Mechanism-based active site modification of the soybean sterol methyltransferase by 26,27-dehydrocycloartenol

26,27-dehydrocycloartenol (26,27-DHC) was shown to be a substrate for the soybean sterol methyltransferase (SMT) as well as a mechanism-based inhibitor of enzyme action. The K(m) and k(cat) for 26,27-DHC was 10 microM and 0.018 min(-1), respectively. SMT catalyzed 26,27-DHC to two products tentatively identified as 26-homocholesta-9,19-cyclo-23(24)E,26(26')-dienol and 26-homocholesta-9,19-cyclo-26(26')-en-3beta,24beta-diol by GC-MS. Inhibitor treatment was concentration- and time-dependent (pseudo-first-order kinetics). A replot of the half-lives for inactivation versus the inverse of the inactivator concentrations gave an apparent K(i) of 42 microM and a maximum rate of inactivation of 0.29 min(-1). A partition ratio (k(cat)/k(inact)) was calculated to be 0.06.     

Cytotoxicity of RH1 and related aziridinylbenzoquinones: involvement of activation by NAD(P)H:quinone oxidoreductase (NQO1) and oxidative stress

It is supposed that the main cytotoxicity mechanism of antitumour aziridinyl-substituted benzoquinones is their two-electron reduction to alkylating products by NAD(P)H:quinone oxidoreductase (NQO1, DT-diaphorase, EC 1.6.99.2). However, other possible cytotoxicity mechanisms, e.g., oxidative stress, are studied insufficiently. In the single-electron reduction of quinones including a novel compound RH1 (2,5-diaziridinyl- 3-(hydroxymethyl)-6-methyl-1,4-benzoquinone), by NADPH:cytochrome P-450 reductase (EC 1.6.2.4, P-450R), their reactivity increased with an increase in the redox potential of quinone/semiquinone couple (E(1)7), reaching a limiting value at E(1)7> or =-0.1V. The reactivity of quinones towards NQO1 did not depend on their E(1)7. The cytotoxicity of aziridinyl-unsubstituted quinones in bovine leukemia virus-transformed lamb kidney fibroblasts (line FLK) mimics their reactivity in P-450R-catalyzed reactions, exhibiting a parabolic dependence on their E(1)7. The toxicity of aziridinyl-benzoquinones, although being higher, also followed this trend and did not depend on their reactivity towards NQO1. The action of aziridinylbenzoquinones in FLK cells was accompanied by an increase in lipid peroxidation, their toxicity decreased by desferrioxamine and the antioxidant N,N'-diphenyl-p-phenylene diamine, and potentiated by 1,3-bis-(2-chloroethyl)-1-nitrosourea. The inhibitor of NQO1, dicumarol, protected against the toxicity of aziridinyl-benzoquinones except of 2,5-bis-(2'-hydroxyethylamino)-3,6-diaziridinyl-1,4-benzoquinone (BZQ), which was almost inactive as NQO1 substrate. The same events except the absence of pronounced effect of dicumarol were characteristic in the cytotoxicity of aziridinyl-unsubstituted quinones. These findings indicate that in addition to the activation by NQO1, the oxidative stress presumably initiated by single-electron transferring enzymes may be an important factor in the cytotoxicity of aziridinylbenzoquinones. The information obtained may contribute to the understanding of the molecular mechanisms of aziridinylquinone cytotoxicity and may be useful in the design of future bioreductive drugs.     

Prooxidant cytotoxicity of chromate in mammalian cells: the opposite roles of DT-diaphorase and glutathione reductase

The geno- and cytotoxicity of chromate, an important environmental pollutant, is partly attributed to the flavoenzyme-catalyzed reduction with the concomitant formation of reactive oxygen species. The aim of this work was to characterize the role of NAD(P)H:quinone oxidoreductase (NQO1, DT-diaphorase, EC 1.6.99.2) and glutathione reductase (GR, EC 1.6.4.2) in the mammalian cell cytotoxicity of chromate, which was evidenced controversially so far. The chromate reductase activity of NQO1 was higher than that of GR, but lower than that of lipoamide dehydrogenase (EC 1.6.4.3), ferredoxin:NADP+ reductase (EC 1.18.1.2), and NADPH: cytochrome P-450 reductase (EC 1.6.2.4). The reduction of chromate by NQO1 was accompanied by the formation of reactive oxygen species. The concentration of chromate for 50% survival of bovine leukemia virus-transformed lamb kidney fibroblasts (line FLK) during a 24-h incubation was (22 +/- 4) microM. The cytotoxicity was partly prevented by desferrioxamine, the antioxidant N,N'-diphenyl-p-phenylene diamine and by an inhibitor of NQO1, dicumarol, and potentiated by 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU), which inactivates GR. The NADPH-dependent chromate reduction by digitonin-permeabilized FLK cells was partly inhibited by dicumarol and not affected by BCNU. Taken together, these data indicate that the oxidative stress-type cytotoxicity of chromate in FLK cells may be partly attributed to its reduction by NQO1, but not by GR. The effect of BCNU on the chromate cytotoxicity may indicate that the general antioxidant action of reduced glutathione is more important than its prooxidant activities arising from the reactions with chromate.     

The relation of glutathione reductase and diaphorase activity of glutathione reductase from Saccharomyces cerevisiae

Glutathione reductase from S. cerevisiae (EC 1.6.4.2) catalyzes the NADPH oxidation by glutathione in accordance with a "ping-pong" scheme. The catalytic constant kcat) is 240 s-1 (pH 7.0, 25 degrees C); kcat for the diaphorase reaction is 4-5 s-1. The enzyme activity does not change markedly at pH 5.5-8.0. At pH less than or equal to 7.0, NADP+ acts as a competitive inhibitor towards NADPH and as a noncompetitive inhibitor towards glutathione. NADP+ increases the diaphorase activity of the enzyme. The maximal activity is observed, when the NADP+/NADPH ratio exceeds 100. At pH 8.0, NADP+ acts as a mixed type inhibitor during the reduction of glutathione. High concentrations of NADP+ also inhibit the diaphorase activity due to the reoxidation of the reduced enzyme by NADP+ at pH 8.0. The redox potential of glutathione reductase calculated from the inhibition data is--306 mV (pH 8.0). Glutathione reductase reduces quinoidal compounds in an one-electron way. The hyperbolic dependence of the logarithm of the oxidation constant on the one electron reduction potential of quinone is observed. It is assumed that quinones oxidize the equilibtium fraction of the two-electron reduced enzyme containing reduced FAD.     

Further characterization of cysteine proteinase inhibitors purified from rat and human epidermis

Cysteine proteinase inhibitors isolated from rat and human epidermis were purified to homogeneity and had isoelectric points of pH 4.31 and pH 5.10, respectively, Both inhibitors caused noncompetitive inhibition to the same degree against papain (EC 3.4.22.2), but the activity of human inhibitor against rat liver cathepsins B (EC 3.4.22.1), H (EC 3.4.22.16), and L (EC 3.422.-) was more effective than that of rat inhibitor. Dependency on pH was observed with rat inhibitor for cathepsins B and H, and with human inhibitor for cathepsin L. The reaction of the inhibitors with papain and cathepsins H and L occurred immediately, while the inhibition reaction of cathepsin B increased progressively during a preincubation time up to 40 min. Incubation at pH 7.0 maximized the progressive inhibitory activity. These findings demonstrate that cysteine proteinase inhibitors from rat and human epidermis inhibited a variety of cysteine proteinases. However, the inhibitor and enzyme interaction depends upon the enzyme, inhibitor source, and experimental conditions such as pH and preincubation time.     

An oxysterol biomarker for 7-dehydrocholesterol oxidation in cell/mouse models for Smith-Lemli-Opitz syndrome

The level of 7-dehydrocholesterol (7-DHC) is elevated in tissues and fluids of Smith-Lemli-Opitz syndrome (SLOS) patients due to defective 7-DHC reductase. Although over a dozen oxysterols have been identified from 7-DHC free radical oxidation in solution, oxysterol profiles in SLOS cells and tissues have never been studied. We report here the identification and complete characterization of a novel oxysterol, 3β,5α-dihydroxycholest-7-en-6-one (DHCEO), as a biomarker for 7-DHC oxidation in fibroblasts from SLOS patients and brain tissue from a SLOS mouse model. Deuterated (d₇)-standards of 7-DHC and DHCEO were synthesized from d₇-cholesterol. The presence of DHCEO in SLOS samples was supported by chemical derivatization in the presence of d₇-DHCEO standard followed by HPLC-MS or GC-MS analysis. Quantification of cholesterol, 7-DHC, and DHCEO was carried out by isotope dilution MS with the d₇-standards. The level of DHCEO was high and correlated well with the level of 7-DHC in all samples examined (R = 0.9851). Based on our in vitro studies in two different cell lines, the mechanism of formation of DHCEO that involves 5α,6α-epoxycholest-7-en-3β-ol, a primary free radical oxidation product of 7-DHC, and 7-cholesten-3β,5α,6β-triol is proposed. In a preliminary test, a pyrimidinol antioxidant was found to effectively suppress the formation of DHCEO in SLOS fibroblasts.     

Cytotoxicity of nitroaromatic explosives and their biodegradation products in mice splenocytes: implications for their immunotoxicity

Nitroaromatic explosives like 2,4,6-trinitrotoluene (TNT) and 2,4,6-trinitrophenyl-N-methyl-nitramine (tetryl) comprise an important group of toxic environmental pollutants, whose toxicity is mainly attributed to the flavoenzyme electrontransferase-catalyzed redox cycling of their free radicals (oxidative stress) and DT-diaphorase [NAD(P)H:quinone oxidoreductase, NQO1, EC 1. 6.99.2]-catalyzed formation of alkylating nitroso and/or hydroxylamine metabolites. Because of the incomprehensive data on the immunotoxic effects of nitroaromatic explosives, we have studied the structure-cytotoxicity relationships in the action of tetryl, TNT as well as its amino and hydroxylamino metabolites, and related nitroaromatic compounds towards mouse splenocyte cells. The protective effects of desferrioxamine and the antioxidant N,N'-diphenyl-p-phenylene diamine against the cytotoxicity of TNT and other nitroaromatics showed that the oxidative stress-type cytotoxicity mechanism takes place. In addition, the cytotoxicity of nitroaromatics is also partly prevented by an inhibitor of NQO1, dicumarol. The cytotoxicity of the amino metabolites of TNT is also partly prevented by alpha-naphthoflavone and isoniazide, which points to the involvement of cytochromes P-450 in their activation. In general the cytotoxicity of nitroaromatics in splenocytes increases with an increase in their single-electron reduction potential, E1(7). This points to the prevailing mechanism of the oxidative stress-type cytotoxicity. The obtained structure-activity relationship and the studies of other mammalian cell lines showed that the immunotoxic potential of nitroaromatic explosives may decrease in the order tetryl > or = TNT > or = hydroxylamino metabolites of TNT > amino and diamino metabolites of TNT.     

A conformational change in phosphoglycerate dehydrogenase induced by a shift in pH.

The fluorescence of NADH bound to phosphoglycerate dehydrogenase (3-phosphoglycerate: NAD+ oxidoreductase, EC 1.1.1.95) decreased by 42% between pH 8.5 and 7.0 Serine, an allosteric inhibitor, quenched the fluorescence of enzyme-bound NADH by 29% at pH 8.5, but not at all at pH 7.0. The kinetics of the fluorescence change which occurred when the pH of an enzyme-NADH solution was rapidly shifted from 8.5 to 7.0 was measured using stopped-flow fluorimetry. The kinetics were first order, with a rate constant of 2.83 s-1. This rate constant was similar in magnitude to the rate constants for fluorescence quenching at pH 8.5 by saturating concentrations of serine and glycine, another allosteric inhibitor (Dubrow, R. and Pizer, L.I. (1977) J. Biol. Chem. 252, 1527-1538). These results indicate that the conformation of phosphoglycerate dehydrogenase at pH 7.0 is similar to, but not identical with, the serine-induced conformation at pH 8.5.     

L-[2,5-H2]phenylalanine, an alternate substrate for rat liver phenylalanine hydroxylase

The phenylalanine analogue L-[2,5-H2]phenylalanine (1) was found to be a viable substrate (KM = 0.45 mM, kcat = 8 s-1) for L-phenylalanine-activated, rat liver phenylalanine hydroxylase (EC 1.14.16.1) (PAH). The PAH-catalyzed oxidation of 1 was stoichiometric with the oxidation of cofactor, 6-methyl-tetrahydropterin. Spectral and chromatographic data of the product from the oxidation of 1 by PAH were found to be in accord with a 3,4-epoxide. The enzymatic epoxidation of 1 is consistent with the hypothesis of an intermediate arene oxide on the reaction coordinate for PAH hydroxylation of L-phenylalanine.     

Specificity and stability of the chymotrypsin inhibitor from winged bean seed (Psophocarpus tetragonolobus (L) Dc)

The specificity of the winged bean chymotrypsin inhibitor is restricted to the chymotrypsins (EC 3.4.21.1 and EC 3.4.21.2). Trypsins (EC 3.4.21.4), elastase (EC 3.4.21.11), subtilisins (EC 3.4.21.14), proteinase K (EC 3.4.21.14) and Pronase (EC 3.4.24.4) are not inhibited. The inhibitor reacts with two molecules of chymotrypsin to form a stable complex (Mr approx. 70 0000) which was isolated by gel filtration on Sephadex G-100. When mixed with substrate, the interaction of the inhibitor with alpha-chymotrypsin is characterized by substrate-induced dissociation of the complex. In contrast, the interaction with chymotrypsin B is quantitative with no substrate-induced dissociation. The inhibitor reacts with alpha-chymotrypsin to form a 1 : 2 molar complex at all ratios of [I]/[E]; however, the interaction with chymotrypsin B is characterized by the formation of initially of a 1 : 1 molar complex at [I] greater than [E] followed by the formation of the 1 : 2 molar complex at [I] less than 2[E]; an intermediate species of Mr approx. 48 000 was demonstrated by gel filtration on Sephadex G-100. The inhibitor is stable over the pH range 2.0-11.5 and to heating up to 70 degrees C at pH 4.1 and 8.0, and up to 90 degrees C at pH 3.0. The inhibitor resists denaturation in 8.0 M urea at pH 8.0 and 4.0, is stable in 0.12 M beta-mercaptoethanol at pH 8.0; however, reduction in 8.0 M urea results in a loss of inhibitory activity. The inhibitor resists digestion with pepsin at pH 2.0, being only slowly degraded over a period of 7 days with an equimolar amount of pepsin.     

Influence of Ethephon and 2,5-Norbornadiene on Antioxidative Enzymes and Proline Content in Salt-Stressed Spinach Leaves

The effects of ethephon, an ethylene generating compound, and 2,5-norbornadiene (NBD), an inhibitor of ethylene action, on peroxidase (POD; EC 1.11.1.7), catalase (CAT; EC 1.11.1.6), polyphenol oxidase (PPO; EC 1.14.18.1) activities and proline content in salt-stressed spinach leaves were investigated. POD and PPO activities were increased by NaCl + ethephon + NBD combination and reduced by NBD. Also, ethephon increased the CAT activity while ethephon + NBD reduced CAT activity. NaCl + ethephon increased proline content. The antagonistic effect of ethephon and NBD was seen on POD and PPO activity and proline accumulation, but was not on CAT activity.     

Inhibition of adrenodoxin reductase by NADP+ and NADPH

Adrenodoxin reductase (EC 1.18.1.2) catalyzes the oxidation of NADPH by 1.4-benzoquinone. The catalytic constant of this reaction at pH 7.0 is equal to 25-28 s-1. NADP+ acts as the mixed-type nonlinear inhibitor of enzyme increasing Km of NADPH and decreasing catalytic constant. NADP+ and NADPH act as mutually exclusive inhibitors relative to reduced adrenodoxin reductase. The patterns of 2',5'-ADP inhibition are analogous to that of NADP+. These data support the conclusion about the existence of second nicotinamide coenzyme binding centre in adrenodoxin reductase.     

Electrophoretic variation of aconitase and phosphoglucomutase detected in Microtus californicus.

Four electrophoretic variants of cytoplasmic aconitase (citrate (isocitrate) hydro-lyase, EC 4.2.1.3) were detected in a population of Microtus californicus when samples were screened by starch gel electrophoresis using Tris/citrate buffers at pH 7.0 and pH 8.7. Variation at what is presumed to be the phosphoglucomutase-3 locus (alpha-D-glucose-1,6-diphosphate:alpha-D-glucose-1-phosphate phosphotransferase, EC 2.7.5.1) was also detected with liver samples but was not detected in kidney samples or red blood cells lysates. This nongenetic variation is due to oxidation of free sulfhydryl groups.     

Effect of pH on organic acid production byClostridium propionicum in test tube and fermentor cultures

Summary Clostridum propionicum is a chemical autotroph that metabolizes alanine to propionic acid (reduction product) and acetic acid (oxidation product). The ratio of propionate/acetate predicted by the electron balance is 2:1. This study reports the effect of pH on growth and organic acid production by this organism when grown in both test tube cultures initially buffered from pH 7.0 to 5.0, and in fermentors maintained at pH 7.0 and 6.5. Highest growth and organic acid production was found at pH 7.0 in both cases. HPLC analysis showed that at pH 7.0, the ratios of propionate to acetate were 0.45:1 (stationary tube, 24 h). The highest ratio observed was 1.8:1 (stationary tube, pH 6.0, 24h). This tube produced 8.5% of the acids produced in the pH 7.0 culture tube. The identify of the major portion of the reduction products of the organism remains unknown.     

Effect of cholesta-3,5-dien-7-one on human liver aldehyde dehydrogenase

A recently isolated cholesterol oxidation product, cholesta-3,5-dien-7-one, which was present at high concentrations in fatty/cirrhotic alcoholic liver was identified as a potent endogenous inhibitor of the cytosolic, E1, isozyme of aldehyde dehydrogenase (EC 1.2.1.3). The oxysterol was a less potent inhibitor of mitochondrial, E2, isozyme. The inhibition of the E1 isozyme was irreversible on the IEF gels, upon dilution and with 33 microM 2-mercaptoethanol during activity assay. The calculated 1-50% values from the inhibition curves for the E1 isozyme were 5-10 microM and approx. 180 microM for the E2 isozyme. The E3 isozyme was not sensitive to the oxysterol. Judging from the Lineweaver-Burk plot, the inhibition of the E1 isozyme with a constant concentration of cholesta-3,5-dien-7-one (52 microM) appeared to be noncompetitive.     

Autooxidation and hydroxylation reactions of oxygenated cytochrome P-450cam.

Oxy-ferrous substrate-bound cytochrome P-450cam (mrsO2) autooxidizes in the absence of its specific effector protein, putidaredoxin, without hydroxylating the substrate, camphor. The autooxidation is first order with an activation energy of 17 kcal mol-1 at 25 degrees, pH 7.0. Substrate removal and low pH accelerate the reaction. The product, 5-exo-OH camphor, and a nonhydroxylated pseudosubstrate, norcamphor, stabilize the complex in a manner similar to camphor. Increased oxidation rate of mrsO2 and substrate hydroxylation are induced by putidaredoxin, rebredoxin, cytochrome b5, and the apoproteins of the latter two. Dihydrolipoic acid and other dithiols also replace putidaredoxin as effector molecules, but 1000-fold higher concentrations are required. Effector molecules do not increase the autooxidation rate of mrsO2 unless camphor, norcamphor, or another pseudosubstrate is present. Kinetic evidence is presented showing that an active complex between mrsO2 and effector is a required intermediate in mixed function oxidation.     

Preliminary crystallographic investigations of glycinamide ribonucleotide transformylase

Crystals of glycinamide ribonucleotide transformylase have been grown from 0.4 to 1 M ammonium sulfate, 0.6 to 1 M sodium-potassium phosphate, or 0.65 to 1 M citrate in the pH range 4.5-7.0. The single crystals display variable morphology with varying pH. The crystals belong to the orthorhombic space group C222 with cell dimensions a = 141.4 A, b = 98.2 A, c = 103.5 A. Co-crystals have also been obtained in the presence of the inhibitor 5,8-dideazafolate (KI = 18 microM) under similar crystallization conditions. Crystals of a chemically modified enzyme, iodinated at Cys-21, were grown under similar conditions within the pH range 6.5-7.0. These crystals are isomorphous with the unmodified enzyme. Crystals suitable for high resolution (less than 2.5 A) x-ray diffraction studies have been obtained for each of the above.     

7-Dehydrocholesterol enhances ultraviolet A-induced oxidative stress in keratinocytes: roles of NADPH oxidase, mitochondria, and lipid rafts

Long wavelength solar UVA radiation stimulates formation of reactive oxygen species (ROS) and prostaglandin E(2) (PGE(2)), which are involved in skin photosensitivity and tumor promotion. High levels of 7-dehydrocholesterol (7-DHC), the precursor to cholesterol, cause exaggerated photosensitivity to UVA in patients with Smith-Lemli-Opitz syndrome (SLOS). Partially replacing cholesterol with 7-DHC in keratinocytes rapidly (<5 min) increased UVA-induced ROS, intracellular calcium, phospholipase A(2) activity, PGE(2), and NADPH oxidase activity. UVA-induced ROS and PGE(2) production were inhibited in these cells by depleting the Nox1 subunit of NADPH oxidase using siRNA or using a mitochondrial radical quencher, MitoQ. Partial replacement of cholesterol with 7-DHC also disrupted membrane lipid raft domains, although depletion of cholesterol, which also disrupts lipid rafts, did not affect UVA-induced increases in ROS and PGE(2). Phospholipid liposomes containing 7-DHC were more rapidly oxidized by a free radical mechanism than those containing cholesterol. These results indicate that 7-DHC enhances rapid UVA-induced ROS and PGE(2) formation by enhancing free radical-mediated membrane lipid oxidation and suggests that this mechanism might underlie the UVA photosensitivity in SLOS.     

Inhibition of phenylalanine hydroxylase by p-chlorophenylalanine; dependence on cofactor structure

The reported discrepancy between the in vitro and in vivo properties of p-chlorophenylalanine as an inhibitor of phenylalanine hydroxylase (E.C.1.14. 3.1) was investigated. It was demonstrated that the lack of inhibition, in vitro, was not due to (1) non-physiological pH or temperature of the in vitro assay system, (2) inhibition by m-chlorotyrosine, a product of the enzymatic hydroxylation of p-chlorophenylalanine, or (3) a slow irreversible reaction of p-chlorophenylalanine with enzyme. However, when the inhibitory properties of p-chlorophenylalanine were determined using the natural cofactor, tetrahydrobiopterin, instead of the pseudocofactor 6,7-dimethyltetrahydropterin, which had been utilized in the reported in vitro studies, it was shown that p-chlorophenylalanine is a potent inhibitor of the enzymatic hydroxylation of phenylalanine. The apparent K_i is 0.03mM with tetrahydobiopterin as cofactor, compared to 1.5mM with 6.7-dimethyltetrahydropterin. The dependence of the inhibitory properties of an aromatic amino acid analog on the structure of the cofactor may be a general phenomenon with all tetrahydrobiopterin dependent aromatic amino acid hydroxylases.