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M Sugumaran

Publications and source records attributed to M Sugumaran.

At least 37 records · Page 2Linked to original sources
PubMed1995

A caution about the azide inhibition of enzymes associated with electrophilic metabolites.

Azide is often used as an inhibitor to detect active site metal ions present in enzymes such as tyrosinases and laccases. Azide is not only a good chelator for metal ions, but also a powerful nucleophile. Consequently, some of the observed inhibition of tyrosinase by azide can be explained by the reaction of enzymatically generated quinones with azide to form azido catechol. In the light of this finding, routine use of inhibition studies with azide to detect the metal ions present at the active site of enzymes generating and/or consuming electrophilic metabolites is discouraged.

Azides↗
PubMed1995

Model sclerotization studies. 3. Cuticular enzyme catalyzed oxidation of peptidyl model tyrosine and dopa derivatives.

Incubation of N-acetyltyrosine methyl ester with cuticular enzymes, isolated from the wandering stages of Calliphora sp larvae, resulted in the generation of N-acetyldopa methyl ester when the reaction was carried out in the presence of ascorbate which prevented further oxidation of the o-diphenolic product. Enzymatic oxidation of N-acetyldopa methyl ester ultimately generated dehydro N-acetyldopa methyl ester. The identity of enzymatically produced N-acetyldopa methyl ester and dehydro N-acetyldopa methyl ester has been confirmed by comparison of the ultraviolet and infrared spectral and chromatographic properties with those of authentic samples as well as by nuclear magnetic resonance studies. Since N-acetyldopaquinone methyl ester was also converted to dehydro N-acetyldopa methyl ester and tyrosinase was responsible for the oxidation of N-acetyldopa methyl ester, a scheme for the cuticular phenoloxidase catalyzed conversion of N-acetyltyrosine methyl ester to dehydro N-acetyldopa methyl ester involving the intermediary formation of the quinone and the quinone methide is proposed to account for the observed results. The conversion of N-acetyldopa methyl ester to dehydro derivative remarkably resembles the conversion of the sclerotizing precursor, N-acetyldopamine, to dehydro-N-acetyldopamine observed in the insect cuticle. Based on these comparative studies, it is proposed that peptidyl dopa derivatives could also serve as the sclerotizing precursors for the sclerotization of the insect cuticle.

Animals↗
PubMed1995

Complex formation between mushroom tyrosinase and Manduca dopachrome isomerase.

Melanin biosynthesis in animals is initiated by the ubiquitously present tyrosinase and is aided by dopachrome isomerase. We have characterized a novel dopachrome isomerase (decarboxylating) from the hemolymph of Manduca sexta that generates a new quinone methide intermediate during melanogenesis (Sugumaran, M. and Semensi, V. (1991) J. Biol. Chem. 266, 6073-6078). This enzyme has the ability to form a complex with mushroom tyrosinase as judged by a number of physicochemical studies. The isomerase exhibited a marked inhibitory effect on tyrosinase and tyrosinase reciprocated by inhibiting the isomerase. While the isomerase showed no activity toward preformed dopaminechrome, it readily influenced the stability of dopaminechrome generated in situ by tyrosinase. Moreover, mushroom tyrosinase, which lacked specific binding to Concanavalin A Sepharose column, after complexing with the isomerase exhibited binding to this column. The complex formation also affected the pI value as well as mobility on a size exclusion column of these enzymes. Enzymes executing sequential metabolic transformation are known to form complexes called metabolons. Based on these above studies, it is concluded that both the enzymes involved in insect melanogenic pathway--phenoloxidase and dopachrome isomerase--are able to form a metabolon complex.

Animals↗
PubMed1995

Oxidation of 3,4-dihydroxybenzylamine affords 3,4-dihydroxybenzaldehyde via the quinone methide intermediate.

Dopamine and related compounds are known to be toxic to melanoma cells. Some of their toxicity may be related, in part, to the oxidation products generated from them upon their interaction with melanogenic enzymes. In this paper, we present our studies on the oxidation chemistry of 3,4-dihydroxybenzylamine, the lower homolog of dopamine. Mushroom tyrosinase catalyzed oxidation of 3,4-dihydroxybenzylamine rapidly generated the corresponding quinone. However, aminomethyl-o-benzoquinone thus formed did not accumulate in the reaction mixture, but readily transformed to another product that exhibited absorbance maxima at 280 and 310 nm. This compound was identified to be 3,4-dihydroxybenzaldehyde based on its HPLC elution profile, cochromatography with authentic sample and UV spectral properties. Possible mechanism for the formation 3,4-dihydroxybenzaldehyde from 3,4-dihydroxybenzylamine and the nature of cytotoxic quninonoid intermediates formed are discussed.

Basidiomycota↗
PubMed1994

1,2-dehydro-N-beta-alanyldopamine as a new intermediate in insect cuticular sclerotization.

Sclerotization of insect cuticle is an extremely important biochemical process for the successful survival of most insects. N-Acetyldopamine and N-beta-alanyldopamine are two widely used sclerotizing precursors. N-Acetyldopamine is converted by phenoloxidase, quinone isomerase, and quinone methide isomerase to generate the reactive intermediates quinone, quinone methide, and quinone methide imine amide for use in quinone tanning, quinone methide sclerotization, and alpha,beta-sclerotization, respectively. N-beta-Alanyldopamine has been claimed to be used only by phenoloxidase and quinone isomerase for quinone tanning and quinone methide sclerotization thereby attributing biochemical diversity to the coloration and strength of different cuticles. However, we demonstrate here that cuticular enzymes isolated from the larvae of Calliphora possess the capacity to generate 1,2-dehydro-N-beta-alanyldopamine from N-beta-alanyldopamine. Chemical synthesis of 1,2-dehydro-N-beta-alanyldopamine and its further oxidation are reported for the first time. Comparative biochemical studies confirm that both the sclerotizing precursors, N-acetyldopamine and N-beta-alanyldopamine, are used by all three different mechanisms of the unified theory of sclerotization.

Animals↗
PubMed1994

The ontogeny of dopachrome isomerase isozyme patterns in the tobacco hornworm Manduca sexta.

Melanogenesis in animals is initiated by tyrosinase and augmented by recently discovered dopachrome isomerase. Mushroom tyrosinase embedded 5% polyacrylamide gels are used to specifically stain for dopachrome isomerase activity after electrophoretic separation. Using this new procedure, the ontogenic changes in dopachrome isomerase are demonstrated for the first time in any organism. While there is no isozyme variation within the same stage of development, there are profound variations in isozyme patterns during the developmental stages from egg to adult, indicating differential expression and/or processing of dopachrome isomerase genes to match the metabolic needs at these stages.

Animals↗
PubMed1994

Detection of dopachrome isomerase activity on gels.

Dopachrome isomerase is a recently discovered enzyme associated with the melanogenesis process occurring in most vertebrates and invertebrates. It catalyzes the conversion of dopachrome to 5,6-dihydroxyindole(s). Based on the fact that 5,6-dihydroxyindoles are rapidly oxidized to melanochrome pigment by tyrosinases, we have developed a rapid, sensitive, and specific staining procedure for detection of dopachrome isomerase activity after gel electrophoresis. The method employs the use of commercially available mushroom tyrosinase entrapped in polyacrylamide gels for electrophoretic separation of dopachrome isomerase. Staining is achieved by the use of dopa solution. The dopachrome formed by the action of mushroom tyrosinase entrapped in the gel is converted to 5,6-dihydroxyindole(s) by dopachrome isomerase initially. The latter compound is subsequently oxidized by tyrosinase to purple-colored melanochrome. Therefore, dopachrome isomerase appears as a bluish-purple band against a pale orange-red background within 10 min. With the use of the new detection technique, the presence of hitherto unknown isozymes of dopachrome isomerase could be readily detected in polyacrylamide gels. This procedure is more sensitive than silver staining for detection of dopachrome isomerase and as little as 15 ng of purified protein could be easily detected on gels.

Animals↗
PubMed1993

Biological and toxicological consequences of quinone methide formation.

Quinone methides are a class of reactive, electrophilic compounds which are capable of alkylating cellular macromolecules. They are formed during xenobiotic biotransformation reactions and are hypothesized to mediate the toxicity of a large number of quinone antitumor drugs as well as several alkylphenols. In addition, oxidation of specific endogenous alkylphenols (e.g. coniferyl alcohol) and alkylcatechols (e.g. N-acetyldopamine, dopa) to quinone methides plays an important role in the synthesis of several complex plant and animal polymers, including lignin, cuticle and melanin. The role of quinone methides in these various processes is reviewed.

Animals↗
PubMed1992

Evidence for the formation of a quinone methide during the oxidation of the insect cuticular sclerotizing precursor 1,2-dehydro-N-acetyldopamine.

1,2-Dehydro-N-acetyldopamine (dehydro-NADA) is an important catecholamine derivative involved in the cross-linking of insect cuticular components during sclerotization. Since sclerotization is a vital process for the survival of insects, and is closely related to melanogenesis, it is of interest to unravel the chemical mechanisms participating in this process. The present paper reports on the mechanism by which dehydro-NADA is oxidatively activated to form reactive intermediate(s) as revealed by pulse radiolysis, electron spin resonance spectroscopy, high performance liquid chromatography, and ultraviolet-visible spectroscopic analysis. Pulse radiolytic one-electron oxidation of dehydro-NADA by N3. (k = 5.3 x 10(9) M-1 s-1) or Br2.- (k = 7.5 x 10(8) M-1 s-1) at pH6 resulted in the rapid generation of the corresponding semiquinone radical, lambda max 400 nm, epsilon = 20,700 M-1 cm-1. This semiquinone decayed to form a second transient intermediate, lambda max 485 nm, epsilon = 8000 M-1 cm-1, via a second order disproportionation process, k = 6.2 x 10(8) M-1 s-1. At pH 6 in the presence of azide, the first order decay of this second intermediate occurred over milliseconds; the rate decreases at higher pH. At pH 6 in the presence of bromide, the intermediate decayed much more slowly over seconds, k = 0.15 s-1. Under such conditions, the dependence of the first order decay constant upon parent dehydro-NADA concentration led to a second order rate constant of 8.5 x 10(2) M-1 s-1 for reaction of the intermediate with the parent, probably to form benzodioxan "dimers." (The term dimer is used for convenience; the products are strictly bisdehydrodimers of dehydro-NADA (see "Discussion" and Fig. 11)) Rate constants of 5.9 x 10(5), 4.5 x 10(5), 2.8 x 10(4) and 3.5 x 10(4) M-1 s-1 were also obtained for decay of the second intermediate in the presence of cysteine, cysteamine, o-phenylenediamine, and p-aminophenol, respectively. By comparison with the UV-visible spectroscopic properties of the two-electron oxidized species derived from dehydro-NADA and from 1,2-dehydro-N-acetyldopa methyl ester, it is concluded that the transient intermediate exhibiting absorbance at 485 nm is the quinone methide tautomer of the o-quinone of dehydro-NADA. Sclerotization of insect cuticle is discussed in the light of these findings.

Animals↗
PubMed1992

Mechanistic studies on tyrosinase-catalysed oxidative decarboxylation of 3,4-dihydroxymandelic acid.

Mushroom tyrosinase, which is known to convert a variety of o-diphenols into o-benzoquinones, has been shown to catalyse an unusual oxidative decarboxylation of 3,4-dihydroxymandelic acid to 3,4-dihydroxybenzaldehyde [Sugumaran (1986) Biochemistry 25, 4489-4492]. The mechanism of this reaction was re-investigated. Although visible-region spectral studies of the reaction mixture containing 3,4-dihydroxymandelic acid and tyrosinase failed to generate the spectrum of a quinone product during the steady state of the reaction, both trapping experiments and non-steady-state kinetic experiments provided evidence for the transient formation of unstable 3,4-mandeloquinone in the reaction mixture. The visible-region spectrum of mandeloquinone resembled related quinones and exhibited an absorbance maximum at 394 nm. Since attempts to trap the second intermediate, namely alpha,2-dihydroxy-p-quinone methide, were in vain, mechanistic studies were undertaken to provide evidence for its participation. The decarboxylative quinone methide formation from 3,4-mandeloquinone dictates the retention of a proton on the alpha-carbon atom. Hence, if we replace this proton with deuterium, the resultant 3,4-dihydroxybenzaldehyde should retain the deuterium present in the original substrate. To test this hypothesis, we chemoenzymically synthesized alpha-deuterated 3,4-dihydroxymandelic acid and examined its enzymic oxidation. Our studies reveal that the resultant 3,4-dihydroxybenzaldehyde retained nearly 90% of the deuterium, strongly indicating the transient formation of quinone methide. On the basis of these findings it is concluded that the enzymic oxidation of 3,4-dihydroxymandelic acid generates the conventional quinone product, which, owing to its unstability, is rapidly decarboxylated to generate transient alpha,2-dihydroxy-p-quinone methide. The coupled dienone-phenol re-arrangement and keto-enol tautomerism of this quinone methide produce the observed 3,4-dihydroxybenzaldehyde.

Basidiomycota↗
PubMed1992

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.

Animals↗
PubMed1991

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.

Animals↗
PubMed1991

The mechanism of tyrosinase-catalysed oxidative decarboxylation of alpha-(3,4-dihydroxyphenyl)-lactic acid.

Mushroom tyrosinase, which is known to catalyse the conversion of o-diphenols into o-benzoquinones, has been shown to catalyse the oxidative decarboxylation of 3,4-dihydroxymandelic acid [Sugumaran (1986) Biochemistry 25, 4489-4492]. To account for this unusual reaction, a quinone methide intermediate has been proposed. Since all attempts to trap this intermediate ended in vain, mechanistic studies were designed to support the formation of this transient product. Replacement of the alpha-proton in 3,4-dihydroxymandelic acid with a methyl group generates alpha-(3,4-dihydroxyphenyl)-lactic acid, the enzymic oxidation of which should produce 3,4-dihydroxyacetophenone as the end product if the oxidative decarboxylation proceeds through the quinone methide intermediate. Accordingly, chemically synthesized alpha-(3,4-dihydroxyphenyl)-lactic acid on enzymic oxidation produced 3,4-dihydroxyacetophenone as the major isolatable product. Non-steady-state kinetic analysis of the enzyme reaction attested to the transient formation of the conventional quinone product. Thus the enzymic oxidation of alpha-(3,4-dihydroxyphenyl)-lactic acid seems to generate the conventional quinone, which, owing to its instability, is rapidly decarboxylated to yield the transient quinone methide. The coupled dieneonephenol re-arrangement and ketol-enol tautomerism transforms the quinone methide into 3,4-dihydroxyacetophenone.

Basidiomycota↗
PubMed1991

Lysolecithin--a potent activator of prophenoloxidase from the hemolymph of the lobster, Homarus americanas.

The phenoloxidase system, which is involved in encapsulation and melanization of foreign objects in crustacean, is found to be present in an inactive proenzyme form in the hemocytes of the lobster, Homarus americanas. Activation of the enzyme could be achieved either by treatment with an anionic detergent such as sodium dodecyl sulfate, or by a cationic detergent such as cetylpyridinium chloride, but not by either nonionic detergent or zwitterionic detergent. In addition, a number of fatty acids also activated the proenzyme. However, phospholipids, especially lysolecithin proved to be the most potent activator of prophenoloxidase. Therefore, it is proposed that apart from the well established proteolytic mode of activation, prophenoloxidase can also be activated by this alternative mode involving lipids.

Animals↗
PubMed1991

Quinone methide as a new intermediate in eumelanin biosynthesis.

The conversion of dopachrome to dihydroxyindole(s), a key reaction in eumelanin biosynthetic pathway, has been shown to be under the control of dopachrome conversion factor. Dopachrome conversion factor isolated from the hemolymph of Manduca sexta larvae, which is devoid of any tyrosinase activity, exhibits a narrow substrate specificity and readily bleaches the iminochromes derived from the oxidation of L-dopa, L-dopa methyl ester, and alpha-methyl-L-dopa, but failed to attack the corresponding D-isomers. The product formed in the case of L-dopachrome was identified to be 5,6-dihydroxyindole. Therefore, aromatization of dopachrome seems to accompany its decarboxylation as well. However, the enzyme also converts L-dopachrome methyl ester to an indole derivative indicating that it can deprotonate the alpha-hydrogen when the carboxyl group is blocked. These results are accounted for by the transient formation and further transformation of a reactive quinone methide intermediate during the dopachrome conversion factor-catalyzed reaction. The fact that the enzyme-catalyzed conversion of alpha-methyl dopachrome methyl ester (where both decarboxylation and deprotonation are blocked) resulted in the generation of a stable quinone methide in the reaction mixture confirms this contention and supports our recent proposal that quinone methide and not indolenine is the key transient intermediate in the conversion of dopachrome to dihydroxyindole observed during melanogenesis.

Animals↗
PubMed1991

Quinone methide as a reactive intermediate formed during the biosynthesis of papiliochrome II, a yellow wing pigment of papilionid butterflies.

Mushroom tyrosinase and the recently identified, 4-alkyl-o-benzoquinone: 2-hydroxy-p-quinone methide isomerase were used to investigate the mechanism of biosynthesis of papiliochrome II pigment found in the yellow scales of the papilionid butterflies. Incubation of N-beta-alanyldopamine (NBAD) and L-kynurenine with mushroom tyrosinase resulted in the formation of adducts tentatively characterized as NBAD quinone-L-kynurenine adducts. If quinone isomerase was included in this reaction mixture, the formation of two new products could be witnessed. These two products exhibited the same retention time and the same UV and visible spectral properties as those of papiliochrome II diastereoisomers. Since quinone isomerase catalyzes the conversion of quinones to quinone methides, the above studies indicate that papiliochrome II biosynthesis involves non-enzymatic and hence non-stereoselective condensation of enzymatically generated NBAD quinone methide with L-kynurenine.

Animals↗
PubMed1991

Oxidation of 3,4-dihydroxybenzyl alcohol: a sclerotizing precursor for cockroach ootheca.

The oxidation of 3,4-dihydroxybenzyl alcohol, one of the sclerotizing precursors for the tanning of the ootheca of cockroach Periplaneta americana, is reported for the first time. Mushroom tyrosinase catalyzed oxidation of 3,4-dihydroxybenzyl alcohol generated the corresponding quinone which was found to be unstable and readily transformed to produce 3,4-dihydroxybenzaldehyde as the stable product probably through the intermediary formation of a quinone methide. Phenoloxidase isolated from the left collateral gland of P. americana also catalyzed this new reaction. When the enzymatic oxidation of 3,4-dihydroxybenzyl alcohol was performed in the presence of a test protein such as lysozyme, the reactive species formed, caused the oligomerization of test protein. Similar studies with collateral gland proteins, failed to generate oligomers, but produced insoluble polymeric proteins. The probable fate of 3,4-dihydroxybenzyl alcohol for the tanning of cockroach ootheca is discussed.

Animals↗