Model sclerotization studies. 3. Cuticular enzyme catalyzed oxidation of peptidyl model tyrosine and dopa derivativesкод для вставкиСкачать
Archives of Insect Biochemistry and Physiology 28:17-32 (1 995) Model Sclerotization Studies. 3. Cuticular Enzyme Catalyzed Oxidation of Peptidyl Model Tyrosine and Dopa Derivatives M a n i c k a m Sugumaran and D e a n Ricketts Deparfmriifof Biology, Unizwsity of Massackiisetfs nt Boston, Harbor Cnmpus, Boston, Massncliiisetts 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-acetyldoparnine 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. o 1995 Witey-Loss. inr. Key words: Calliphora, tyrosine hydroxylation, dopa oxidation, dehydrodopa, quinone reactivity Acknowledgments: We thank Dr. Kaliappan Nellaiappan for his help in preparation of the enzyme and Dr. Han Li Sun for providing some of the chemicals used in the study. Funds for this project were provided by U . Mass/Boston and National Institutes of Health (grant R 0 1 -Al-l4753). Received January 13, 1994; accepted May 6, 1994. Address reprint requests t o Manickam Sugumaran, Department of Biology, University of Massachusetts at Boston, Harbor Campus, Boston, M A 021 25. Part two of this series i s Sugumaran M, Hennigan B, O’Brien J (1987): Tyrosinase catalyzed protein polymerization as an in vitro model for quinone tanning of insect cuticle. Arch Insect Biochem Physiol 6:9-25. 0 1995 Wiley-Liss, Inc. 18 Sugurnaran and Ricketts INTRODUCTION Sclerotization of insect cuticle, a vital process that protects soft parts of insects, has been extensively studied for over five decades. Studies carried out in this laboratory and others culminated in the presentation of three biochemical mechanisms to account for sclerotization reactions. Pryor's quinone tanning hypothesis calls for the enzymatic generation of quinones and their subsequent interaction with cuticular components to account for the hardening of cuticle (Pryor, 1940a,b). Andersen's p-sclerotization invokes the generation of 1,2-dehydro-N-acetyldopamine from N-acetyldopamine (NADA") by the action of a specific NADA desaturase. The quinone of this compound was proposed to be the reactive intermediate that cross-links cuticular components (Andersen, 1979, 1985; Andersen and Roepstorff, 1982). Quinone methide sclerotization mechanism advocated by this laboratory calls for the liberation of reactive quinone methides and their subsequent reactions in cuticle (Sugumaran, 1987,1988a,b; Sugumaran and Lipke, 1983). Recent studies from this laboratory on the enzymology of cuticular sclerotization have resulted in the discovery of two new enzymes-namely, quinone isomerase and quinone methide isomerase-participating in the hardening process apart from the well-established phenoloxidases (Saul and Sugumaran 1988,1989a,b, 1990a,b). Our findings have eventually led to the unification of the three different mechanisms for sclerotization which is summarized in Figure 1. Taking NADA as the specific case, the unified mechanism can be elaborated as follows: Enzymatic oxidation of NADA by cuticular phenoloxidases (both laccase and o-diphenoloxidase) generates the corresponding o-quinone that participates in quinone tanning. Quinone isomerase converts NADA quinone to NADA quinone methide and provides it for quinone methide sclerotization (Saul and Sugumaran, 1988, 1990a,b). Alternatively, quinone methide is converted to dehydro NADA by quinone methide isomerase (Saul and Sugumaran, 1989b, 1990b). The resultant dehydro NADA upon enzymatic oxidation produces the reactive quinone that is rapidly isomerized to reactive quinone methide imine amide (Sugumaran et al., 1992) which uses both the side chain carbon atoms for cross-linking purposes. The unified mechanism can also use other N-acyldopamine derivatives, such as N-Palanyldopamine (Ricketts and Sugumaran, 1994). Diversity in hardness, color, and flexibility of the cuticle calls for differential use of sclerotization mechanisms and sclerotizing precursors. Thus, NADA seems to be used for making colorless cuticle as in the case of locusts, while N-P-alanyldopamine is believed to make brown cuticle of pupal cases of Manduca sexta (Andersen, 1989).Alternative modes of sclerotization are therefore possible. Nearly 45 years ago, Brown (1950, 1952) proposed the occurrence of autotanning mechanism in marine molluscs. Subsequently Waite and his collaborators confirmed this hypothesis and demonstrated that peptidyl derived dopa units are the sclerotizing precursors in these organisms (for *Abbreviations used: NADA = N-acetyldoparnine; NAcDME = N-acetyldopa methyl ester; NAcTME = N-acetyltyrosine methyl ester. O ~ N H 0 DEHYDRO QUINONE ( V ) C O H R D o ~ r n NCOR! 0' DEHYDRO QUINONE METHIDE (VI) D ( OUINONE METHIDE SCLEROTIZATION p -SCLEROTIZATfON 1 Fig. 1. Unified mechanism for sclerotization. Sclerotizing precursors such as NADA (R = H; R1 = CHI) and N-P-alanyldopamine ( R = H; R1 = CHlCHrNH2) are oxidized by cuticular phenoloxidases (A: both laccases and o-diphenol oxidases) to the corresponding quinones that participate i n quinone tanning. Quinone isomerase (B) converts these quinones to quinone methides (nonenzymatic transformation is also possible and provides them for quinone methide sclerotization. NADA quinone methide has been shown to be specifically converted to dehydro NADA by quinone methide isomerase (C), although other quinone methides are known to form dehydro derivatives nonenzymatically. Dehydro NADA i s further oxidized by phenoloxidases to dehydro quinone which rapidly isomerizes to quinone methide and forms cross-links with both the side chain carbon atoms. D = nonenzymatic reactions. review see Waite, 1990).Oxidative coupling of these protein-bound sclerotizing precursors seems to cause the hardening reaction in molluscs. Recently conducted model oxidation studies to mimic this system with N-acetyldopa ethyl ester and mushroom tyrosinase have revealed the formation of dehydro-Nacetyldopa ethyl ester in the reaction mixture (Rzepecki and Waite, 1991; Rzepecki et al., 1991).N-acetyldopa ethyl ester quinone and quinone methide intermediates as observed in the case of NADA conversion to dehydro NADA have been proposed to account for the observed reaction (see Fig. 1; conversion of catecholamine I to dehydrocatecholamine IV; R = COOC2H5and R, = CH,; tyrosinase initiates the oxidation of catecholamine; the rest of the reactions u p to dehydrocatecholamine are nonenzymatic). In insects, little attention has been paid to the possible occurrence of dopyl proteins and their participation in cuticular hardening. During our studies on the cuticular sclerotization in the sarcophagid system, we have reported the presence of peptides that contain an o-dihydroxyphenolic moiety that seems to be posttranslationally arylated proteins, although the possibility of dopa-containing proteins cannot be ruled out (Lipke et al., 1982). This laboratory has also reported the possible participation of tyrosine-rich proteins in the cross-linking process (Lipke and Henzel, 1981). Arylphorin, a tyrosinerich major serum protein found in numerous insect larvae, is believed to par- 20 Sugumaran and Ricketts ticipate in the tanning process (Peter and Scheller, 1991). Recently, Kramer and his associates have reported the presence of a dopa-containing protein in the cuticle of Manduca sexta (Okot-Kotber et al., 1993). These findings indicate the possible conversion of tyrosine-rich cuticular proteins to dopyl proteins and their participation in the tanning process. In order to see whether cuticular enzymes are capable of converting tyrosine-containing peptide units to dopyl units and then to dehydrodopyl units, we examined the enzymatic oxidation of NAcTME and NAcDME and report in this paper that these model compounds are readily converted to dehydro dopa derivatives by the cuticular enzymes isolated from the last larval instar cuticle of Calliphora. MATERIALS AND METHODS Preparation of Cuticular Enzymes Calliphora larvae were obtained from commercial suppliers (Grub Co., Hamilton, Ohio) and used at wandering stage. The larvae were suspended in 1%sodium borate, pH 8.5, and homogenized in a Waring blender at 30 seconds pulse until their internal contents were extracted in buffer. The transparent cuticle obtained was suspended in 1% sodium borate, p H 8.5, for approximately 18 h in the cold room. The extracted protein was subjected to ammonium sulfate fractionation. The proteins precipitated between 0 and 30% ammonium sulfate saturation were collected by centrifugation at 15,OOOg for 15 min. The pellet obtained was either used immediately or stored at -80°C until use (within 3 4 weeks). The ammonium sulfate pellet was dissolved in a minimum amount of 10 mM sodium phosphate buffer, pH 6.0 and desalted on a Sephadex G 25 column (2.5 x 20 cm) using the same buffer. The protein fraction was used as the enzyme source. Chemicals NAcDME and dehydro NAcDME were kindly donated by Dr. Han Li Sun (Department of Biology, University of Massachusetts at Boston, Boston, MA). NAcTME was synthesized from N-acetyltyrosine, methanol, and HC1 gas (87% yield) and was purified by Bio Gel P-2 column chromatography using 0.2 M acetic acid as the eluant. N-acetyltyrosine, ascorbic acid, mushroom tyrosinase (specific activity 6,300 units/mg protein) were obtained from Sigma Chemical Co (St. Louis, MO). All other reagents used were of analytical grade. HPLC Analysis HPLC analyses of the reaction mixtures were carried out using a Beckman (Berkeley, CA) model 332 liquid chromatography system equipped with two model llOB pumps, a model 420 controller, a model 160 absorbance detector, and a model 427 integrator. Separations were achieved on a Beckman CIB-IP ultrasphere reverse phase column (5 pm, 4.6 x 250 mm) with two different isocratic solvent systems at a flow rate of 0.6 ml/min. Solvent A consisted of 50 mM acetic acid containing 0.2 mM sodium octylsulfonate in 20% methanol, while solvent B had the same ingredients with the exception of 30% methanol. Dopa Derivatives in Sclerotization 21 Other Procedures Estimation of o-dihydroxyphenolic compounds was carried out using Arnow’s reagent (Arnow, 1937; Waite and Tanzer, 1981). Typically, the following reagents were added to 1 ml of the assay mixture in succession: 1 ml of 1 N HC1,l ml of 10% sodium nitrite-10% sodium molybdate reagent, and 1 ml of 2 N NaOH. After the addition of each reagent, the contents were mixed thoroughly. As soon as NaOH was added, absorbance of the assay mixture at 495 nm was monitored against a blank reaction mixture which did not contain the o-diphenol (in the case of enzymatic reaction, zero time reaction was taken as the blank). Ultraviolet and visible spectral studies were carried out using a Milton Roy (Rochester, NY) spectronic 3000 array spectrophotometer. FT-IR spectra were recorded using a Perkin Elmer 1600 series IR spectrophotometer (Oak Brook, IL) in KBr pellets. A Brucker AC 250 MHz NMR spectrometer (Billerica, MA) was used to take the NMR of spectra of isolated products. RESULTS The cuticular enzyme fraction obtained from the wandering stages of Cnlliplzova larvae contained all the three enzyme activities responsible for sclerotization of insect cuticle-namely, phenoloxidase, quinone isomerase and quinone methide isomerase-as reported in the case of the related dipteran larvae, Snrcoplmga bidlafa (Saul and Sugumaran, 1989a,b, 1990b). It readily hydroxylated NAcTME in the presence of a reducing agentsuch as ascorbic acid. Figure 2 shows the time course of hydroxylation of NAcTME. The product formed by the action of cuticular enzymes on NAcTME could be confirmed to be NAcDME in a number of ways. First of all, the enzymatic product responded positively to the nitrite-molybdate reagent of Arnow-a common reaction widely exhibited by o-dihydroxy compounds (Arnow, 1937; Waite and Tanzer, 1981).Secondly, when the reaction mixture was subjected to HPLC analysis (Fig. 3) formation of a new product eluting at 4.3 min could be observed. This product formation increased with time and was dependent upon the presence of both the enzyme and ascorbic acid. The elution time of the product matched with that of an authentic sample of NAcDME. To further confirm the formation of NAcDME from NAcTME, a large-scale reaction was conducted, and the product formed was isolated by preparative chromatography on a Sephadex LH-20 column (Fig. 4). The product (peak B, Fig. 4) exhibited the same elution time as that of NAcDME, and its UV absorbance spectra matched that of the authentic sample in both 0.2 M acetic acid and 3% sodium borate (Fig. 5). That the enzyme responsible for this conversion is phenoloxidase can be confirmed by the specific inhibition of the hydroxylation reaction. Thus phenylthiourea, a potent inhibitor of phenoloxidases at 100 pM level, completely inhibited the hydroxylation (curve C, Fig. 2). From these studies, it was concluded that the primary enzymatic oxidation product arising from the action of cuticular phenoloxidase(s) on NAcTME in the presence of excess ascorbate is indeed NAcDME. This contention was confirmed by comparison of the IR spectrum of both synthetic and isolated products. 22 Sugumaran and Ricketts = A 0) d I L 0 0 7 In 40 80 TIME (mln) Fig. 2. Time course of hydroxylation of NAcTME. A reaction mixture containing 0.1 m M NAcTME, 0.3 m M ascorbic acid, and 0.5 m g of enzyme protein i n 1.0 m l ot 50 m M sodium phosphate buffer, p H (A) 7.0 or (B) 8.0, was incubated at room temperature, and the amount of catecholic compounds formed in the reaction mixture was estimated by Arnow’s reagent, as outlined in Materials and Methods. Curve C contains the same ingredients as curve A except the reaction mixture also contained 100 pM phenylthiourea. E C 0 a3 w IU W 0 z 4 m K 0 v) m 4 0 5 10 ELUTION TIME (mln) Fig. 3. HPLC analysis of NAcTME-enzyme reaction. Conditions of the reaction mixture are as outlined for Figure 2. At the indicated time intervals, 1 5 PI aliquots of the assay mixture were subjected to HPLC analysis, as outlined in Materials and Methods, using solvent system B. The large broken peak i s due to ascorbate and salts. Trace A, 5 min reaction; Trace €3, 15 min reaction; Trace C, 30 min reaction. Dopa Derivatives in Sclerotization 23 E C g 0. cu I- U W 0 z U m K 0 v) rn 4 0 10 20 FRACTION NUMBER 30 Fig. 4. Sephadex LH-20 column chromatography of NAcTME-enzyme reaction. A reaction mixture containing 0.1 m M NAcTME, 0.25 m M ascorbic acid, and 10 mg of enzyme protein i n 20 m l of 50 m M sodium phosphate buffer, pH 6.2, was incubated at room temperature for 45 min and concentrated by lyophilization. The contents were then dissolved in a minimum amount of 0.2 M acetic acid and subjected to chromatographic separation on a Sephadex LH-20 column (22.5 x 3 cm) using 0.2 M acetic acid as the eluant. Fractions of 6 ml were collected. Peak A, unreacted NAcTME; peak B, product corresponding to NAcDME. 0.3 B 0.2 W 0 Z U rn 0.0 U 0 v) m U 0.1 0.0 250 300 WAVELENGTH 350 400 (nm) Fig. 5. U V absorbance spectrum of NAcDME. Authentic sample (1 1 and isolated product (2) in 0.2 M acetic acid (A) and 3% sodium borate (6). 24 Sugumaran and Ricketts As shown in Figure 6, the IR spectrum of isolated product matched peak to peak that of the synthetic sample, confirming that they are one and the same compound. Moreover, the NMR spectrum of the isolated product in DMSOd, exhibited the following proton signals at 6 = 8.8 (2H, br s, -OH), 8.25 (lH, d, J = 7.5, -NH), 6.6 (lH, d, J = 8, ArH5), 6.55 (lH, d, J = 2, ArH2), 6.4 (lH, dd, J = 8 and 2, ArHG), 4.3 (lH, m, a-H), 3.55 (3H, s, -OCH3),2.8 (lH, dd, J = 14 and 6, P-H), 2.65 (lH, dd, J = 14 and 8.5, P-H), 1.8 (3H, s, -NAc), which confirmed the assigned structure. To further study the metabolism of NAcDME, two types of experiments were carried out. In the first case, the enzymatic oxidation of NAcDME was examined, while in the second case, the enzymatic oxidation of NAcTME was studied in the presence of trace amounts of catechol (specifically NADA). Figure 7 shows the UV spectral changes associated with the oxidation of NAcDME by cuticular enzymes. The marked increase in absorbance observed at about 330 nm can be ascribed to the formation of a catechol with a conjugated double bond. Structural considerations indicated the generation of dehydro NAcDME in the reaction mixture. To confirm this contention, a largescale reaction was conducted, and the entire reaction mixture was chromatographed for product isolation. Figure 8 shows the chromatographic pattern of the reaction mixture on a Sephadex LH-20 column. Along with several minor products, formation of a major product (labelled peak B) can be witnessed in the chromatogram. This product eluted with (at the same 4000 3000 ' 20b0 1500 1000 51 WAVENUMBER (cm -1 I Fig. 6. I R spectrum of NAcDME. Solid line, authentic sample; broken line, isolated product. Dopa Derivatives in Sclerotization 25 W 0 z a m K zm a 250 300 350 WAVELENGTH 400 (nm) Fig. 7. U V spectral changes associated with the enzymatic oxidatioti of NAcDME. A reaction mixture containing 0.1 m M NAcDME and 0.5 mg of enzyme protein i n 1.0 ml of 50 m M sodium phosphate buffer, pH 6.2, was incubated at room temperature, and the spectral changes associated with the oxidation were periodically monitored at 2 min intervals. A, zero time reaction; T, 40 min reaction. 0 a) - @l * I- A 6 0.5- FRACTION NUMBER Fig. 8. Sephadex LH-20 column chromatography of NAcDME enzyme reaction. A reaction mixture containing 0.1 m M NAcDME and 10 mg of enzyme protein in 20 ml of 50 m M sodium phosphate butter, p H 6.2, was incubated at room temperature for 30 min and concentrated by lyophilization. The contents were then dissolved in a minimum amount of 0.2 M acetic acid and subjected to chromatographic separation on a Sephadex LH-20 column (50 x 3 cm) using 0.2 M acetic acid as the eluant. Fractions of 6 ml were collected. Peak A, unreacted NAcDME; peak B, product corresponding to dehydro NAcDME. 26 Sugumaran and Ricketts elution volume) an authentic sample of dehydro NAcDME. In addition, both exhibited the same retention time (7.8 min; the retention time of NAcDME is 7.5 min) when subjected to HPLC in solvent A. Furthermore, both compounds showed the same UV absorbance maxima in both 0.2 M acetic acid and 3% sodium borate (Fig. 9). Finally, the IR spectrum of the product (Fig. 10) matched peak to peak the authentic sample, confirming that they are one and the same compounds. NMR spectrum of the isolated product in DMSO-d6 exhibited the following proton signals at 6 = 9.4 (lH, s, -NH), 7.15 (lH, d, J = 2, ArH2), 7.05 (lH, d, P-H), 6.9 (lH, dd, J = 8 and 2, ArH6), 6.75 (lH, d, J = 8, ArH5), 3.65 (3H, s, -OCH,), 2.0 (3H, s, -NAc), which confirmed the assigned structure. When the enzymatic oxidation of NAcTME (Fig. 11)was carried out in the absence of ascorbic acid, and in the presence of a catalytic amount of NADA, similar UV spectral changes as those observed for the oxidation of NAcDME (Fig. 7) were obtained. The observed increase in absorbance at 330 nm indicated again the formation of dehydro NAcDME in the reaction. HPLC analysis of the reaction and the UV spectral properties of the isolated product exhibited the same properties as those of authentic dehydro NAcDME, confirming this contention. The above results can be explained as follows: Trace amounts of NADA serve as the electron donor for the hydroxylation of NAcTME. NAcDME thus formed is then enzymatically converted to the dehydro compound. 260 320 380 WAVELENGTH (nm) Fig. 9. U V absorbance spectrum of dehydro NAcDME. Authentic sample (1) and isolated product (2) in 0.2 M acetic acid (A) and 3% sodium borate (B). 27 Dopa Derivatives in Sclerotization b 4000 3000 2000 1500 I000 500 WAVENUMBER (cm" Fig. 10. IR spectrum of dehydro NAcDME. Solid line, authentic sample; broken line, isolated product. 300 400 WAVELENGTH (nm) Fig. 11. U V spectral changes associated with the enzymatic oxidation of NAcTME in presence of trace amounts of NADA. A reaction mixture containing 0.1 m M NAcTME, 1 pM NADA, and 0.5 mg of enzyme protein in 1 .O ml of 50 rnM sodium phosphate buffer, p H 6.2, was incubated at room temperature, and the spectral changes assoc-iated with the oxidation were periodically monitored at 2 tnin intervals. A, zero time reaction; T, 40 min reaction. 28 Sugumaran and Ricketts The formation of dehydro NAcDME from NAcDME can occur either by a direct desaturation of the L-dopa side chain or by an indirect route involving transient formation of NAcDME quinone and NAcDME quinone methide. In order to identify the operational mechanism in the formation of dehydro NAcDME, two different experiments were carried out. In the first experiment attempts were made to identify the NAcDME quinone in the reaction mixture. When the enzymatic oxidation of NAcDME is carried out in sodium phosphate buffer, there are no significant absorbance changes in the visible region of the spectrum. However, if the reaction is carried out in 50 mM Tris maleate buffer, pH 6.0, a buffer which has been shown to inhibit the further transformation of the quinone (Sugumaran et al., 1989b), the accumulation of quinone in the reaction mixture could be easily witnessed, as evidenced by the marked absorbance increase at about 400 nm (Fig. 12). Furthermore, when NAcDME quinone was synthesized and mixed with the enzyme, facile conversion to dehydro NAcDME could be observed (Fig. 13). From these studies, it was concluded that NAcDME is converted to dehydro NAcDME through the intermediary formation of quinone and possibly quinone methide intermediates. In order to check whether dehydro NAcDME could serve as the substrate for the cuticular phenoloxidase(s), spectral studies of the reaction mixture containing the dehydro compound and the enzyme were carried out. As shown in Figure 14, cuticular phenoloxidase(s) readily oxidized dehydro 300 400 500 600 WAVELENGTH (nm) Fig. 12. Visible spectral changes associated with the enzymatic oxidation of NAcDME i n Tris maleate buffer. A reaction mixture containing 0.1 m M NAcDME and 0.5 mg of enzyme protein in 1.0 ml of 50 m M Tris maleate buffer, pH 6.0, was incubated at room temperature, and the spectral changes associated with the oxidation were periodically monitored at 1 min intervals. A, zero time reaction; T, 20 min reaction. Dopa Derivatives in Sclerotization 29 2.0 M W 0 z U m 1.0 a zm U " ' I " ' 50 450 350 WAVELENGTH (nm) Fig. 1 3 . U V and visible spectral changes associated with the transformation of NAcDME quinone. A reaction mixture containing 0.1 m M NAcDME quinone and 0.5 mg of enzyme protein in 1 .0 ml of 50 tnM sodium phosphate buffer, pH 6.2, was incubated at room temperature, and the spectral changes associated vvith the transformation were periodically monitored at 1 min intervals. A, zero time reaction; M, 1 3 min reaction. W 0 1.0 z U m [r 0 0.5 U 0 .o 300 400 501 WAVELENGTH (nrn) Fig. 14. U V spectral changes associated with the enzymatic oxidation of dehydro NAcDME. A reaction mixture containing 0.1 n i M dehydro NAcDME and 0.5 mg of enzyme protein in 1 .O ml of 50 mM 3odium phosphate buffer, p t i 6.2, was incubated at room temperature, and the spectral changes associated with the oxidation were periodically monitored at 1 min intervals. A, zero time reaction; T, 20 min reartion. 30 Sugumaran and Ricketts NAcDME to the corresponding quinone, which rapidly accumulated in the reaction mixture and slowly decomposed to unidentifiable products. DISCUSSION Novel transformation of carboxy protected dihydrocaffeiate to caffeiate derivatives during the oxidation with tyrosinase was first reported by our laboratory (Sugumaran et al., 1989a,c). To account for this unusual reaction, we proposed the sequence catechol, quinone, quinone methide, and dehydro catechol. Subsequently, we established that the enzymatic oxidation of NADA in insect cuticle proceeded by a similar route to generate dehydro NADA and discovered two new enzymes involved in sclerotization reactions: quinone isomerase and quinone methide isomerase (Saul and Sugumaran, 1989a,b, 1990a,b).The dehydro-N-acetyldopamine formed is highly reactive and could generate potential cross-links after oxidation to quinone and quinone methide, accounting for the hardening of insect cuticle (Sugumaran et al., 1992). Following our studies, Waite and his associates examined the mushroom tyrosinase catalyzed oxidation of N-acetyltyrosine ethyl ester and N-acetyldopa ethyl ester as a model for sclerotization of dopyl proteins and reported that the fungal enzyme converted N-acetyldopa ethyl ester to N-acetyldehydro dopa ethyl ester via quinone and quinone methide (Rzepecki and Waite, 1991; Rzepecki et al., 1991). Thus the peptidyl dopa oxidation resembles remarkably the mechanistic transformation of NADA in insect cuticle. In molluscs, the sclerotization of periostracum and hardening of byssal threads are achieved by autotanning (Brown, 1950,1952) caused by tyrosinase-catalyzed oxidative coupling of dopa-containing peptides (Waite, 1990). The dopyl proteins upon enzymatic oxidation seem to undergo similar transformations as shown in Figure 1 and account for the unusual hardening of periostracum and byssal threads in molluscs. In the case of insects, widespread use of small molecular weight sclerotizing precursors such as NADA and N-P-alanyldopamine and the presence of a complex array of cuticular proteins have precluded a careful examination of dopa protein involvement in the tanning process. Tyrosine-rich larval serum proteins readily exhibit polymerization reaction upon oxidation with tyrosinase (Peter and Scheller, 1991; Grun and Peter, 1983). This, coupled with the fact that some tyrosine-rich proteins are associated with cuticular sclerotization (Lipke and Henzel, 1981; Lipke et al., 1982; Peter and Scheller, 1991; Grun and Peter, 1983), calls for a greater attention to the role of tyrosine-containing proteins in the sclerotization process. Recent studies from Kramer’s laboratory support the possible presence of dopa-containing proteins in insect cuticle (Okot-Kotber et al., 1993). The present studies constitute our attempt to demonstrate the conversion of model peptidyl tyrosine and dopa derivatives into oxidation products that can cross-link cuticular proteins. Since cuticular phenoloxidases are nonspecific and attack a variety of substrates, we used NAcTME and NAcDME as model compounds and confirmed that cuticular enzymes are capable of converting them to dehydro dopa derivatives and further oxidizing them to possible reactive intermediates that could serve as sclerotizing agents. Therefore, Dopa Derivatives in Sclerotization 31 apart from low molecular weight catecholamine derivatives such as NADA and N-P-alanyldopamine, high molecular weight protein-bound tyrosines and dopa derivatives could also function as useful sclerotizing agents in insect cuticle. LITERATURE CITED Andersen SO (1979): Biochemistry of insect cuticle. Ann Rev Entomol24:29-61. Andersen SO (1985): Sclerotization and tanning of the cuticle. In Kerkut GA, Gilbert LI (eds): Comprehensive Insect Physiology Biochemistry and Pharmacology, vol. 3. New York: Pergamon Press, p p 59-74. Andersen SO (1989): Oxidation of N-P-alanyldopamine by insect cuticles and its role in cuticular sclerotization. Insect Biochem 19:581-586. Andersen SO, Roepstorff P (1982): Sclerotization of insect cuticle. 111. An unsaturated derivative of N-acetyldopamine and its role in sclerotization. Insect Biochem 12269-276. 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