Dopa quinone

Some regulatory mechanisms have been revealed for the TPHs, such as activation by phosphorylation-dependent interaction with 14-3-3 proteins (71,72). Catecholamines are also reported to be inhibitors of TPH (73). l-DOPA and L-DOPA-quinone derivatives, but not the end product serotonin, have also been shown to be inhibitors of TPH activity (see Martinez et al. 2001 (74) for the physiological and pharmacological implications of the inhibitory effects by catechol derivatives). 

In his classical studies on melanin formation from DOPA (3), Raper proposed the following scheme for the formation of the red pigment now known to be the aminochrome dopachrome (4). The first stage involved the oxidation of the catechol nucleus of-3 to give the quinone dopa-quinone (16). The second stage was the non-oxidative intramolecular cyclization of 16 to leuco-dopachrome (17), which was in turn oxidized to dopachrome (4).72,73 Since... 

More definite evidence for the transient existence of the un-cyclized l-(jS-aminoethyl)-3,4-benzoquinones has been obtained recently by Kodja and Bouchilloux,77 78 who noted that a transient yellow color (Amax ca. 385 mp) was occasionally observed during the enzymic oxidations of catecholamines (particularly in unbuffered systems at low temperatures). This phenomenon was probably due to the formation of the transient o-quinones. (The absorption maximum of o-benzoquinone, the effective chromophore of the open-chain quinones, is known to occur at ca. 390 mp.79) An absorption maximum at 390 mp is characteristic of the formation of the dopa-quinone chromophore during oxidation of small C -terminal tyrosine peptides in the presence of tyrosinase.37 48 Similar spectroscopic features were observed when the oxidations were carried out with lead dioxide in sulfuric acid solutions (pH> 1). If the initial oxidation was carried out for a short period of time, it was possible to regenerate the original catecholamines by reduction (e.g. with sodium bisulfite, potassium iodide, and zinc powder) and to show that the 385 mp peak disappeared.77,78 Kodja and Bouchilloux were also able to identify 2,4-dinitrophenylhydrazones of several of the intermediate non-cyclized quinones by paper chromatography and spectroscopy (Amax n weakly acid solution ca. 350 mp with a shoulder at ca. 410 mp).77,78... 

Dopa quinone is converted to norepinephrine and epinephrine (Fig. 15-11) in the adrenal medulla. 

The calculated mass for these tautomers is 393.1801 (monoisotopic mass) or 393.45 (average mass), which shows excellent agreement with the experimental mass of 393.1830 (monoisotopic mass from accurate mass measurement, sample 2) or 393.45 (average mass from LC/ESIMS experiment, sample 1) for the derivatized active site cofactor. The underivatized cofactor itself has a structure of the type shown as 3. This is consistent with the strong evidence for a quinone-like structure in LO (6, 8, 9, 23), and with the conclusion that the cofactor is comprised of a crosslink between a tyrosine derivative and a lysine residue. This structure could arise from an initial hydroxylation of Tyr to form dopa, followed by the oxidation of dopa to dopa quinone, and subsequent nucleophilic attack by the e-amino group of a lysine side chain to generate an aminoquinol (13). 

In normal melaninogenesis, the amino acid tyrosine is hydroxylated to form 3,4-dihydroxyphenylalanine ( dopa ), which is then oxidized to dopa-quinone. The latter moiety is polymerized to form melanin, thereafter combining with melanoprotein to form a stable complex within premelanosomes and melanosomes. Tyrosinase plays a central role in this process, by catalyzing the first step in the stated sequence. As such, it is a specific marker for melanocytic differentiation. This premise has been affirmed by studies showing that tyrosinase gene transcripts are strictly confined to melanin-producing cells. 

Structure of the active center. The active centers of this dimeric enzyme are so well embedded into its protein structure that they are inaccessible to the solvent. The two centers are situated approximately 30 A apart from each other but connected by /3-strands. The active center consists of a type 2 copper center and a cofactor. Sequence comparisons have established that the residues His 8, His 246, and His 357 coordinate the copper ions in both yeast and plants (e.g., lentil seeds) [120,122]. The participating cofactor is typical for amine oxidases, diamine oxidases, and lysyl oxidases but has not yet been found in any other protein - 2,4,5-trihydroxy-phenylalanine quinone [123, 124] (also known as TOPA-quinone, TPQ or 6-hydroxy-DOPA quinone), an internal cofactor which is created by post-translational modification of the tyrosine in position 387 [120]. The consensus sequence of the amino acids neighboring the TOPA cofactor are conserved in all known amine oxidases - Asn-TOPA-Asp/Glu [113,120, 123,125-127]. The positions of the histidine ligands relative to TOPA quinone are conserved in all known amine oxidases as well. The chain lengths of the amine oxidase monomers vary according to the organism of origin 692 residues in yeast [128], 762 in bovine serum amine oxidase [128,129] and 569 in the enzyme from lentil seeds [120,130]. 

EXAMPLE 14.19 Tyrosine, itself a degradation product of phenylalanine, is initially converted to 3,4-dihydroxyphe-nylalanine (DOPA) and the corresponding DOPA quinone by the copper-containing enzyme tyrosinase. Tyrosinase is located in melanocytes and is a mixed-function oxidase. 

Fig. 14-32 Tyrosine is oxidized to DOPA and then to DOPA quinone.

DOPA-quinone is a precursor of norepinephrine and epinephrine in the adrenal medulla. 

The different biocatalytic transformations probed with QDs have led to the implementation of a FRET process to follow the reaction progress. The ET quenching of QDs represents an alternative mechanism for probing biocatalytic transformations a typical example was the biocatalytic function of two enzymes, tyrosinase and thrombin, which were probed by using CdSe/ZnS QDs [196]. In this case, the CdSe/ ZnS QDs were capped with a monolayer of methyl ester tyrosine (34). A subsequent tyrosinase-induced oxidation of tyrosine to dopa-quinone led to the generation of ET quencher units that suppressed the luminescence of the QDs (Figure 6.28a). The... 

Dopa quinone imine conversion factor M,sexta Pharate pupal cuticle 11,63... 

To summarize, dopa quinone imine conversion factors have been detected in cuticle and/or hemolymph from only three species of Lepidoptera. Other kinds of regulatory factors such as dihydroxy-indole blocking factor have not been detected in insect tissues. The precise physiological roles played by conversion factors that generate indoles is unknown. They may be modulators of reactions associated primarily with melanization. 

The enzyme/substrate (tyrosinase/wool hydrolysate) mixture (20 pL) was diluted widi 980 pL of distilled water, 70 pL of ethylenediamine, and 50 pL of 2M etfaylenediainine dihydrochloride (pH 11). The mixture was incubated at 50°C for 2 h in die dark, and then the fluorescence intensity was measured using a UVA IS spectrofluorom o (Tecan). The excitation and emission wavelengdis were at 420 and 543 nm, respectively. The concentrations of the DOPA residue in the preparations were estimated using a standard fluorescence curve of DOPA [8]. For the Dopa Quinone (DQ) quantification M6TH (3-methyl-2-benzothiazolinone hydrazone hydrochlcnide monohydrate) was used. MBTH reacts with DQ to form a pink pigmoit widi Xnm at 505 nm. The assay solution was prepared by mixing 480 pL of the en me/substrate reaction mixture, 980 pL of 4.3% (v/v) DMF (dimethyl formamide) in distiUed water, and 580 pL of 20.7 mM MBTH. The total volume was 2 mL. Tbe reacticm mixture was incubated at 25 C for 10 min before the absorbance measurement (at 505 nm) [8]. 

Copper is present in a number of mammalian proteins and the characteristics of these have been summarized by Scheinberg and Sternlieb [3]. Amongst the copper proteins identified in man, but of unknown function, are cerebro-cuprein I, a protein extracted from normal human brain by Porter and Ainsworth [4], liver copper-protein [5], and erythrocuprein [6]. Tyrosinase, a protein of 0.25% copper content is to be found wherever melanin is present in the body, it catalyses the oxidation of tyrosine to dopa and accelerates the conversion of dopa to dopa quinone, the initicJ stages in the conversion of tyrosine to melanin. Lack of this enzyme is not, however, associated with deficient production of pressor amines, another pathway being present in the adreneil gletnd for the hydroxylation of tyrosine [7]. 

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