the esterase reaction occurs after the formation of 4-nitrophenoxide ion. Arguments are presented for the existence of distinct ester- and aldehyde-binding sites.     

Human liver aldehyde dehydrogenase. Esterase activity.

Human liver aldehyde dehydrogenase has been found to be capable of hydrolyzing p-nitrophenyl esters. Esterase and dehydrogenase activities exhibited identical ion exchange and affinity properties, indicating that the same protein catalyzes both reactions. Competitive inhibition of esterase activity by glyceraldehyde and chloral hydrate furnished evidence that p-nitrophenyl acetate was hydrolyzed at the aldehyde binding site for dehydrogenase activity. Pyridine nucleotides modified esterase activity; NAD+ accelerated the rate of p-nitrophenyl acetate hydrolysis more that 5-fold, whereas NADH increased activity by a factor of 2. Activation constants of 117 muM for NAD+ and 3.5 muM for NADH were obtained from double reciprocal plots of initial rates as a function of modifier concentration at pH 7. The kinetics of activation of ester hydrolysis were consistent with random addition of pyridine nucleotide modifier and ester substrate to this enzyme.