On the mechanism of side chain oxidation of N-beta-alanyldopamine by cuticular enzymes from Sarcophaga bullata

The metabolism of N-beta-alanyldopamine (NBAD) by Sarcophaga bullata was investigated. Incubation of NBAD with larval cuticular preparations resulted in the covalent bindings of NBAD to the cuticle and generation of N-beta-alanyl-norepinephrine (NBANE) as the soluble product. When the reaction was carried out in presence of a powerful quinone trap viz., N-acetylcysteine, NBANE formation was totally abolished; but a new compound characterized as NBAD-quinone-N-acetylcysteine adduct was generated. These results indicate that NBAD quinone is an obligatory intermediate for the biosynthesis of NBANE in sarcophagid cuticle. Accordingly, phenylthiourea--a well-known phenoloxidase inhibitor--completely inhibited the NBANE production even at 5 microM level. A soluble enzyme isolated from cuticle converted exogenously supplied NBAD quinone to NBANE. Chemical considerations indicated that the enzyme is an isomerase and is converting NBAD quinone to its quinone methide which was rapidly and nonenzymatically hydrated to form NBANE. Consistent with this hypothesis is the finding that NBAD quinone methide can be trapped as beta-methoxy NBAD by performing the enzymatic reaction in 10% methanol. Moreover, when the reaction was carried out in presence of kynurenine, two diastereoisomeric structures of papiliochrome II-(Nar-[alpha-3-aminopropionyl amino methyl-3,4-dihydroxybenzyl]-L-kynurenine) could be isolated as by-products, indicating that the further reactions of NBAD quinone methide with exogenously added nucleophiles are nonenzymatic and nonstereoselective. Based on these results, it is concluded that NBAD is metabolized via NBAD quinone and NBAD quinone methide by the action of phenoloxidase and quinone isomerase respectively. The resultant NBAD quinone methide, being highly reactive, undergoes nonenzymatic and nonstereoselective Michael-1,6-addition reaction with either water (to form NBANE) or other nucleophiles in cuticle to account for the proposed quinone methide sclerotization.     

Nonenzymatic transformations of enzymatically generated N-acetyldopamine quinone and isomeric dihydrocaffeiyl methyl amide quinone

We have recently demonstrated that the side chain hydroxylation of N-acetyldopamine and related compounds observed in several insects is caused by a two-enzyme system catalyzing the initial oxidation of catecholamine derivatives and subsequent isomerization of the resultant quinones to isomeric quinone methides, which undergo rapid nonenzymatic hydration to yield the observed products [Saul, S.J. and Sugumaran, M. (1989) FEBS Lett. 249, 155-158]. During our studies on o-quinone/p-quinone methide tautomerase, we observed that quinone methides are also produced nonenzymatically slowly, under physiological conditions. The quinone methide derived from N-acetyldopamine was hydrated to yield N-acetylnorepinephrine as the stable product as originally shown by Senoh and Witkop [(1959) J. Am. Chem. Soc. 81, 6222-6231], while the isomeric quinone methide from dihydrocaffeiyl methylamide exhibited a new reaction to form caffeiyl amide as the stable product. The identity of this product was established by UV and IR spectral studies and by chemical synthesis. We could not find any evidence of intramolecular cyclization of N-acetyldopamine quinone to iminochrome-type compound(s). The importance of quinone methides in these reactions is discussed.     

4-alkyl-o-quinone/2-hydroxy-p-quinone methide isomerase from the larval hemolymph of Sarcophaga bullata. I. Purification and characterization of enzyme-catalyzed reaction

An enzyme which catalyzes the conversion of certain 4-alkyl-o-benzoquinones to 2-hydroxy-p-quinone methides has been purified to apparent homogeneity from the hemolymph of Sarcophaga bullata by employing conventional protein purification techniques. The purified enzyme migrated with an approximate molecular weight of 98,000 on gel filtration chromatography. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it migrated as a single band with a molecular weight of 46,000, indicating that it is made up of two identical subunits. It exhibited a pH optimum of 6.0 and readily converted chemically synthesized as well as enzymatically generated quinones derived from N-acetyldopamine, N-beta-alanyldopamine, and 3,4-dihydroxyphenethyl alcohol to highly unstable 2-hydroxy-p-quinone methides. The quinone methides thus formed were rapidly and nonenzymatically hydrated to form side chain hydroxylated o-diphenols as the stable product. In support of this proposition, when the enzyme reaction with N-acetyldopamine quinone was conducted in the presence of 10% methanol, racemic beta-methoxy-N-acetyldopamine was recovered as an additional product. The quinones of N-acetylnorepinephrine, N-beta-alanylnorepinephrine, and 3,4-dihydroxyphenylglycol were also attacked by the isomerase, resulting in the formation of N-acetylarterenone, N-beta-alanylarterenone and 2-hydroxy-3',4'-dihydroxyacetophenone, respectively as the stable products. The isomerase converted the dihydrocaffeiyl methyl amide quinone to its quinone methide analog which rapidly tautomerized to yield caffeiyl methyl amide. The importance of quinone isomerase in insect immunity and sclerotization of insect cuticle is discussed.     

Molecular mechanisms for mammalian melanogenesis. Comparison with insect cuticular sclerotization.

Melanogenesis is an important biochemical process for the production of skin pigments which protect many animals from the damage of solar radiation. The abnormalities in melanogenesis are associated with albinism, vitiligo, as well as malignant melanoma in humans. In the lower forms of animals viz., insects, the exoskeleton is hardened to protect their soft bodies by a process called sclerotization, which is often accompanied by melanization. Recent advances in the biochemistry of sclerotization and melanization reveal remarkable similarity between these two processes. The seven stages of sclerotization are: (a) enzymatic oxidation of N-acyldopamine, (b) Michael-1,4-addition reactions of N-acyldopamine quinone, (c) tautomerization of quinone to quinone methide, (d) Michael-1,6-addition of quinone methides, (e) tautomerization of N-acyldopamine quinone methide to 1,2-dehydro-N-acyldopamine, (f) enzymatic oxidation of 1,2-dehydro-N-acyldopamine, and (g) the reactions of resultant quinonoid compounds. Amazingly, striking similarities in the reaction sequences are found in the melanization process starting from dopa. These comparisons predict a central role for quinone methides as reactive intermediates during melanization. Accordingly, recent studies provide increasing evidence in favor of this proposition.     

Quinone methides—and not dehydrodopamine derivatives—as reactive intermediates of β-sclerotization in the puparia of flesh fly Sarcophaga bullata

Cuticular phenoloxidase(s) from Sarcophaga bullata larvae oxidized a variety of o-diphenolic compounds. While catechol, 3,4-dihydroxybenzoic acid, dopa, dopamine, and norepinephrine were converted to their corresponding quinone derivatives, other catechols such as 3,4-dihydroxyphenylacetic acid, 3,4-dihydroxyphenethyl alcohol, 3,4-dihydroxyphenyl glycol, 3,4-dihy-droxymandelic acid, and N-acetyldopamine were oxidized to their side-chain oxygenated products. In addition, the enzyme-catalyzed oxidation of the latter group of compounds accompanied the formation of colorless catecholcuticle adducts consistent with the operation of β-sclerotization. Radioactive trapping experiments failed to support the participation of 1,2-dehydro-N-acetyldopamine as a freely formed intermediate during phenoloxidase-mediated oxidation of N-acetyldopamine. When specifically tritiated substrates were provided, cuticular enzyme selectively removed tritium from [7-³H]N-acetyldopamine and not from either [8-³H] or [ring-³H]N-acetyldopamine during the initial phase of oxidation. The above results are consistent with the generation and subsequent reactions of quinone methides as the initial products of enzyme-catalyzed N-acetyldopamine oxidation and confirm our hypothesis that quinone methides and not 1,2-dehydro-N-acetyldopamine are the reactive intermediate of β-sclerotization of sarcophagid cuticle. Quinone methide sclerotization resolves a number of conflicting observations made by previous workers in this field.     

Biosynthesis of dehydro-N-acetyldopamine by a soluble enzyme preparation from the larval cuticle of Sarcophaga bullata involves intermediary formation of N-acetyldopamine quinone and N-acetyldopamine quinone methide

The enzymes involved in the side chain hydroxylation and side chain desaturation of the sclerotizing precursor N-acetyldopamine (NADA) were obtained in the soluble form from the larval cuticle of Sarcophaga bullata and the mechanism of the reaction was investigated. Phenylthiourea, a well-known inhibitor of phenoloxidases, drastically inhibited both the reactions, indicating the requirement of a phenoloxidase component. N-acetylcysteine, a powerful quinone trap, trapped the transiently formed NADA quinone and prevented the production of both N-acetylnorepinephrine and dehydro NADA. Exogenously added NADA quinone was readily converted by these enzyme preparations to N-acetylnorepinephrine and dehydro NADA. 4-Alkyl-o-quinone:2-hydroxy-p-quinone methide isomerase obtained from the cuticular preparations converted chemically synthesized NADA quinone to its quinone methide. The quinone methide formed reacted rapidly and nonenzymatically with water to form N-acetylnorepinephrine as the stable product. Similarly 4-(2-hydroxyethyl)-o-benzoquinone was converted to 3,4-dihydroxyphenyl glycol. When the NADA quinone-quinone isomerase reaction was performed in buffer containing 10% methanol, beta-methoxy NADA was obtained as an additional product. Furthermore, the quinones of N-acetylnorepinephrine and 3,4-dihydroxyphenyl glycol were converted to N-acetylarterenone and 2-hydroxy-3',4'-dihydroxyacetophenone, respectively, by the enzyme. Comparison of nonenzymatic versus enzymatic transformation of NADA to N-acetylnorepinephrine revealed that the enzymatic reaction is at least 100 times faster than the nonenzymatic rate. Resolution of the NADA desaturase system on Benzamidine Sepharose and Sephacryl S-200 columns yielded the above-mentioned quinone isomerase and NADA quinone methide:dehydro NADA isomerase. The latter, on reconstitution with mushroom tyrosinase and hemolymph quinone isomerase, catalyzed the biosynthesis of dehydro NADA from NADA with the intermediary formation of NADA quinone and NADA quinone methide. The results are interpreted in terms of the quinone methide model elaborated by our group [Sugumaran: Adv. Insect Physiol. 21:179-231, 1988; Sugumaran et al.: Arch. Insect Biochem. Physiol. 11:109, 1989] and it is concluded that the two enzyme beta-sclerotization model [Andersen: Insect Biochem. 19:59-67, 375-382, 1989] is inadequate to account for various observations made on insect cuticle.     

Further studies on the mechanism of oxidation of N-acetyldopamine by the cuticular enzymes from Sarcophaga bullata and other insects

In accordance with our earlier results, quinone methide formation was confirmed to be the major pathway for the oxidation of N-acetyldopamine (NADA) by cuticle-bound enzymes from Sarcophaga bullata larvae. In addition, with the use of a newly developed HPLC separation condition and cuticle prepared by gentle procedures, it could be demonstrated that 1, 2-dehydro-NADA and its dimeric oxidation products are also generated in the reaction mixture containing a high concentration of NADA albeit at a much lower amount than the NADA quinone methide water adduct, viz., N-acetylnorepinephrine (NANE). By using different buffers, it was also possible to establish the accumulation of NADA quinone in reaction mixtures containing NADA and cuticle. That the 1,2-dehydro-NADA formation is due to the action of a NADA desaturase system was established by pH and temperature studies and by differential inhibition of NANE production. Of the various cuticle examined, adult cuticle of Locusta migratoria, presclerotized cuticle of Periplaneta americana, and white puparial cases of Drosophila melanogaster exhibited more NADA desaturase activity than NANE generating activity, while the reverse was observed with the larval cuticle of Tenebrio molitor and pharate pupal cuticle of Manduca sexta. These studies indicate that both NADA quinone methide and 1, 2-dehydro NADA are formed during enzymatic activation of NADA in insect cuticle. Based on these results, a unified mechanism for β-sclerotization involving quinone methides as the reactive species is presented.     

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.     

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.     

Differential mechanism of oxidation of N-acetyldopamine and N-acetylnorepinephrine by cuticular phenoloxidase from Sarcophaga bullata

The mechanism of oxidation of two related sclerotizing precursors—N-acetyldopamine and N-acetylnorepinephrine—by the cuticular phenoloxidase from Sarcophaga bullata was studied and compared with mushroom tyrosinase-mediated oxidation. While the fungal enzyme readily generated the quinone products from both of these catecholamine derivatives, sarcophagid enzyme converted N-acetyldopamine to a quinone methide derivative, which was subsequently bound to the cuticle with the regeneration of o-dihydroxy phenolic function as outlined in an earlier publication [Sugumaran: Arch Insect Biochem Physiol, 8, 73 (1988)]. However, it converted N-acetylnorepinephrine to its quinone and not to the quinone methide derivative. Proteolytic digests of N-acetyldopamine-treated cuticle liberated peptides that had covalently bound catechols, while N-acetylnorepinephrine-treated cuticle did not release such peptides. Acid hydrolysis of N-acetyldopamine-treated cuticle, but not N-acetylnorepinephrine-treated cuticle liberated 2-hydroxy-3′,4′-dihydroxyacetophenone and arterenone. These results further confirm the unique conversion of N-acetyldopamine to its corresponding quinone methide derivative and N-acetylnorepinephrine to its quinone derivative by the cuticular phen-oloxidase. Significance of this differential mechanism of oxidation for sclerotization of insect cuticle is discussed.     

Quinone and quinone methide as transient intermediates involved in the side chain hydroxylation of N-acyldopamine derivatives by soluble enzymes from Manduca sexta cuticle.

Proteins solubilized from the pharate cuticle of Manduca sexta were fractionated by ammonium sulfate precipitation and activated by the endogenous enzymes. The activated fraction readily converted exogenously supplied N-acetyldopamine (NADA) to N-acetylnorepinephrine (NANE). Either heat treatment (70 degrees C for 10 min) or addition of phenylthiourea (2.5 microM) caused total inhibition of the side chain hydroxylation. If chemically prepared NADA quinone was supplied instead of NADA to the enzyme solution containing phenylthiourea, it was converted to NANE. Presence of a quinone trap such as N-acetylcysteine in the NADA-cuticular enzyme reaction not only prevented the accumulation of NADA quinone, but also abolished NANE production. In such reaction mixtures, the formation of a new compound characterized as NADA-quinone-N-acetylcysteine adduct could be readily witnessed. These studies indicate that NADA quinone is an intermediate during the side chain hydroxylation of NADA by Manduca cuticular enzyme(s). Since such a conversion calls for the isomerization of NADA quinone to NADA quinone methide and subsequent hydration of NADA quinone methide, attempts were also made to trap the latter compound by performing the enzymatic reaction in methanol. These attempts resulted in the isolation of beta-methoxy NADA (NADA quinone methide methanol adduct) as an additional product. Similarly, when the N-beta-alanyldopamine (NBAD)-Manduca enzyme reaction was carried out in the presence of L-kynurenine, two diastereoisomers of NBAD quinone methide-kynurenine adduct (= papiliochrome IIa and IIb) could be isolated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quinone methide sclerotization: A revised mechanism for β-sclerotization of insect cuticle

Molecular mechanisms responsible for the stiffening and tanning of insect cuticle are reviewed. Two mechanisms, viz., quinone tanning and β-sclerotization, both involving catecholamine derivatives as sclerotizing precursors, are known to strengthen the cuticle. Quinone tanning mechanism invokes the generation and reactions of o-benzoquinones as the sclerotizing agents, whereas β-sclerotization dictates the activation of catecholamine side chains prior to their incorporation into cuticle. The reactive intermediate for the latter process was proposed by other workers to be 1,2-dehydro-N-acetyldopamine and its quinone. The role of these two compounds in β-sclerotization is critically evaluated. Based on our observation that incubation of cuticular enzyme from Sarcophaga bullata with 4-alkylcatechols results in the production of soluble side chain oxygenated compounds and the formation of catechol-cuticle adducts, an alternate mechanism for β-sclerotization is proposed. This mechanism calls for the generation of quinone methides, tautomers of 4-alkyl-quinones, as the initial products of oxidation of catecholamine derivatives in cuticle. Quinone methides formed spontaneously react with available nucleophiles in cuticle, resulting in the generation of catechol-cuticle adducts and side chain hydroxylated products. Further oxidation of adducts and coupling to other structural units ensure crosslinking of cuticular components. The proposed quinone methide sclerotization accounts for all the chemical observations made on the β-sclerotized cuticle.     

Studies on the enzymes involved in puparial cuticle sclerotization in Drosophila melanogaster.

The properties of cuticular enzymes involved in sclerotization of Drosophila melanogaster puparium were examined. The cuticle-bound phenoloxidase from the white puparium exhibited a pH optimum of 6.5 in phosphate buffer and oxidized a variety of catecholic substrates such as 4-methylcatechol, N-beta-alanyldopamine, dopa, dopamine, N-acetyldopamine, catechol, norepinephrine, 3,4-dihydroxyphenylglycol, 3,4-dihydroxybenzoic acid, and 3,4-dihydroxyphenylacetic acid. Phenoloxidase inhibitors such as potassium cyanide and sodium fluoride inhibited the enzyme activity drastically, but phenylthiourea showed marginal inhibition only. This result, coupled with the fact that syringaldazine served as the substrate for the insoluble enzyme, confirmed that cuticular phenoloxidase is of the "laccase" type. In addition, we also examined the mode of synthesis of the sclerotizing precursor, 1,2-dehydro-N-acetyldopamine. Our results indicate that this catecholamine derivative is biosynthesized from N-acetyldopamine through the intermediate formation of N-acetyldopamine quinone and N-acetyldopamine quinone methide as established for Sarcophaga bullata [Saul, S. and Sugumaran, M., F.E.B.S. Letters 251, 69-73 (1989)]. Accordingly, successful solubilization and fractionation of cuticular enzymes involved in the introduction of a double bond in the side chain of N-acetyldopamine indicated that they included o-diphenoloxidase, 4-alkyl-o-quinone:p-quinone methide isomerase, and N-acetyldopamine quinone methide:dehydro N-acetyldopamine isomerase and not any side chain desaturase.     

Characterization of quinone tautomerase activity in the hemolymph of Sarcophaga bullata larvae

The hemolymph of Sarcophaga bullata larvae was activated with either zymosan or proteolytic enzymes such as chymotrypsin or subtilisin and assayed for phenoloxidase activity by two different assays. While oxygen uptake studies readily attested to the wide specificty of activated phenoloxidase, visible spectral studies failed to confirm the accumulation of quinone products in the case of 4-alkyl substituted catechols such as N-acetyldopamine and N-β-alanyldopamine. Sepharose 6B column chromatography of the activated hemolymph resolved phenoloxidase activity into two fractions, designated as A and B. Peak A possessed typical o-diphenoloxidase (o-diphenol, oxygen oxidoreductase EC 1.10.3.1) activity, while peak B oxidized physiologically important catecholamine derivatives such as N-acetyldopamine, N-acetylnorepinephrine, and N-β-alanyldopamine into N-acetylnorepinephrine, N-acetylarterenone, and N-β-alanylnorepinephrine, respectively, and converted 3,4-dihydroxyphenylacetic acid, 3,4-dihydroxymandelic acid, and 3,4-dihydroxyphenylglycol into 3,4-dihydroxymandelic acid, 3,4-dihydroxybenzaldehyde, and 2-hydroxy-3′,4′-dihydroxyacetophenone, respectively. These transformations are consistent with the conversion of phenoloxidase-generated quinones to quinone methides and subsequent non-enzymatic transformations of quinone methides. Accordingly, Peak B contained both o-diphenoloxidase activity and quinone tautomerase activity. Sepharose 6B column chromatography of unactivated hemolymph resulted in the separation of quinone tautomerase from prophenoloxidase. The tautomerase rapidly converted both chemically made and mushroom tyrosinase-generated quinones to quinone methides. Thus the failure to observe the accumulation of quinones with N-acyl derivatives of dopamine and related compounds in the whole hemolymph is due to the rapid conversion of these long lived toxic quinones to short lived quinone methides. The latter, being unstable, undergo rapid non-enzymatic transformations to form side-chain-oxygenated products that are non-toxic. The possible roles of quinone isomerase and its reaction products—quinone methides—as essential components of sclerotization of cuticle and defense reaction of Sarcophaga bullata are discussed.     

Oxidation chemistry of 1,2-dehydro-N-acetyldopamines: direct evidence for the formation of 1,2-dehydro-N-acetyldopamine quinone

Two-electron oxidation of catecholamines either by phenol oxidase or by chemical oxidants such as sodium periodate produces their corresponding o-quinones as observable products. But, in the case of 1,2-dehydro-N-acetyldopamine, an important insect cuticular sclerotizing precursor, phenol oxidase catalyzed oxidation has been reported to generate a quinone methide analog as a transient, but first observable product. ?Sugumaran, M., Semensi, V., Kalyanaraman, B., Bruce, J. M., and Land, E. J. (1992) J. Biol. Chem. 267, 10355-10361. The corresponding quinone has escaped detection until now. However, in this paper, for the first time, we present direct evidence for the formation of dehydro-N-acetyldopamine quinone and show that it can readily be produced from the tautomeric quinone methide imine amide during the chemical oxidation of dehydro-N-acetyldopamine under acidic conditions. This situation is in sharp contrast to other known alkyl-substituted catechol oxidations, where quinone is the first observable product and quinone methide is the subsequently generated product. Dehydro-N-acetyldopamine quinone thus formed is also highly unstable. Semiempirical molecular orbital calculation also indicates that quinone methide imine amide is more stable than the quinone. Chemical considerations indicate that the quinone methide tautomer, and not the dehydro-N-acetyldopamine quinone, is responsible for crosslinking the structural proteins and chitin polymer in the insect cuticle. Therefore, the quinone methide tautomer, and not the quinone, is the key reactive intermediate aiding the hardening of insect cuticle.     

Mechanism of activation of 1,2-dehydro-N-acetyldopamine for cuticular sclerotization

The mechanism of oxidation of 1,2-dehydro-N-acetyldopamine (dehydro NADA) was examined to resolve the controversy between our group and Andersen's group regarding the reactive species involved in β-sclerotization. While Andersen has indicated that dehydro NADA quinone is the β-sclerotizing agent [Andersen, 1989], we have proposed quinone methides as the reactive species for this process [Sugumaran, 1987; Sugumaran, 1988]. Since dehydro NADA quinone has not been isolated or identified till to date, we studied the enzymatic oxidation of dehydro NADA in the presence of quinone traps to characterize this intermediate. Accordingly, both N-acetylcysteine and o-phenylenediamine readily trapped the transiently formed dehydro NADA quinone as quinone adducts. Interestingly, when the enzymatic oxidation was performed in the presence of o-aminophenol or different catechols, adduct formation between the dehydro NADA side chain and the additives had occurred. The structure of the adducts is in conformity with the generation and reactions of dehydro NADA quinone methide (or its radical). This, coupled with the fact that 4-hydroxyl or amino-substituted quinones instantly transformed into p-quinonoid structure, indicates that dehydro NADA quinone is only a transient intermediate and that it is the dehydro NADA quinone methide that is the thermodynamically stable product. However, since this compound is chemically more reactive due to the presence of both quinone methide and acylimine structure on it, the two side chain carbon atoms are “activated.” Based on these considerations, it is suggested that the quinone methide derived from dehydro NADA is the reactive species responsible for cross-link formation between dehydro NADA and cuticular components during β-sclerotization.     

Conjugation of a hairpin pyrrole-imidazole polyamide to a quinone methide for control of DNA cross-linking

A series of quinone methide precursors designed for DNA cross-linking were prepared and conjugated to a pyrrole-imidazole polyamide for selective association to the minor groove. Although reaction was only observed for DNA containing the predicted recognition sequence, yields of strand alkylation were low. Interstrand cross-linking was more efficient than alkylation but still quite modest and equivalent to that generated by a comparable conjugate containing the N-mustard chlorambucil. Varying the length of the linker connecting the polyamide and quinone methide derivative did not greatly affect the yield of DNA cross-linking. Instead, intramolecular trapping of the quinone methide intermediate by nucleophiles of the attached polyamide appears to be the major determinant that limits its reaction with DNA. Self-adducts of the quinone methide conjugate form readily and irreversibly as detected by a combination of chromatography and mass spectroscopy. This result is unlike comparable self-adducts observed for oligonucleotide conjugates that form more slowly and remain reversible. Equivalent intramolecular alkylation of a polyamide by its attached chlorambucil mustard was not observed under similar condition. The presence of DNA, however, did facilitate hydrolysis of this mustard conjugate.     

A new mechanism for the control of phenoloxidase activity: inhibition and complex formation with quinone isomerase

Insect phenoloxidases participate in three physiologically important processes, viz., cuticular hardening (sclerotization), defense reactions (immune reaction), and wound healing. Arrest or even delay of any of these processes compromises the survival of insects. Since the products of phenoloxidase action, viz., quinones, are cytotoxic, uncontrolled phenoloxidase action is deleterious to the insects. Therefore, the activity of this important enzyme has to be finely controlled. A novel inhibition of insect phenoloxidases, which serves as a new regulatory mechanism for control of its activity, is described. The activity of phenoloxidases isolated from both Sarcophaga bullata and Manduca sexta is drastically inhibited by quinone isomerase (isolated from Calliphora), an enzyme that utilizes the phenoloxidase-generated 4-alkylquinones. In turn, phenoloxidase reciprocated the inhibition of isomerase. By forming a complex and controlling each other's activity, these two enzymes seem to regulate the levels of endogenously quinones. In support of this contention, an endogenous complex consisting of phenoloxidase, quinone isomerase, and quinone methide isomerase was characterized from the insect, Calliphora. This sclerotinogenic complex was isolated and purified by borate extraction of the larval cuticle, ammonium sulfate precipitation, and Sepharose 6B column chromatography. The complex exhibited a molecular mass of about 620-680 kDa, as judged by size-exclusion chromatography on Sepharose 6B and HPLC and did not even enter 3% polyacrylamide gel during electrophoresis. The phenoloxidase activity of the complex exhibited a wide substrate specificity. Incubation of the complex with N-acetyldopamine rapidly generated N-acetylnorepinephrine, dehydro-N-acetyldopamine, and its dimers. In addition, transient accumulation of N-acetyldopamine quinone was also observed. These results confirm the presence of phenoloxidase, quinone isomerase, and quinone methide isomerase in the complex. Attempts to dissociate the complex with even trace amounts of SDS ended in the total loss of quinone isomerase activity. The complex does not seems to be made up of stoichiometric amounts of individual enzymes as the ratio of phenoloxidase to quinone isomerase varied from preparation to preparation. It is proposed that the complex formation between sequential enzymes of sclerotinogenic pathway is advantageous for the organism to effectively channel various reactive intermediates during cuticular hardening.     

Characterization of a new enzyme system that desaturates the side chain of N-acetyldopamine

A novel enzyme system that desaturates the side chain of the catecholamine derivative, N-acetyldopamine (NADA), was isolated and characterized from the larval cuticle of Sarcophaga bullata. The NADA desaturase system which converts NADA to 1,2-dehydro-NADA, surprisingly, does not resemble dehydrogenases such as succinate dehydrogenase. It uniquely performs the desaturation reaction by oxidizing NADA to its corresponding quinone and subsequently converting the resultant quinone to 1,2-dehydro-NADA via NADA quinone methide. Accordingly, desaturase enzyme preparation contained both o-diphenoloxidase activity and NADA quinone:NADA quinone methide isomerase activity. In addition, inhibition studies as well as trapping experiments also confirmed the obligatory formation of NADA quinone as the transient intermediate of the NADA desaturation. It is the first report of a cell-free system causing the side chain desaturation of any catecholamine derivative.     

o-quinone/quinone methide isomerase: a novel enzyme preventing the destruction of self-matter by phenoloxidase-generated quinones during immune response in insects

Melanization and encapsulation of invading foreign organisms observed during the immune response in insects is known to be due to the action of activated phenoloxidase. Phenoloxidase-generated quinones are deposited either directly or after self-polymerization on foreign objects accounting for the observed reactions. Since the reactions of quinones are nonenzymatic, they do not discriminate self from nonself and hence will also destroy self-matter. In this report we present evidence for the presence of a novel quinone/quinone methide isomerase in the hemolymph of Sarcophaga bullata which destroys long-lived quinones and hence acts to protect the self-matter. Quinone methides, formed by the action of this enzyme on physiologically important quinones, being unstable undergo rapid hydration to form nontoxic metabolites.