On the mechanism of formation of N-acetyldopamine quinone methide in insect cuticle

The mechanism of formation of quinone methide from the sclerotizing precursor N-acetyldopamine (NADA) was studied using three different cuticular enzyme systems viz. Sarcophaga bullata larval cuticle, Manduca sexta pharate pupae, and Periplaneta americana presclerotized adult cuticle. All three cuticular samples readily oxidized NADA. During the enzyme-catalyzed oxidation, the majority of NADA oxidized became bound covalently to the cuticle through the side chain with the retention of o-diphenolic function, while a minor amount was recovered as N-acetylnorepinephrine (NANE). Cuticle treated with NADA readily released 2-hydroxy-3′,4′-dihydroxyacetophenone on mild acid hydrolysis confirming the operation of quinone methide sclerotization. Attempts to demonstrate the direct formation of NADA-quinone methide by trapping experiments with N-acetylcysteine surprisingly yielded NADA-quinone-N-acetylcysteine adduct rather than the expected NADA-quinone methide-N-acetylcysteine adduct. These results are indicative of NADA oxidation to NADA-quinone and its subsequent isomerization to NADA-quinone methide. Accordingly, all three cuticular samples exhibited the presence of an isomerase, which catalyzed the conversion of NADA-quinone to NADA-quinone methide as evidenced by the formation of NANE—the water adduct of quinone methide. Thus, in association with phenoloxidase, newly discovered quinone methide isomerase seems to generate quinone methides and provide them for quinone methide sclerotization.     

Trapping of transiently formed quinone methide during enzymatic conversion of N-acetyldopamine to N-acetylnorepinephrine

We have demonstrated that quinone methide formation is an important aspect of insect physiology and proposed that enzymatically generated quinone methides react nonenzymatically with water or other nucleophiles to form Michael-1,6-addition products [(1988) Adv. Insect Physiol. 21, 179-231; (1989) J. Cell. Biochem. suppl. 13C, 58]. Using a purified o-quinone isomerase from the larval cuticle of Sacrophaga bullata and mushroom tyrosinase, we now demonstrate that transiently formed N-acetyldopamine quinone methide from N-acetyldopamine can be trapped by methanol to produce beta-methoxy N-acetyldopamine. The methanol adduct thus formed was found to be a racemic mixture and can be resolved into the optical isomers on cyclodextrin chiral column. These results confirm our contention that enzymatically generated quinone methides are nonenzymatically and nonstereoselectively transformed to Michael-1,6-adducts by reaction with water or other nucleophiles.     

N-acetyldopamine quinone methide/1,2-dehydro-N-acetyl dopamine tautomerase. A new enzyme involved in sclerotization of insect cuticle

The enzyme system causing the side chain desaturation of the sclerotizing precursor, N-acetyldopamine (NADA), was solubilized from the larval cuticle of Sarcophaga bullata and resolved into three components. The first enzyme, phenoloxidase, catalyzed conversion of NADA to NADA quinone and provided it for the second enzyme (NADA quinone isomerase), which makes the highly unstable NADA quinone methide. Quinone methide was hydrated rapidly and nonenzymatically to form N-acetylnorepinephrine. In addition, it also served as the substrate for the last enzyme, quinone methide tautomerase, which converted it to 1,2-dehydro-NADA. Reconstitution of NADA side chain desaturase activity was achieved by mixing the last enzyme fraction with NADA quinone isomerase, obtained from the hemolymph of the same organism, and mushroom tyrosinase. Therefore, NADA side chain desaturation observed in insects is caused by the combined action of three enzymes rather than the action of a single specific NADA desaturase, as previously thought.     

Nonenzymatic cleavage of proteins by reactive oxygen species generated by dithiothreitol and iron

Many enzymes, represented by yeast glutamine synthetase, are inactivated and degraded in the presence of dithiothreitol (DTT), oxygen, and catalytic amounts of iron salts. The roles of DTT and iron can be replaced by ascorbate and copper, respectively. Experimental data suggest that reactive oxygen species, likely hydroxyl radicals, are generated locally around irons bound at specific sites on enzymes, and these species are responsible for the inactivation and degradation. Since many biochemicals are contaminated with metal salts in quantities sufficient for some hydroxyl radical formation to occur, the possibility of oxidative modification and degradation should be considered when an enzyme is exposed to DTT.     

A relationship between amide hydrogen bond strength and quinone reduction potential: implications for photosystem I and bacterial reaction center quinone function

A series of 11 simple phylloquinone derivatives, each lacking the extended phytyl side chain but featuring H-bond donor amides at one or both peri positions, were prepared and some salient physical properties were measured. A correlation between both IR frequency and NMR peak position, as indicators of internal H-bond strength, and the quinone half-wave reduction potential, was observed. These data are consistent with the prevailing hypothesis that quinone carbonyl H-bonding in general, and stronger H-bonds in particular, favorably bias the endogenous quinone's electrochemical potential toward easier reduction.     

The reactions of horseradish peroxidase, lactoperoxidase, and myeloperoxidase with enzymatically generated superoxide

The formation and decay of intermediate compounds of horseradish peroxidase, lactoperoxidase, and myeloperoxidase formed in the presence of the superoxide/hydrogen peroxide-generating xanthine/xanthine oxidase system has been studied by observation of spectral changes in both the Soret and visible spectral regions and both on millisecond and second time scales. It is tentatively concluded that in all cases compound III is formed in a two-step reaction of native enzyme with superoxide. The presence of superoxide dismutase completely inhibited compound III formation; the presence of catalase had no effect on the process. Spectral data which indicate differences in the decay of horseradish peroxidase compound III back to the native state in comparison with compounds III of lactoperoxidase and myeloperoxidase are also presented.     

Demonstration of N-acetyldopamine in human kidney and urine

Free and conjugated dopamine and N-acetyldopamine concentrations were measured in human urine and kidneys by reversed phase high performance liquid chromatography with single-electrode electrochemical detection. Conjugated N-acetyldopamine was found to occur in urine from six normal humans and in four out of six human kidneys. Unconjugated N-acetyldopamine was detected in only one urine sample and in three of seven kidneys. Urinary excretion of total N-acetyldopamine averaged 0.485 micromoles/day. This compares to a total dopamine excretion of 4.69 micromoles/day in the same subjects. In the kidneys, total N-acetyldopamine concentration averaged 1.46 nanomoles/gram. Total dopamine in the same tissues averaged 5.48 nanomoles/gram. N-acetyldopamine was not detected in human caudate nucleus, mouse whole brain, or liver from Rhesus monkey. When daily urinary excretion rates of N-acetyldopamine were determined in six individuals by both single-and dual-electrode electrochemical detection, the results were highly correlated for both free and total N-acetyldopamine (r>0.97,p<0.001). Using dual-electrode electrochemical detection, conjugated N-acetyldopamine accounted for 96.4% of the total N-acetyldopamine excretion. This value was 95.8% in the same individuals using single-electrode detection.     

Hydroxyl radical production from hydrogen peroxide and enzymatically generated paraquat radicals: Catalytic requirements and oxygen dependence

The ability of paraquat radicals (PQ^+.) generated by xanthine oxidase and glutathione reductase to give H₂O₂-dependent hydroxyl radical production was investigated. Under anaerobic conditions, paraquat radicals from each source caused chain oxidation of formate to CO₂, and oxidation of deoxyribose to thiobarbituric acid-reactive products that was inhibited by hydroxyl radical scavengers. This is in accordance with the following mechanism derived for radicals generated by γ-irradiation [H. C. Sutton and C. C. Winterbourn (1984) Arch. Biochem. Biophys.235, 106–115] PQ^+. + Fe³⁺ (chelate) → Fe²⁺ (chelate) + PQ⁺⁺ H₂O₂ + Fe²⁺ (chelate) → Fe³⁺ (chelate) + OH^? + OH.. Iron-(EDTA) and iron-(diethylenetriaminepentaacetic acid) (DTPA) were good catalysts of the reaction; iron complexed with desferrioxamine or transferrin was not. Extremely low concentrations of iron (0.03 μm) gave near-maximum yields of hydroxyl radicals. In the absence of added chelator, no formate oxidation occurred. Paraquat radicals generated from xanthine oxidase (but not by the other methods) caused H₂O₂-dependent deoxyribose oxidation. However, inhibition by scavengers was much less than expected for a reaction of hydroxyl radicals, and this deoxyribose oxidation with xanthine oxidase does not appear to be mediated by free hydroxyl radicals. With O₂ present, no hydroxyl radical production from H₂O₂ and paraquat radicals generated by radiation was detected. However, with paraquat radicals continuously generated by either enzyme, oxidation of both formate and deoxyribose was measured. Product yields decreased with increasing O₂ concentration and increased with increasing iron(DTPA). These results imply a major difference in reactivity between free and enzymatically generated paraquat radicals, and suggest that the latter could react as an enzyme-paraquat radical complex, for which the relative rate of reaction with Fe³⁺ (chelate) compared with O₂ is greater than is the case with free paraquat radicals.     

Nonenzymatic methylation of DNA by the intracellular methyl group donor S-adenosyl-L-methionine is a potentially mutagenic reaction.

Incubation of DNA with S-adenosyl-L-methionine (SAM) in neutral aqueous solution leads to base modification, with formation of small amounts of 7-methylguanine and 3-methyladenine. The products have been identified by high performance liquid chromatography of DNA hydrolysates and by the selective release of free 3-methyladenine from SAM-treated DNA by a specific DNA glycosylase. We conclude that SAM acts as a weak DNA-alkylating agent. Several control experiments including extensive purification of [3H-methyl]SAM preparations and elimination of the alkylating activity by pretreatment of SAM with a phage T3-induced SAM cleaving enzyme, have been performed to determine that the activity observed was due to SAM itself and not to a contaminating substance. We estimate that SAM, at an intracellular concentration of 4 X 10(-5) M, causes DNA alkylation at a level similar to that expected from continuous exposure of cells to 2 X 10(-8) M methyl methane-sulphonate. This ability of SAM to act as a methyl donor in a nonenzymatic reaction could result in a background of mutagenesis and carcinogenesis. The data provide an explanation for the apparently universal occurrence of multiple DNA repair enzymes specific for methylation damage.     

The differentiation of isomeric biological compounds using collision-induced dissociation of ions generated by fast atom bombardment

Though fast atom bombardment ionization makes possible the ionization and molecular weight determination of polar or thermally labile biological compounds, the resulting mass spectra commonly give few or no fragment ions which would allow detailed structural analysis. In particular, isomeric compounds often give identical spectra. Collision-induced dissociation of ions resulting from fast atom bombardment ionization is shown to be a powerful combination which can differentiate isomeric substances. The technique is applied to isomeric bile acid salts and steroid conjugates and is capable of differentiating structural isomers which have similar fast atom bombardment mass spectra. A range of isomeric cyclic nucleotides is also shown to be amenable to the method. Sensitivity limits are examined and the unequivocal identification of two 3',5'-cyclic nucleotides isolated from living systems is demonstrated.     

Total synthesis of PGC2 methyl ester. Comparison with enzymatically produced material

The synthesis of PGC₂ methyl ester is described. Comparison of the synthetic material with the methyl ester prepared from natural PGC₂ showed the two to be identical, thus confirming the structure assignment. The physical and biological data of PGC₂ methyl ester are presented.Horton and Jones have recently shown that PGA₁ (Ic) and PGA₂ (Ia) are deactivated on incubation with cats blood (1,2). Jones has proposed that this deactivation involves the enzymic conversion of PGA (I) to an 11,12-double bond isomer (PGC, II) which is subsequently isomerized by base to the inactive PGB (III) (3). Structure assignment of the PGC's in the earlier study were based on chromatographic mobilities and uv and mass spectrometric properties. We report here a total synthesis of PGC₂ methyl ester (IIb). Comparison of the biological and physical properties of this material with those of diazomethane-esterified natural material confirms the earlier structure assignment for PGC₂ (IIa).     

Preparation of both enantiomers of methyl 3-benzoyloxypentanoate by enzyme-catalysed hydrolysis of corresponding racemic nitrile and amide

Rhodococcus rhodochrous IFO 15564 enantioselectively hydrolysed racemic 3-benzoyloxypentanenitrile and 3-benzoyloxypentanamide to afford (R)-amide and (S)-car☐ylic acid with high enantiomeric excess (> 90%). In this reaction, both enantiomers of the starting nitrile were converted to the amide by nitrile hydratase, and amidase-catalysed enantioselective hydrolysis of the amide was responsible for the kinetic resolution. The lack of enantioselectivity of the nittile hydratase toward the racemic nitrile forms a marked contrast to the case of previously reported highly enantioselective conversion of prochiral 3-benzoyloxypentanedinitrile by this enzyme. since (R)-amide could be hydrolysed chemically to (R)-car☐ylic acid without any loss of its ee, the present microbial kinetic resolution serves as an effective method for preparing both enantiomers of synthetically useful 3-hydroxypentanoic acid derivatives.     

New approaches to the analysis of enzymatically hydrolyzed methyl cellulose. Part 2. Comparison of various enzyme preparations

In this part of our studies, dealing with new approaches to the analysis of enzymatically hydrolyzed methyl cellulose, five different enzymes or enzyme preparations containing endoglucanases (from Bacillus agaradhaerens Cel 5A, Trichoderma reesei, Trichoderma viride, and two obtained from Trichoderma longibrachiatum) were used to hydrolyze six different methyl celluloses (MCs). The main goal was to investigate whether enzymes could be used for determination of the heterogeneity of the substituent distribution along the cellulose chain. To obtain information about the heterogeneity, it was necessary to gather information on how the enzymes affect hydrolysis. Size exclusion chromatography with multi-angle light scattering and refractive index detection (SEC-MALS/RI) was used to estimate the molar mass distribution of the MCs before and after hydrolysis. A novel internal standard addition method in combination with electrospray ionization ion trap mass spectrometry (ESI-ITMS) was used to determine the amount of formed oligomers. Two MCs, one with a degree of substitution (DS) of 1.8 and one with DS 1.3, were hydrolyzed with all of the five enzymes. The yield of summarized di- and trisaccharides was approximately 2% of the hydrolysis products for the MC with DS 1.8, whereas the product mixture, obtained from a MC with a DS of 1.3, contained 7-16% di- and trisaccharides. By a novel sample preparation method in combination with ESI-IT tandem MS, outlined in part 1 of this work, it was shown that the enzymes produced oligomers with the reducing end bearing no or only one substituent. Comparison of the methyl pattern at the nonreducing ends of the dimers and trimers indicated that the -2 subsite of the active complex is less tolerant than subsites -3 and +1. All enzymes had similar general selectivity toward the methyl substituents but also showed some differences. From both SEC-MALS/RI and ESI-ITMS, differences with respect to substituent distribution of MCs could be recognized but not for each enzyme used. Basic considerations for enzymatic hydrolysis and analysis of methyl cellulose were listed as a consequence of the results from the work.     

Synthesis of two isomeric methyl β-D-xylotriosides containing a (1→2)-β-linkage

Partial hydrolysis with acid, methylation analysis (including uronic acid degradation), Smith degradation, and p.m.r. spectroscopy have been used to determine the primary structure of the capsular polysaccharide of Klebsiella serotype k64. The hexasaccharide repeating-unit, which also contains one O-acetyl substituent, comprises a 4)-α-d-GlcpA-(1 → 3)-α-d-Manp-(1 → 3)-β-d-Glcp-(1 → 4)-α-d-Manp-(1 → chain with a 4,6-O-(l-carboxyethylidene)-β-d-glucopyranosyl and an l-rhamnosyl group attached to the 4-linked d-mannosyl residue at O-2 and O-3, respectively.