Mechanism of inactivation of Escherichia coli and Lactobacillus leichmannii ribonucleotide reductases by 2'-chloro-2'-deoxynucleotides: evidence for generation of 2-methylene-3(2H)-furanone

Incubation of 2'-chloro-2'-deoxy[3'-3H]uridine 5'-diphosphate ([3'-3H]ClUDP) with Escherichia coli ribonucleotide reductase (RDPR) and use of thioredoxin-thioredoxin reductase as reductants result in release of 4.7 equiv of 3H2O/equiv of B1 protomer, concomitant with enzyme inactivation. Inactivation is accompanied by the production of 6 equiv of inorganic pyrophosphate [Stubbe, J. A., & Kozarich, J.W. (1980) J. Am. Chem. Soc. 102, 2505-2507] and by the release of uracil as previously shown [Thelander, L., Larsson, A., Hobbs, J., & Eckstein, F. (1976) J. Biol. Chem. 251, 1398-1405]. Reisolation of RDPR by Sephadex chromatography and analysis by scintillation counting indicate that 0.96 equiv of 3H is bound per protomer of the B1 subunit of the inactivated enzyme. Incubation of [5'-3H]ClUDP with RDPR followed by similar analysis indicates that 4.6 mol of 3H is bound per protomer of the B1 subunit of the inactivated enzyme. No 3H2O is released, and 6 equiv of inorganic pyrophosphate is produced during the inactivation. RDPR is protected against inactivation when dithiothreitol (DTT) is used as a reductant in place of thioredoxin-thioredoxin reductase. Incubation of [5'-3H]ClUDP with RDPR and DTT results in the isolation of CHCl3-extractable material that exhibits infrared absorptions at 1710 and 1762 cm-1. The infrared spectrum and the NMR spectrum of the CHCl3-extracted material are very similar to model compounds prepared by the interaction of 2-methylene-3(2H)-furanone with ethanethiol. Incubation of ribonucleoside-triphosphate reductase (RTPR) from Lactobacillus leichmannii with [3'-3H]ClUTP and 3 mM DTT also results in time-dependent 3H2O release concomitant with enzyme inactivation. Reisolation of the inactive protein by Sephadex chromatography followed by radiochemical analysis indicates that 0.4 equiv of 3H is bound covalently per mol of inactivated enzyme. Similar studies with [5'-3H]ClUTP indicate that 2.9 equiv of 3H is bound covalently per mol of inactivated enzyme. No 3H2O is released. High concentrations of DTT protect the enzyme against inactivation. Extraction of the enzymatic reaction mixture with CHCl3 and analysis of the isolated products result in an infrared spectrum and an NMR spectrum remarkably similar to those observed with the E. coli RDPR. Data presented are consistent with the proposal that both the E. coli and L. leichmannii enzymes are able to catalyze the breakdown of the appropriate 2'-chloro-2'-deoxynucleotide to a 3'-keto-2'-deoxynucleotide that can collapse to form the reactive sugar intermediate 2-methylene-3(2H)-furanone.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mechanism of ribonucleotide reductase from herpes simplex virus type 1. Evidence for 3' carbon-hydrogen bond cleavage and inactivation by nucleotide analogs

Isotope effects of 2.5, 2.1, and 1.0 were measured on the conversion of [3'-3H]ADP, [3'-H]UDP, and [5-3H] UDP to the corresponding 2'-deoxynucleotides by herpes simplex virus type 1 ribonucleotide reductase. These results indicate that the reduction of either purine or pyrimidine nucleotides requires cleavage of the 3' carbon-hydrogen bond of the substrate. The substrate analogs 2'-chloro-2'-deoxyuridine 5'-diphosphate (ClUDP), 2'-deoxy-2'-fluorouridine 5'-diphosphate, and 2'-azido-2'-deoxyuridine 5'-diphosphate were time-dependent inactivators of the herpes simplex virus type 1 ribonucleotide reductase. Incubation of [3'-3H]ClUDP with the enzyme was accompanied by time-dependent release of 3H to the solvent. Reaction of [beta-32P]ClUDP with the reductase resulted in the production of inorganic pyrophosphate. These results are consistent with the enzyme-mediated cleavage of the 3' carbon-hydrogen bond of ClUDP and the subsequent conversion of the nucleotide to 2-methylene-3(2H)furanone, as previously reported with the Escherichia coli ribonucleotide reductase (Harris, G., Ator, M., and Stubbe, J. A. (1984) Biochemistry 23, 5214-5225; Ator, M., and Stubbe, J. A. (1985) Biochemistry 24, 7214-7221).     

Products of the inactivation of ribonucleoside diphosphate reductase from Escherichia coli with 2'-azido-2'-deoxyuridine 5'-diphosphate

Ribonucleoside diphosphate reductase (RDPR) from Escherichia coli was completely inactivated by 1 equiv of the mechanism-based inhibitor 2'-azido-2'-deoxyuridine 5'-diphosphate (N3UDP). Incubation of RDPR with [3'-3H]N3UDP resulted in 0.2 mol of 3H released to solvent per mole of enzyme inactivated, indicating that cleavage of the 3' carbon-hydrogen bond occurred in the reaction. Incubation of RDPR with [beta-32P]N3UDP resulted in stoichiometric production of inorganic pyrophosphate. One equivalent of uracil was eliminated from N3UDP, but no azide release was detected. Analysis of the reaction of RDPR with [15N3]N3UDP by mass spectrometry revealed that the azide moiety was converted to 0.9 mol of nitrogen gas per mole of enzyme inactivated. The tyrosyl radical of the B2 subunit was destroyed during the inactivation by N3UDP as reported previously [Sj?berg, B.-M., Gr?slund, A., & Eckstein, F. (1983) J. Biol. Chem. 258, 8060-8067], while the specific activity of the B1 subunit was reduced by half. Incubation of [5'-3H]N3UDP with RDPR resulted in stoichiometric covalent radiolabeling of the enzyme. Separation of the enzyme's subunits by chromatofocusing revealed that the modification was specific for the B1 subunit.     

Inactivation of the Lactobacillus leichmannii ribonucleoside triphosphate reductase by 2'-chloro-2'-deoxyuridine 5'-triphosphate: stoichiometry of inactivation, site of inactivation, and mechanism of the protein chromophore formation

The ribonucleoside triphosphate reductase (RTPR) of Lactobacillus leichmannii is inactivated by the substrate analogue 2'-chloro-2'-deoxyuridine 5'-triphosphate (ClUTP). Inactivation is due to alkylation by 2-methylene-3(2H)-furanone, a decomposition product of the enzymic product 3'-keto-2'-deoxyuridine triphosphate. The former has been unambiguously identified as 2-[(ethylthio)methyl]-3(2H)-furanone, an ethanethiol trapped adduct, which is identical by 1H NMR spectroscopy with material synthesized chemically. Subsequent to rapid inactivation, a slow process occurs that results in formation of a new protein-associated chromophore absorbing maximally near 320 nm. The terminal stages of the inactivation have now been investigated in detail. The alkylation and inactivation stoichiometries were studied as a function of the ratio of ClUTP to enzyme. At high enzyme concentrations (0.1 mM), 1 equiv of [5'-3H]ClUTP resulted in 0.9 equiv of 3H bound to protein and 83% inactivation. The amount of labeling of RTPR increased with increasing ClUTP concentration up to the maximum of approximately 4 labels/RTPR, yet the degree of inactivation did not increase proportionally. This suggests that (1) RTPR may be inactivated by alkylation of a single site and (2) decomposition of 3'-keto-dUTP is not necessarily enzyme catalyzed. The formation of the new protein chromophore was also monitored during inactivation and found to reach its full extent upon the first alkylation. Thus, out of four alkylation sites, only one appears capable of undergoing the subsequent reaction to form the new chromophore. While chromophore formation was prevented by NaBH4 treatment, the chromophore itself is resistant to reduction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of ribonucleoside diphosphate reductase from Escherichia coli. Evidence for 3'-C--H bond cleavage

Incubation of the pyrimidine [3'-3H]UDP with ribonucleotide reductase resulted in an isotope effect on the conversion to dUDP which varied as a function of pH and allosteric effectors (pH, kH/kT, effector): 6.6, 4.7, ATP; 7.6, 3.3, ATP; 7.6, 2.6, dATP; 7.6, 2.0, TTP; 8.4, 2.8, ATP. During this reaction 3H2O was also released. The lower the pH of the reaction, the larger the isotope effect, and the smaller the amount of 3H2O produced. At 50% conversion of UDP to dUDP and at pH 7.6, approximately 0.5% of total 3H present in solution was volatilized, while at pH 8.4, approximately 0.9% was volatilized. Similar experiments in which the purine [3'-3H]ADP was incubated with ribonucleotide reductase also resulted in an isotope effect on its conversion to dATP which varied as a function of pH (pH, kH/kT with dGTP as an effector); 6.6, 1.9; 7.6, 1.7; 8.6, 1.4. Furthermore, 3H2O was also released as a function of the extent of the reaction. At 50% turnover and pH 7.6, approximately 0.6% of 3H2O was volatilized, while at pH 8.6 approximately 1.25% was released. Two control experiments in which either the B1 subunit of ribonucleotide reductase was inactivated with 2'-chloro-2'-deoxyuridine 5'-diphosphate or the B2 subunit of ribonucleotide reductase was inactivated with 2'-azido-2'-deoxyuridine 5'-diphosphate and then the enzyme incubated with [3'-3H]ADP or [3'-3H]UDP indicated that in neither case was 3H released. Both B1 and B2 subunits are required for cleavage of the 3'-C--H bond. Incubation of [3'-3H]dADP or [3'-3H]dUDP with ribonucleotide reductase produced no measurable release of 3H. These data clearly indicate that conversion of a purine or pyrimidine diphosphate to a deoxynucleotide diphosphate by Escherichia coli ribonucleotide reductase requires cleavage of the 3'-C--H bond of the substrate. The fate of the 3'-H of the substrate was also determined. Incubation of [3'-2H]UDP with ribonucleotide reductase resulted in the production of [3'-2H]dUDP.     

Interaction of C225SR1 mutant subunit of ribonucleotide reductase with R2 and nucleoside diphosphates: tales of a suicidal enzyme.

Ribonucleotide reductase (RDPR) from Escherichia coli is composed of two subunits, R1 and R2, both of which are required to catalyze the conversion of nucleotides to deoxynucleotides. This reduction process is accompanied by oxidation of two cysteines within the active site to a disulfide. One of these putative active site cysteines, C225, has been mutated to a serine, and the properties of this mutant (C225SR1) have been investigated in detail. Incubation of C225SR1 and R2 with [3'-3H,U-14C]UDP results in time-dependent inactivation of the enzyme! This inactivation is accompanied by production of 2.4 uracils, 3H2O, and 3H,14C-labeled protein with an absorbance change at 320 nm. There is an isotope effect (kH/k3H) on uracil production of 3.2. In addition, the tyrosyl radical on R2 is reduced. The observation of 3H2O, indicative of 3' carbon-hydrogen bond cleavage and loss of the tyrosyl radical, provides a direct test of our mechanistic hypothesis that cleavage of this bond occurs concomitantly with tyrosyl radical reduction. Incubation of [3'-2H]UDP with C225SR1 and R2 resulted in a V and V/K isotope effect on loss of the radical of 2.0 and 2.0, respectively. These studies provide the first direct evidence for protein radical involvement in catalysis. Reduction of the tyrosyl radical on R2 is accompanied by a stoichiometric cleavage of the R1 polypeptide into two new polypeptides of 26 and 61 kDa. The 26-kDa polypeptide is the N-terminus of R1, and hence cleavage of the polypeptide is occurring in the region of the mutation. The N-terminus of the 61-kDa polypeptide is blocked.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluoride, pyrophosphate, and base release from 2'-deoxy-2'-fluoronucleoside 5'-diphosphates by ribonucleoside-diphosphate reductase

Ribonucleoside-diphsophate reductase from Escherichia coli catalyzes release of fluoride, inorganic pyrophosphate, and base from 2'-deoxy-2'-fluoronucleoside diphosphates. This reaction is accompanied by inactivation of the enzyme and an increase in absorbance at 314 nm of the inactivated protein. 2'-Deoxy-2'-fluoroadenosine 5'-diphosphate requires two turnovers per inactivation, whereas 2'-deoxy-2'-fluorocytidine 5'-diphosphate requires 100 turnovers per inactivation.     

Inactivation of S-adenosyl-L-homocysteine hydrolase with fluorinated analogs of 2'- and 3'-deoxy-5'-methylthioadenosine

Fluorinated analogs of 2'- and 3'-deoxy-5'-methylthioadenosine 1-4 caused irreversible inactivation of AdoHcy hydrolase. Based on the ESI-Mass spectra analysis of the inactivated enzyme with the fluorinated analog 1 a mechanism of inactivation is proposed.     

Interaction of 3'-[3H]2'-Chloro-2'-deoxyuridine 5'-triphosphate with ribonucleotide reductase from Lactobacillus leichmannii

Incubation of [3'-3H]2'-chloro-2'-deoxyuridine 5'-triphosphate (CldUTP) with adenosylcobalamin (AdoCbl), reductant, and ribonucleotide reductase from Lactobacillus leichmannii results in the production of 3H2O, uracil, tripolyphosphate, 5'-deoxyadenosine, and cob(II)alamin. The rate of 3H2O release (0.19 mumol/min/mg) is almost identical with the rate of UTP reduction (0.24 mumol/min/mg). The amount of 3H2O release is dependent upon the enzyme to cofactor ratio. With a ribonucleotide reductase: AdoCbl ratio of 1:1000, approximately 500 eq of 3H2O are released. At this time the enzyme is still active, but further destruction of cofactor and turnover of CldUTP is prevented by competitive inhibition of Co(II) + 5'-deoxyadenosine with AdoCbl for binding to ribonucleotide reductase. The 5'-deoxyadenosine and AdoCbl reisolated during incubation of [3'-3H]CldUTP and ribonucleotide reductase contains no detectable radioactivity.     

Differences in adult and foetal human cytochrome P-450 forms recognized by monoclonal antibodies with specificity for the P450III family.

The biosynthesis of 9-[5'-deoxy-5'-(methylthio)-beta-D-xylofuranosyl]adenine (xylosyl-MTA), a naturally occurring analogue of 5'-deoxy-5'-methylthioadenosine (MTA) recently characterized, was studied in the nudibranch mollusc Doris verrucosa. Experiments performed in vivo with putative labelled precursors such as [8-14C]adenine, [Me-14C]methionine and [Me-14C]MTA indicate that xylosyl-MTA originates from MTA. Experiments with MTA double-labelled at critical positions are consistent with a 3'-isomerization of the nucleoside through the formation of a 3'-oxo intermediate. In addition, experiments with the newly synthesized [3'-3H]xylosyl-MTA are indicative for a very low turnover rate of this molecule, which therefore accumulates in the mollusc.     

Biosynthesis of 9-beta-D-arabinofuranosyladenine: hydrogen exchange at C-2' and oxygen exchange at C-3' of adenosine

The data presented here describe new findings related to the bioconversion of adenosine to 9-beta-D-arabinofuranosyladenine (ara-A) by Streptomyces antibioticus by in vivo investigations and with a partially purified enzyme. First, in double label in vivo experiments with [2'-18O]- and [U-14C]adenosine, the 18O:14C ratio of the ara-A isolated does not change appreciably, indicating a stereospecific inversion of the C-2' hydroxyl of adenosine to ara-A with retention of the 18O at C-2'. In experiments with [3'-18O]- and [U-14C]-adenosine, [U-14C]ara-A was isolated; however, the 18O at C-3' is below detection. The adenosine isolated from the RNA from both double label experiments has essentially the same ratio of 18O:14C. Second, an enzyme has been isolated and partially purified from extracts of S. antibioticus that catalyzes the conversion of adenosine, but not AMP, ADP, ATP, inosine, guanosine, or D-ribose, to ara-A. In a single label enzyme-catalyzed experiment with [U-14C]adenosine, there was a 9.9% conversion to [U-14C]ara-A; with [2'-3H]-adenosine, there was a 8.9% release of the C-2' tritium from [2'-3H]adenosine which was recovered as 3H2O. Third, the release of 3H as 3H2O from [2'-3H]adenosine was confirmed by incubations of the enzyme with 3H2O and adenosine. Ninety percent of the tritium incorporated into the D-arabinose of the isolated ara-A was in C-2 and 8% was in C-3. The enzyme-catalyzed conversion of adenosine to ara-A occurs without added cofactors, displays saturation kinetics, a pH optimum of 6.8, a Km of 8 X 10(-4) M, and an inhibition by heavy metal cations. The enzyme also catalyzes the stereospecific inversion of the C-2' hydroxyl of the nucleoside antibiotic, tubercidin to form 7-beta-D-arabinofuranosyl-4-aminopyrrolo[2,3-d]pyrimidine. The nucleoside antibiotic, sangivamycin, in which the C-5 hydrogen is replaced with a carboxamide group, is not a substrate. On the basis of the single and double label experiments in vivo and the in vitro enzyme-catalyzed experiments, two mechanisms involving either a 3'-ketonucleoside intermediate or a radical cation are proposed to explain the observed data.     

The mechanism of inhibition of S-adenosyl-L-homocysteine hydrolase by fluorine-containing adenosine analogs

(Z)-4',5'-Didehydro-5'-deoxy-5'-fluoroadenosine (I), 5'-deoxy-5'-difluoroadenosine (II), and 4',5'-didehydro-5'-deoxy-5'-fluoroarabinosyl-adenosine (III) are inhibitors of rat liver S-adenosyl-L-homocysteine hydrolase. Compounds I and II are time-dependent and irreversible inhibitors of the enzyme. Both I and II are oxidized by E.NAD to produce E.NADH, and fluoride anion is formed in the inactivation reaction (0.7 to 1.0 mole fluoride/mole of enzyme subunit, and 1.7 moles fluoride/mole of enzyme subunit from I and II, respectively). The enzyme is stoichiometrically labeled with [8-3H]-I, but the label is lost upon denaturation of the protein either with or without treatment of the labeled complex with sodium borohydride. The compound III, the arabino derivative of I, is a competitive inhibitor of the enzyme. The mechanism of the inhibition of S-adenosyl-L-homocysteine hydrolase by these inhibitors is discussed.     

Identification of two alpha-ketoglutarate-dependent dioxygenases in extracts of Rhodotorula glutinis catalyzing deoxyuridine hydroxylation

Attempts to isolate deoxyuridine 2'-hydroxylase from Rhodotorula glutinis by the procedure of Warn-Cramer et al. (Warn-Cramer, B. J., Macrander, L. A., and Abbott, M. T. (1983) J. Biol. Chem. 258, 10551-10557) have led to the identification and partial purification of a newly recognized alpha-ketoglutarate-requiring oxygenase. This activity, designated deoxyuridine (uridine) 1'-hydroxylase, in the presence of iron and ascorbate, catalyzes the conversion of deoxyuridine (uridine), O2, and alpha-ketoglutarate to uracil, deoxyribonolactone (ribonolactone), CO2, and succinate. Incubation of [1'-3H]uridine with this activity results in time-dependent formation of uracil concomitant with production of CO2 and 3H2O. No Vmax/Km isotope effect is observed on this reaction. Uracil production is accompanied by stoichiometric production of ribonolactone identified by NMR spectroscopy. Also reported in this paper is the partial purification and characterization of the alpha-ketoglutarate-requiring enzyme, deoxyuridine 2'-hydroxylase. Incubation of [2'-alpha-3H]deoxyuridine with this activity results in concomitant production of uridine and 3H2O. Incubation with [2'-beta-3H] deoxyuridine results in the production of uridine whose specific activity is identical to that of the starting material. This enzyme catalyzes the conversion of deoxyuridine to uridine with retention of configuration. No isotope effect is observed on this transformation.     

Affinity labeling of bovine adrenal 3 beta-hydroxysteroid dehydrogenase/steroid isomerase by 5'-[p-(fluorosulfonyl)benzoyl]adenosine

Incubation of bovine adrenal 3 beta-hydroxysteroid dehydrogenase/steroid isomerase with 5'-[p-(fluorosulfonyl)benzoyl]adenosine (5'-FSBA) results in the inactivation of the 3 beta-hydroxysteroid dehydrogenase enzyme activity following pseudo-first-order kinetics. A double-reciprocal plot of 1/kobs versus 1/[5'-FSBA] yields a straight line with a positive y intercept, indicative of reversible binding of the inhibitor prior to an irreversible inactivation reaction. The dissociation constant (Kd) for the initial reversible enzyme-inhibitor complex is estimated at 0.533 mM, with k2 = 0.22 min-1. The irreversible inactivation could be prevented by the presence of NAD+ during the incubation, indicating that 5'-FSBA inactivates the 3 beta-hydroxysteroid dehydrogenase activity by reacting at the NAD+ binding site. Although the enzyme was inactivated by incubation with 5'-FSBA, no incorporation of the inhibitor was found in labeling studies using 5'-[p-(fluorosulfonyl)benzoyl] [14C]adenosine. However, the inactivation of 3 beta-hydroxysteroid dehydrogenase activity caused by incubation with 5'-FSBA could be completely reversed by the addition of dithiothreitol. This indicates the presence of at least two cysteine residues at or in the vicinity of the NAD+ binding site, which may form a disulfide bond catalyzed by the presence of 5'-FSBA. The intramolecular cysteine disulfide bridge was found between the cysteine residues in the peptides 274EWGFCLDSR282 and 18IICLLVEEK26, by comparing the [14C]iodoacetic acid labeling before and after recovering the enzyme activity upon the addition of dithiothreitol.     

Activation of phospholipase C associated with isolated rabbit platelet membranes by guanosine 5''-[gamma-thio]triphosphate and by thrombin in the presence of GTP.

Rabbit platelets were labelled with [3H]inositol and a membrane fraction was isolated in the presence of ATP, MgCl2 and EGTA. Incubation of samples for 10 min with 0.1 microM-Ca2+free released [3H]inositol phosphates equivalent to about 2.0% of the membrane [3H]phosphoinositides. Addition of 10 microM-guanosine 5'-[gamma-thio]triphosphate (GTP[S]) caused an additional formation of [3H]inositol phosphates equivalent to 6.6% of the [3H]phosphoinositides. A half-maximal effect was observed with 0.4 microM-GTP[S]. The [3H]inositol phosphates that accumulated consisted of 10% [3H]inositol monophosphate, 88% [3H]inositol bisphosphate ([3H]IP2) and 2% [3H]inositol trisphosphate ([3H]IP3). Omission of ATP and MgCl2 led to depletion of membrane [3H]polyphosphoinositides and marked decreases in the formation of [3H]inositol phosphates. Thrombin (2 units/ml) or GTP (4-100 microM) alone weakly stimulated [3H]IP2 formation, but together they acted synergistically to exert an effect comparable with that of 10 microM-GTP[S]. The action of thrombin was also potentiated by 0.1 microM-GTP[S]. Guanosine 5'-[beta-thio]diphosphate not only inhibited the effects of GTP[S], GTP and GTP with thrombin, but also blocked the action of thrombin alone, suggesting that this depended on residual GTP. Incubation with either GTP[S] or thrombin and GTP decreased membrane [3H]phosphatidylinositol 4-phosphate ([H]PIP) and prevented an increase in [3H]phosphatidylinositol 4,5-bisphosphate ([3H]PIP2) observed in controls. Addition of unlabelled IP3 to trap [3H]IP3 before it was degraded to [3H]IP2 showed that only about 20% of the additional [3H]inositol phosphates that accumulated with GTP[S] or thrombin and GTP were derived from the action of phospholipase C on [3H]PIP2. The results provide further evidence that guanine-nucleotide-binding protein mediates signal transduction between the thrombin receptor and phospholipase C, and suggest that PIP may be a major substrate of this enzyme in the platelet.     

The synthesis of some branched-chain-sugar nucleoside analogues.

1-(2,3-Epoxy-5-O-trityl-beta-D-lyxofuranosyl)uracil was treated with a number of carbon nucleophiles. Ethynyl lithium gave 3'-deoxy-3'-ethynyl-5'-O-trityl-ara-uridine, which was reduced to the corresponding 3'-ethenyl compound. Sodium cyanide gave 3'-cyano-3'-deoxy-5'-O-trityl-ara-uridine which upon alkaline hydrolysis gave the corresponding 3'-carboxamido compound. 1,3-Dithian-2-yl lithium gave 3'-deoxy-3'-(1,3-dithian-2-yl)-5'-O-trityl-ara-uridine. The trityl group was removed from each of these compounds by mild acidic hydrolysis. Treatment of 2 with 0.1M H2sO4 and mercury (II) acetate afforded 3'-acetyl-3'-deoxy-ara-uridine which upon reduction with NaBH4 gave 3'-deoxy-3'-(1-hydroxyethan-1-yl)-ara-uridine. Acetylation of 6 yielded 5'-O-acetyl-3'-acetyl-2',3'-didehydro-2',3'-dideoxyuridine which upon reduction with NaBH4 produced a mixture of 5'-O-acetyl-2',3'-didehydro-2',3'-dideoxy-3'-(1-hydroxyethan -1-yl)uridine and 1-(R)[5-(S)-acetoxymethyl-4-(1-hydroxyethan-1-yl)-tetrahydrofuran- 2-yl]- uracil. Reduction of 14 with Raney nickel followed by removal of the trityl group gave 3'-deoxy-3'-methyl-ara-uridine.     

Alpha-pyrones and a 2(5H)-furanone from Hyptis pectinata

Three pyrones and a 2(5H)-furanone, designated pectinolides D-G, have been isolated from the dichloromethane extract of Hyptis pectinata. The metabolites were characterized on the basis of 1D and 2D NMR spectroscopic techniques. The pyrones were identified as 6S-[3S,6S-(diacetoxy)-5R-hydroxy-1Z-heptenyl]-5S-hydroxy-5,6-dihydro-2H-pyran-2-one (1)- pectinolide D, 6S-[3S,5R,6S-(triacetoxy)-1Z-heptenyl]-5S-acetoxy-5,6-dihydro-2H-pyran-2-one (2)- pectinolide E and 6S-[3S,5R,6S-(triacetoxy)-1Z-heptenyl]-5S-acetoxy-4R-methoxy-3,4,5,6-tetrahydro-4H pyran-2-one (3)- pectinolide F. The furanone was identified as [2'Z,5(1')Z] 5-(4'S,6'R,7'S-triacetoxy-2-octenylidene)-2(5H)-furanone (4)-pectinolide G.     

On the mechanism of oxidation of cholesterol at C-7 in a lipoxygenase system.

Incubation of [7-2H2]cholesterol with soybean lipoxygenase and linoleic acid in the presence of oxygen gave a mixture of 5-cholestene-3 beta,7 alpha-diol, 5-cholestene-3 beta,7 beta-diol, 3 beta-hydroxy-5-cholesten-7-one,5 alpha,6 alpha-epoxycholestan-3 beta-ol, and 5 beta,6 beta-epoxycholestan-3 beta-ol. The conversion into the 7-oxygenated products was associated with a very high intermolecular isotope effect (KH/KD = 15-17), suggesting that the rate-limiting step in the overall conversion is likely to be the abstraction of hydrogen at C-7 in a radical reaction. Evidence that linoleic acid is to some extent directly involved was obtained with the use of [7-3H]cholesterol. Incubation of [7-3H]cholesterol resulted in a significant incorporation of 3H in the reisolated linoleic acid fraction. The isotope effect associated with conversion of [7 alpha-2H]cholesterol into 7-oxygenated products in the lipoxygenase system was 2-3, indicating that the extraction of hydrogen is nonstereospecific. Incubation of [7-2H2]cholesterol with 13-hydroperoxy-9,11-octadecadienoic acid gave the above 7-oxygenated products with relatively small isotope effects (KH/KD = 3-4). It is concluded that the most important mechanism for oxidation of cholesterol at C-7 in the lipoxygenase system involves participation of radicals and that a carbon-centered linoleic acid radical can extract hydrogen directly from cholesterol. Fatty acid hydroperoxides and their secondary products seem to be less important as initiators in connection with oxidation of cholesterol.     

Inactivation of gamma-aminobutyric acid aminotransferase by (Z)-4-amino-2-fluorobut-2-enoic acid

(Z)-4-Amino-2-fluorobut-2-enoic acid (1) is shown to be a mechanism-based inactivator of pig brain gamma-aminobutyric acid aminotransferase. Approximately 750 inactivator molecules are consumed prior to complete enzyme inactivation. Concurrent with enzyme inactivation is the release of 708 +/- 79 fluoride ions; transamination occurs 737 +/- 15 times per inactivation event. Inactivation of [3H]pyridoxal 5'-phosphate ([3H]PLP) reconstituted GABA aminotransferase by 1 followed by denaturation releases [3H]PMP with no radioactivity remaining attached to the protein. A similar experiment carried out with 4-amino-5-fluoropent-2-enoic acid [Silverman, R. B., Invergo, B. J., & Mathew, J. (1986) J. Med. Chem. 29, 1840-1846] as the inactivator produces no [3H]PMP; rather, another radioactive species is released. These results support an inactivation mechanism for 1 that involves normal catalytic isomerization followed by active site nucleophilic attack on the activated Michael acceptor. A general hypothesis for predicting the inactivation mechanism (Michael addition vs enamine addition) of GABA aminotransferase inactivators is proposed.     

2-[(4-Bromo-2,3-dioxobutyl)thio]-1,N6-ethenoadenosine 5'-diphosphate. A new fluorescent affinity label of a tyrosyl residue in the active site of rabbit muscle pyruvate kinase

A new reactive fluorescent ADP analog has been synthesized: 2-[(4-bromo-2,3-dioxobutyl)thio]-1,N6-ethenoadenosine 5'-diphosphate (2-BDB-T epsilon A-5'-DP). Rabbit muscle pyruvate kinase is inactivated by 200 microM 2-BDB-T epsilon A-5'-DP in a biphasic manner, with an initial loss of 75% activity followed by a slow total inactivation. The rate constants for both phases exhibit nonlinear dependence on reagent concentration, consistent with reversible formation of an enzyme-reagent complex (KI = 133 microM) prior to irreversible reaction. Loss of activity is prevented by substrates. The best protection against inactivation is provided by phosphoenolpyruvate (PEP), KCl, and MnSO4, suggesting that the reaction occurs in the region of the PEP binding site. Incorporation of 1.7 mol/mol enzyme subunit accompanies 90% inactivation by 200 microM 2-BDB-T epsilon A-5'-DP in 80 min. However, in the presence of PEP, KCl, and MnSO4, 1.0 mol of reagent is incorporated when the enzyme is only 14% inactivated. These results indicate that 2-BDB-T epsilon A-5'-DP reacts with two groups on the enzyme, one of which is at or near the PEP binding site. Incubation of pyruvate kinase with related nucleotide analogs lacking a 5'-diphosphate or a diketo group suggests that the diketo group, but not the diphosphate, is essential for inactivation. The enolized form of the bromodioxobutyl group resembles phosphoenolpyruvate and probably directs the reagent to the PEP binding site. Modified enzyme, prepared by incubating pyruvate kinase with 200 microM 2-BDB-T epsilon A-5'-DP in the absence and presence of phosphoenolpyruvate, KCl, and MnSO4, was reduced with [3H]NaBH4, carboxymethylated, and digested with trypsin. Nucleotidyl peptides were isolated by chromatography on phenylboronateagarose followed by reverse phase high pressure liquid chromatography. Two radioactive peptides were identified: Asn162-Ile-Cys-Lys165 and Ile141-Thr-Leu-Asp-Asn-Ala-Tyr-Met-Glu-Lys150. Only the tetrapeptide was modified in the presence of PEP, KCl, and Mn+ when the enzyme retained most of its activity. Cys164 is thus designated the nonessential modified residue, while modification of Tyr147 near the active site of pyruvate kinase is responsible for loss of enzymatic activity. The observed biphasic kinetics of inactivation are due to the negatively cooperative reaction of 2-BDB-T epsilon A-5'-DP with Tyr147 in the tetramer. The new compound, 2-BDB-T epsilon A-5'-DP, may have general application as an affinity label of ADP and PEP sites in other proteins.