Olefin oxygenation and N-dealkylation by dopamine beta-monooxygenase: catalysis and mechanism-based inhibition

In an initial communication [May, S. W., Mueller, P. W., Padgette, S. R., Herman, H. H., & Phillips, R. S. (1983) Biochem. Biophys. Res. Commun. 110, 161-168], we reported that 1-phenyl-1-(aminomethyl)ethene hydrochloride (PAME) is an olefinic substrate for dopamine beta-monooxygenase (DBM; EC 1.14.17.1) which inactivates the enzyme in an apparent mechanism-based manner. The present study further characterizes this reaction. The inactivation reaction yields kinact = 0.23 min-1 at pH 5.0 and 37 degrees C and is strictly dependent on reductant (ascorbate) and oxygen. The DBM/PAME substrate reaction (apparent kcat = 14 s-1), shown to be stimulated by fumarate, gives the corresponding epoxide as product, identified by derivatization with 4-(p-nitrobenzyl)pyridine. However, the lack of DBM inhibition by alpha-methylstyrene oxide, and the observation of identical PAME/DBM inactivation rates in the absence and presence of preformed enzymatic PAME epoxide, indicates that free epoxide is not the inactivating species. A structure-activity study revealed that 4-hydroxylation of PAME (to give 4-HOPAME) increases both kinact (0.81 min-1) and apparent kcat (56 s-1) values, while 3-hydroxylation (to give 3-HOPAME) greatly diminishes inactivation activity while retaining substrate activity (apparent kcat = 47 s-1). 4-Hydroxy-alpha-methylstyrene was found to be a DBM inhibitor (kinact = 0.53 min-1) with weak substrate activity (apparent kcat = 0.71 s-1), while 3-hydroxy-alpha-methylstyrene and alpha-(cyanomethyl) styrene were found not to exhibit detectable DBM substrate activity and only weak inhibitory activity. 3-Phenylpropargylamine hydrochloride showed no detectable DBM substrate activity but rapidly inactivated the enzyme. A new substrate activity for DBM was discovered, N-dealkylation of N-phenylethylenediamine and N-methyl-N-phenylethylenediamine, and the lack of O-dealkylation activity with phenyl 2-aminoethyl ether and 4-hydroxyphenyl 2-aminoethyl ether indicates that DBM N-dealkylation proceeds via initial one-electron abstraction from the benzylic nitrogen heteroatom. With this new substrate and inhibitor reactivity information in hand, along with the other known substrate reactions, a DBM oxygenation mechanism analogous to that for cytochrome P-450 is proposed.     

In vitro potency and selectivity of the non-steroidal androgen aromatase inhibitor CGS 16949A compared to steroidal inhibitors in the brain.

A sensitive in vitro 3H2O microassay for aromatase activity was used to evaluate the potency and selectivity of three aromatase inhibitors in mammalian (gerbil) and avian (ring dove) hypothalamus. The steroidal inhibitors, 1,4,6-androstatrien-3,17-dione (ATD) and 4-hydroxy-androstenedione (4-OH-A) were compared with a new non-steroidal imidazole inhibitor, CGS 16949A [4-(5,6,7,8-tetrahydroimidazo-[1,5-a]-pyridin-5-yl)benzonitrile HCl]. Adult male dove hypothalamic aromatase is highly active [Vmax = 5.3 pmol testosterone (T) converted/h/mg protein], has high substrate binding affinity (Km = 4.0 nM), and direct involvement in control of sexual behaviour. With [1 beta-3H]T or [1 beta-3H]A as substrate, male dove preoptic aromatase activity was inhibited more effectively and selectively by CGS 16949A. Thus, Kis and IC50s for aromatization were approximately 50 times lower for the non-steroidal inhibitor, and inhibition of the other major androgen-metabolizing enzymes (5 alpha/beta-reductase) occurred at concentrations at least one order of magnitude greater than for ATD and 4-OH-A. Neonatal male gerbil hypothalamic aromatase activity (Vmax = 1.3 pmol T converted/h/mg protein) was lower than in the dove. Aromatase inhibition by CGS 16949A is more potent in the neonatal gerbil than in the dove (Kis of 0.03 and 0.60 nM, respectively, with A as substrate). We conclude that the imidazole is an effective aromatase inhibitor in both the adult and developing brain.     

Synthesis and antibacterial activity of novel and potent DNA gyrase inhibitors with azole ring

The 4-piperidyl moiety and the pyrazole ring in 1-(3-chlorophenyl)-5-(4-phenoxyphenyl)-3-(4-piperidyl)pyrazole 2, which has previously shown improved DNA gyrase inhibition and target-related antibacterial activity, were transformed to other groups and the in vitro antibacterial activity of the synthesized compounds was evaluated. The selected pyrazole, oxazole and imidazole derivatives showed moderate inhibition against DNA gyrase and topoisomerase IV with similar IC(50) values (IC(50)=9.4-25 microg/mL). In addition, many of the pyrazole, oxazole and imidazole derivatives synthesized in this study exhibited potent antibacterial activity against quinolone-resistant clinical isolates and coumarin-resistant laboratory isolates of Gram-positive bacteria with minimal inhibitory concentration values equivalent to those against susceptible strains.     

Protein-serine kinase from rat epididymal adipose tissue which phosphorylates and activates acetyl-CoA carboxylase. Possible role in insulin action.

1. Most of the cyclic-nucleotide-independent acetyl-CoA carboxylase kinase activity in an extract of rat epididymal adipose tissue was evaluated from a Mono Q column by 0.175 M-NaCl at pH 7.4. The activity of the kinase in this fraction (fraction 1) was increased after exposure of intact tissue to insulin. 2. Incubation of purified adipose-tissue acetyl-CoA carboxylase with [gamma-32P]ATP and samples of fraction 1 led to the incorporation of up to 0.4 mol of 32P/mol of enzyme subunit. Most of the phosphorylation was on serine residues within a single tryptic peptide. This peptide, on the basis of two-dimensional t.l.c. analysis, h.p.l.c. and Superose 12 chromatography, appeared to be the same as the acetyl-CoA carboxylase peptide ('I'-peptide) which exhibits increased phosphorylation in insulin-treated tissue. 3. Phosphorylation of purified acetyl-CoA carboxylase by the kinase in fraction 1 was found to be associated with a parallel 4-fold increase in activity. However, increases in both phosphorylation and activity were much diminished if fraction 1 was treated by Centricon centrifugation to remove low-Mr components. Among these components was a potent inhibitor of acetyl-CoA carboxylase activity which appeared to be necessary for the kinase in fraction 1 to be fully active. 4. The inhibitor remains to be identified, but inhibition requires MgATP, although the inhibitor itself does not cause any phosphorylation of the carboxylase. No effects of insulin were observed on the activity of the inhibitor. 5. It is concluded that the kinase probably plays an important role in the mechanism whereby insulin brings about the well-established increases in phosphorylation and activation of acetyl-CoA carboxylase in adipose tissue.     

3-trifluoromethyl-4-nitro-5-arylpyrazoles are novel K(ATP) channel agonists

This communication describes the discovery and synthesis of a series of 3-trifluoromethyl-4-nitro-5-arylpyrazoles as potent K(ATP) channel agonists. The most potent compound reported is ca. 100-fold more potent than diazoxide and exhibits selectivity for the SUR1 K(ATP) channel subtype. The 4-nitro substitutent on the pyrazole ring was required for activity, and limited SAR suggests that the de-protonated pyrazole maybe the active species.     

Active prorenin: Evidence for the formation of a conformational variant of recombinant human prorenin

Using highly purified recombinant human prorenin, we report the first evidence for the formation of a stable, partially active, conformational variant of the recombinant proenzyme. The enzymatically active prorenin exhibits the following characteristics: (1) the proenzyme N-terminal sequence and molecular weight are maintained; (2) the active proenzyme is capable of cleaving a novel fluorogenic peptide substrate based on the sequence of human angiotensinogen and exhibits about 30% of mature renin specific activity for the fluorogenic substrate; (3) the active proenzyme conformation binds to, and can be eluted from, a pepstatin affinity column; and (4) the activity of the active proenzyme can be inhibited by a novel peptidomimetic renin inhibitor.     

Inhibition of juvenile hormone III biosynthesis by fluoromevalonolactone and citronellyl phenyl imidazoles in isolated corpora allata from the cockroach Periplaneta americana

1-Citronellyl-5-phenyl imidazole (1,5-CPI), 1-citronellyl-4-phenyl imidazole (1,4-CPI) and 1-citronellyl-2-phenyl imidazole (1,2-CPI) were tested as inhibitors of JH-III biosynthesis in vitro. 1,5-CPI was found to be most active followed by 1,2-CPI. The least active isomer was 1,4-CPI. Inhibition of JH biosynthesis by 1,5-CPI resulted in no significant accumulation of the epoxidation substrate methyl farnesoate, and piperonyl butoxide, a known microsomal epoxidase inhibitor, produced only a slight increase in methyl farnesoate. Topical application of fluoromevalonolactone resulted in reduced biosynthetic capability of subsequently excised corpora allata.     

The major piscine liver alcohol dehydrogenase has class-mixed properties in relation to mammalian alcohol dehydrogenases of classes I and III.

The major alcohol dehydrogenase of cod liver has been purified, enzymatically characterized, and structurally analyzed in order to establish original functions and relationships among the deviating classes of the enzyme in mammalian tissues. Interestingly, the cod enzyme exhibits mixed properties--many positional identities with a class III protein, but functionally a class I enzyme--blurring the distinction among the classes of alcohol dehydrogenase. The two domain interfaces, affected by movements upon coenzyme binding, both exhibit substitutions in a manner thus far unique to the cod enzyme. In contrast, coenzyme-binding residues are highly conserved. At the active site, inner and outer parts of the substrate pocket show different extents of amino acid replacement. In total, no less than 7-10 residues of 11 in the substrate binding pocket differ from those of all the mammalian classes, explaining the substrate specificities. However, the inner part of the substrate pocket is very similar to that of the class I enzymes, which is compatible with the observed characteristics of the cod enzyme: ethanol is an excellent substrate (Km = 1.2 mM) and 4-methylpyrazole is a strong inhibitor (Ki = 0.1 microM). These values are about as low as those typical for the ethanol-active class I mammalian enzyme and do not at all resemble those for class III, for which ethanol is hardly a substrate and pyrazole is hardly an inhibitor. Further out in the substrate pocket, several residues differ from the mammalian classes, affecting large substrates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Correlation between the predicted and the observed biological activity of the symmetric and nonsymmetric cyclic urea derivatives used as HIV-1 protease inhibitors. A 3D-QSAR-CoMFA method for new antiviral drug design

The predicted inhibition constant (Ki) and the predicted inhibitor concentration (IC90) of the HIV-1 protease (HIV-1 PR) inhibitors: symmetric and nonsymmetric - benzyl, ketone, oxime, pyrazole, imidazole, and triazole cyclic urea derivatives, were obtained by the 3D-CoMFA (Comparative Molecular Field Analysis) method. The CoMFA statistical parameters: cross-validate correlation coefficient (q2), higher than 0.5, and the fitted correlation coefficient (r2), higher than 0.90 validated the predicted biological activities. The best predictions were found for the trifluoromethyl ketoxime derivative (log 1/Ki predict = 8.42), the m-pyridineCH2 pyrazole derivative (log 1/Ki predict = 9.77) and the 1,2,3 triazole derivative (log 1/Ki predict = 7.03). We attempted to design a new potent HIV-1 protease inhibitor by addition of o-benzyl to the (p-HOPhCH2) pyrazole 12f derivative inhibitor. A favorable steric area surrounded the o-benzyl, suggesting a possible new potent HIV-1 protease inhibitor.     

4-Aminopyrazolo[3,4-d]pyrimidine (4-APP) as a novel inhibitor of the RNA and DNA depurination induced by Shiga toxin 1

Shiga toxin 1 (Stx1) catalyses the removal of a unique and specific adenine from 28S RNA in ribosomes (RNA-N-glycosidase activity) and the release of multiple adenines from DNA (DNA glycosylase activity). Added adenine behaves as an uncompetitive inhibitor of the RNA-N-glycosidase reaction binding more tightly to the Stx1–ribosome complex than to the free enzyme. Several purine derivatives and analogues have now been assayed as inhibitors of Stx1. Most of the compounds showed only minor differences in the rank order of activity on the two enzymatic reactions catalysed by Stx1. The survey highlights the importance of the amino group in the 6-position of the pyrimidine ring of adenine. Shifting (2-aminopurine) or substituting (hypoxanthine, 6-mercaptopurine, 6-methylpurine) the group greatly decreases the inhibitory power. The presence of a second ring, besides the pyrimidine one, is strictly required. Substitution, by introducing an additional nitrogen, of the imidazole ring of adenine with triazole leads to loss of inhibitory power, while rearrangement of the nitrogen atoms of the ring from the imidazole to the pyrazole configuration greatly enhances the inhibitory power. Thus 4-aminopyrazolo[3,4-d]pyrimidine (4-APP), the isomer of adenine with the five-membered ring in the pyrazole configuration, is by far the most potent inhibitor of both enzymatic reactions catalysed by Stx1. This finding opens perspectives on therapeutic strategies to protect endothelial renal cells once endocytosis of Stx1 has occurred (haemolytic uraemic syndrome). In the RNA-N-glycosidase reaction 4-APP binds, as adenine, predominantly to the Stx1–ribosome complex (uncompetitive inhibition), while inhibition of the DNA glycosylase activity by both inhibitors is of the mixed type.     

Synthesis and antimicrobial activity of new 3-(1-R-3(5)-methyl-4-nitroso-1H-5(3)-pyrazolyl)-5-methylisoxazoles

A number of new 3-(1-R-3(5)-methyl-4-nitroso-1H-5(3)-pyrazolyl)-5-methylisoxazoles 6a–g (7b–f) were synthesized and tested for antibacterial and antifungal activity. Some of these compounds displayed antifungal activity at non-cytotoxic concentrations. Derivative 6c was 9 times more potent in vitro than miconazole and 20 times more selective against C. neoformans. 6c was also 8- and 125-fold more potent than amphotericin B and fluconazole, respectively. None of the compounds was active against bacteria. Preliminary structure–activity relationship (SAR) studies showed that the NO group at position 4 of the pyrazole ring is essential for the activity. Lipophilicity of the pyrazole moiety, N-alkyl chain length and planarity of the two heterocyclic rings appear to play a decisive role in modulating cytotoxicity and antifungal activity.     

Glycosaparaginase from human leukocytes. Inactivation and covalent modification with diazo-oxonorvaline

The apparent active site of human leukocyte glycoasparaginase (N4-(beta-acetylglucosaminyl)-L-asparaginase EC 3.5.1.26) has been studied by labeling with an asparagine analogue, 5-diazo-4-oxo-L-norvaline. Glycoasparaginase was purified 4,600-fold from human leukocytes with an overall recovery of 12%. The purified enzyme has a Km of 110 microM, a Vmax of 34 mumol x l-1 x min-1, and a specific activity of 2.2 units/mg protein with N4-(beta-N-acetylglucosaminyl)-L-asparagine as substrate. The carbohydrate content of the enzyme is 15%, and it exhibits a broad pH maximum between 7 and 9. The 88-kDa native enzyme is composed of 19-kDa light (L) chains and 25-kDa heavy (H) chains and it has a heterotetrameric structure of L2H2-type. The glycoasparaginase activity decreases rapidly and irreversibly in the presence of 5-diazo-4-oxo-L-norvaline. At any one concentration of the compound, the inactivation of the enzyme is pseudo-first-order with time. The inhibitory constant, K1, is 80 microM and the second-order rate constant 1.25 x 10(3) M-1 min-1 at pH 7.5. The enzyme activity is competitively protected against this inactivation by its natural substrate, aspartylglucosamine, indicating that this inhibitor binds to the active site or very close to it. The covalent incorporation of [5-14C]diazo-4-oxo-L-norvaline paralleled the loss of the enzymatic activity and one inhibitor binding site was localized to each L-subunit of the heterotetrameric enzyme. Four peptides with the radioactive label were generated, purified by high performance liquid chromatography, and sequenced by Edman degradation. The sequences were overlapping and all contained the amino-terminal tripeptide of the L-chain. By mass spectrometry, the reacting group of 5-diazo-4-oxo-L-norvaline was characterized as 4-oxo-L-norvaline that was bound through an alpha-ketone ether linkage to the hydroxyl group of the amino-terminal amino acid threonine.     

Thermodynamic fidelity of the mammalian cytochrome P450 2B4 active site in binding substrates and inhibitors

Structural plasticity of mammalian cytochromes P450 (CYP) has recently been explored in our laboratory and elsewhere to understand the ligand-binding promiscuity. CYP2B4 exhibits very different conformations and thermodynamic signatures in binding the small inhibitor 4-(4-chlorophenyl)imidazole (4-CPI) versus the large bifonazole. Using four key active-site mutants (F296A, T302A, I363A, and V367L) that are involved in binding one or both inhibitors, we dissected the thermodynamic basis for the ability of CYP2B4 to bind substrates and inhibitors of different sizes and chemistry. In all cases, 1:1 binding stoichiometry was observed. The inhibitors 4-CPI, 1-(4-chlorophenyl)imidazole, and 1-(2-(benzyloxy)ethyl)imidazole bind to the mutants with a free energy difference (ΔΔG) of ∼ 0.5 to 1 kcal/mol compared with the wild type but with a large entropy-enthalpy compensation of up to 50 kcal/mol. The substrate testosterone binds to all four mutants with a ΔΔG of ∼ 0.5 kcal/mol but with as much as 40 kcal/mol of entropy-enthalpy compensation. In contrast, benzphetamine binding to V367L and F296A is accompanied by a ΔΔG of ∼ 1.5 and 3 kcal/mol, respectively. F296A, I363A, and V367L exhibit very different benzphetamine metabolite profiles, indicating the different substrate-binding orientations in the active site of each mutant. Overall, the findings indicate that malleability of the active site allows mammalian P450s to exhibit a high degree of thermodynamic fidelity in ligand binding.     

Crystal structure of tryptophan hydroxylase with bound amino acid substrate

Tryptophan hydroxylase (TPH) is a mononuclear non-heme iron enzyme, which catalyzes the reaction between tryptophan, O 2, and tetrahydrobiopterin (BH 4) to produce 5-hydroxytryptophan and 4a-hydroxytetrahydrobiopterin. This is the first and rate-limiting step in the biosynthesis of the neurotransmitter and hormone serotonin (5-hydroxytryptamine). We have determined the 1.9 A resolution crystal structure of the catalytic domain (Delta1-100/Delta415-445) of chicken TPH isoform 1 (TPH1) in complex with the tryptophan substrate and an iron-bound imidazole. This is the first structure of any aromatic amino acid hydroxylase with bound natural amino acid substrate. The iron coordination can be described as distorted trigonal bipyramidal coordination with His273, His278, and Glu318 (partially bidentate) and one imidazole as ligands. The tryptophan stacks against Pro269 with a distance of 3.9 A between the iron and the tryptophan Czeta3 atom that is hydroxylated. The binding of tryptophan and maybe the imidazole has caused the structural changes in the catalytic domain compared to the structure of the human TPH1 without tryptophan. The structure of chicken TPH1 is more compact, and the loops of residues Leu124-Asp139 and Ile367-Thr369 close around the active site. Similar structural changes are seen in the catalytic domain of phenylalanine hydroxylase (PAH) upon binding of substrate analogues norleucine and thienylalanine to the PAH.BH 4 complex. In fact, the chicken TPH1.Trp.imidazole structure resembles the PAH.BH 4.thienylalanine structure more (root-mean-square deviation for Calpha atoms of 0.90 A) than the human TPH1 structure (root-mean-square deviation of 1.47 A).     

1-Halo analogs of dihydroxyacetone 3-phosphate. The effects of the fluoro analog on cytosolic glycerol-3-phosphate dehydrogenase and triosephosphate isomerase.

Fluoro-o-hydorxyacetone phosphate (fluoroacetol phosphate) has been prepared by oxidation of 1-fluoro-3-chloro-2-propanol to 1-fluoro-3-chloroacetone, phosphorylation with silver dibenzylphosphate, and the intermediate isolation of 1-fluoro-3-hydroxyacetone phosphate dibenzyl ester, followed by catalytic hydrogenation and preparation of the stable monosodium salt. The chloro analog as the pure, stable monosodium salt has been prepared by a similar route from 1,3-dichloroacetone. 1-Fluoro-3-hydroxyacetone-P is substrate for cytosolic NAD+-linked glycerol-3-P dehydrogenese (EC 1.1.1.8) from rabbit skeletal muscle with an apparent Km of 50 mM under conditions in which dihydroxyacetone-P exhibits an apparent Km of 0.15 mM. Under these conditions the fluoro analog is 85% hydrated wheras dihydroxyacetone-P has been shown by others to be 44% hydrated. The turnover numbers are 49,000 molecules of NADH oxidized per minute per molecule of enzyme at 25 degrees with the fluoro analog as substrate, and 60,000 with dihydrocyacetone-P as substrate. The product of the reduction of the fluoro analog has been identified as 1-fluorodeoxyglycerol-3-P. 1-Fluoro-3-hydroxyacetone-P is comparatively weak irreversible inhibitor at 4 degrees of rabbit muscle triosephosphate isomerase (EC 5.3.1.1) with second-order rate constant of 2.6 M minus 1 sec minus 1. Inhibition by pyrazole in vivo of alcohol dehydrogenese catalyzed oxidation of 1-fluorodeoxyglecerol-3-P indicates in mice the reduction of 1-fluoro-3-hydroxyacetone-P to -l-1-fluorodexoxyglycerol-3-P is not significant metabolic route, or that an alternative route exists when the alcohol dehydrogenase dependent pathway is inhibited.     

Replacement of non-heme Fe(II) with Cu(II) in the alpha-ketoglutarate dependent DNA repair enzyme AlkB: spectroscopic characterization of the active site

The bacterial DNA repair enzyme AlkB is an alpha-ketoglutarate (alphaKG) dependent non-heme Fe(II) containing dioxygenase. Here we describe, for the first time, the preparation of a Cu(II)-reconstituted form of AlkB in various complexes. Spectroscopic characterization showed correct AlkB folding upon incorporation of Cu(II) in the active site. The Cu site was classified as a type 2 site by EPR spectroscopy. The accessibility of the active site metal was studied using imidazole as a probe. Although addition of imidazole did not change the EPR spectrum of the AlkB-Cu-alphaKG complex, the spectrum of the AlkB-Cu-succinate complex clearly changed, indicating binding of imidazole at the Cu site. Binding of substrate (methylated DNA) to the AlkB-Cu-alphaKG complex did not induce changes in the EPR spectrum, demonstrating that the substrate does not bind in the immediate vicinity of the metal centre. This work provides a basis for advanced EPR approaches aimed at studying the interactions and dynamics of AlkB complexes in solution.     

Dibenzoylmethane Exerts Metabolic Activity through Regulation of AMP-Activated Protein Kinase (AMPK)-Mediated Glucose Uptake and Adipogenesis Pathways

Dibenzoylmethane (DBM) has been shown to exert a variety of beneficial effects on human health. However, the mechanism of action is poorly understood. In this study, DBM increased phosphorylation of AMP-activated protein kinase (AMPK) and stimulated glucose uptake in a skeletal muscle cell line. Both knockdown of AMPK with siRNA and inhibition with AMPK inhibitor blocked DBM-induced glucose uptake. DBM increased the concentration of intracellular calcium and glucose uptake due to DBM was abolished by STO-609 (a calcium/calmodulin-dependent protein kinase inhibitor). DBM stimulated phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK), which was blocked by pretreatment with compound C, an AMPK inhibitor. The expression of glucose transporter type 4 (GLUT4) was increased by DBM. The translocation of GLUT4 to the plasma membrane was also increased by DBM in AMPK dependently. In addition, DBM suppressed weight gain and prevented fat accumulation in the liver and abdomen in mice fed a high-fat diet. In pre-adipocyte cells, DBM decreased the activity of acetyl-CoA carboxylase (ACC), the rate-limiting enzyme of fatty acid synthesis. Expression of the adipogenic gene, fatty acid synthase (FAS), was suppressed by DBM in an AMPK-dependent manner. These results showed that the beneficial metabolic effects of DBM might be due to regulation of glucose uptake via AMPK in skeletal muscle and inhibition of adipogenesis in pre-adipocytes.     

Ascorbate depletion as a consequence of product recycling during dopamine beta-monooxygenase catalyzed selenoxidation

The competence of dopamine beta-monooxygenase (DBM) to process selenide substrates was investigated, in anticipation that the expected selenoxide products would exhibit unique reactivity and redox properties. The prototypical selenide phenyl 2-aminoethyl selenide (PAESe) was synthesized and shown to be a substrate for DBM with the characteristic e/O2 ratio of 2:1 for monooxygenation. The kinetic parameters for oxygenation of PAESe were found to be similar to those for the DBM-catalyzed sulfoxidation of the cognate sulfide phenyl 2-aminoethyl sulfide [May, S. W., & Phillips, R. S. (1980) J. Am. Chem. Soc. 102, 5981-5983], and selenoxidation was stimulated by fumarate in a manner similar to other well-characterized DBM monooxygenation reactions. Identification of phenyl 2-aminoethyl selenoxide (PAESeO) as the enzymatic product was accomplished by the demonstration of coincident elution of authentic PAESeO with the enzymatic product in three significantly different HPLC systems. PAESeO was found to oxidize ascorbic acid with the concomitant and stoichiometric reduction of PAESeO back to the selenide, PAESe. As a consequence of this nonenzymatic reaction, ascorbate-supported DBM turnover was prematurely terminated under standard assay conditions due to depletion of reduced ascorbate. The kinetics of the redox reaction between PAESeO and ascorbate were investigated with a spectrophotometric assay of ascorbate at 300 nm, and a second-order rate constant of 3.4 M-1 s-1 was determined at pH 5.0, 25 degrees C. Spectrophotometric assay of cytochrome c (cyt c) reduction at 550 nm during the oxidation of ascorbate by PAESeO demonstrated that no cyt c trappable semidehydroascorbate was produced in this nonenzymatic reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increase in ovarian kallikrein activity during ovulation in the gonadotrophin-primed immature rat

The ovulatory process was initiated in 25-day-old rats by injecting them with hCG (10 i.u., s.c.) 2 days after the animals had been primed with PMSG (10 i.u., s.c.). At 2-h intervals after hCG, the ovaries were extracted and assayed for glandular kallikrein activity by using a chromogenic substrate (H-D-Val-Leu-Arg-p-nitroanilide) which exhibits optical density (at 405 nm) upon hydrolysis. In 0-h control ovaries the activity was 12.5 x 10(-3) kallikrein units (KU)/mg protein and it increased to a peak of 56.6 x 10(-3) KU/mg at 12 h after hCG, when the follicles first began to rupture. The kallikrein activity was distinguishable from ovarian plasminogen activator activity on the basis of pH optima and response to trypsin inhibitor (SBTI). The activity was inhibited by a s.c. dose of indomethacin of 0.3 mg/rat, or higher, and this dosage inhibited ovulation. The results suggest that kallikrein activity contributes to the degradation of Graafian follicles during ovulation in mammals.     

Crystal structure of the predicted phospholipase LYPLAL1 reveals unexpected functional plasticity despite close relationship to acyl protein thioesterases

Sequence homology indicates the existence of three human cytosolic acyl protein thioesterases, including APT1 that is known to depalmitoylate H- and N-Ras. One of them is the lysophospholipase-like 1 (LYPLAL1) protein that on the one hand is predicted to be closely related to APT1 but on the other hand might also function as a potential triacylglycerol lipase involved in obesity. However, its role remained unclear. The 1.7 Å crystal structure of LYPLAL1 reveals a fold very similar to APT1, as expected, but features a shape of the active site that precludes binding of long-chain substrates. Biochemical data demonstrate that LYPLAL1 exhibits neither phospholipase nor triacylglycerol lipase activity, but rather accepts short-chain substrates. Furthermore, extensive screening efforts using chemical array technique revealed a first small molecule inhibitor of LYPLAL1.