Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.10.3.1 (tyrosinase)
9,065 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Mutations in the gene for the pigment-producing enzyme tyrosinase are responsible for type IA (tyrosinase-negative) oculocutaneous albinism (OCA). Most reported mutations have been single base substitutions. We now report three different frameshift mutations in three unrelated individuals with type IA OCA. The first individual has a single base deletion within a series of five guanidines, resulting in a premature stop codon in exon I on one allele and a missense mutation at codon 382 in exon III on the homologous allele. The second individual is a genetic compound of two separate frameshift mutations, including both the same exon I single base deletion found in the first individual and a deletion of a thymidine-guanidine pair, within the sequence GTGTG, forming a termination codon (TAG) in exon I on the homologous allele. The third individual has a single base insertion in exon I on one allele and a missense mutation at codon 373 in exon III on the homologous allele. The two missense mutations occur within the copper Bbinding region and may interfere with either copper binding to the enzyme or oxygen binding to the copper. These five different mutations disrupt tyrosinase function and are associated with a total lack of melanin biosynthesis.
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PMID:Three different frameshift mutations of the tyrosinase gene in type IA oculocutaneous albinism. 190 79

Both native glue protein from marine mussels and a synthetic nonhydroxylated analog were analyzed by far-uv CD under a variety of conditions. Analysis of the CD spectra using various models strongly suggest a primarily random coil structure for both forms of the protein, a fact also supported by the absence of spectral change for the glue protein upon dilution into 6 M guanidine hydrochloride. The nonhydroxylated analog, which consists of 20 repeats of the peptide sequence Ala-Lys-Pro-Ser-Tyr-Pro-Pro-Thr-Tyr-Lys, was further characterized by enzyme modification using mushroom tyrosinase. Enzymatic hydroxylation of tyrosines was found to be best fit by a model containing two rate constants, 5.6 (+/- 0.6) X 10(-3) and 7.2 (+/- 0.3) X 10(-2) min-1. At equilibrium, HPLC analysis of digests showed nearly 100% conversion of Tyr-9 and only 15 to 35% conversion of Tyr-5. The Chou and Fasman rules for predicting structure were applied to the repeat sequence listed above. The rules predict the absence of alpha helix and beta pleated sheets in the structure of this peptide. On the other hand, beta turns are predicted to be present with Tyr-5 being in the region of highest probability. These data suggest that the protein in solution has only a small amount of secondary structure.
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PMID:Mussel glue protein has an open conformation. 249 14

For the first time, unfolding (6 M guanidine) and refolding of partially proteolysed purified polyphenol oxidase (PPOr) was achieved, with 88% of activity recovered. Optimal refolding conditions consisted in stepwise dialysis of guanidine treated extracts, the dialysis buffers containing 1 M (NH4)2SO4 and 100 microM CuSO4. However, CuSO4 had limited effect on the recovering of PPOr activity, whereas (NH4)2SO4 was essential. Concerning the PPO tertiary structure, denaturing conditions (combinations of boiling and reducing agent) used on SDS-PAGE have shown (i) a compact tertiary structure and (ii) the presence of disulfide bonds in PPOr, accounting for the shift between 27 and 41 kDa, and 41 and 42 kDa, respectively. Resistance to proteolytic cleavage was used to study the conformational changes induced by the denaturing treatments. Folded PPOr was resistant to further proteolysis whereas unfolded PPO was totally digested, indicating the role of tertiary structure of PPOr in the resistance to proteases.
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PMID:Unfolding and refolding of active apple polyphenol oxidase. 984 26

Tyrosinase, with an isoelectric point at pH 4.9, was purified to electrophoretic homogeneity from an extremely thermophilic bacterium, Thermomicrobium roseum. Gel filtration, N-terminal amino acid sequencing and SDS/PAGE analysis indicate that T. roseum tyrosinase is composed of two identical subunits, each with a molecular mass of 43000 Da. The enzyme exhibited high substrate specificity towards catechol, chlorogenic acid, L-3-(3,4-dihydroxyphenyl)-L-alanine (L-DOPA) and pyrogallol. The K(m) value of the enzyme for L-DOPA was 0.18 mM. beta-Mercaptoethanol and sodium diethyldithiocarbamate notably inhibited the enzymic activity. The activity of the enzyme was optimal at pH 9.5 and 70 degrees C, and was increased by addition of 1 mM Mg(2+), K(+) or Cu(2+). The enzyme was highly stable against high temperature and guanidine hydrochloride. The N-terminal amino acid sequence of the enzyme was determined to be Asp-Ile-Asn-Gly-Gly-Gly-Ala-Thr-Leu-Pro-Gln-Lys-Leu-Tyr. These facts indicate that T. roseum tyrosinase appears to be distinct from the tyrosinases so far purified from other sources.
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PMID:Purification and characterization of a highly stable tyrosinase from Thermomicrobium roseum. 1074 56

The unfolding and inhibition study of mushroom tyrosinase have been studied in the presence of different denaturants such as sodium dodecyl sulfate (SDS), guanidine hydrochloride (GdnHCl), and urea. The kinetic two-phase rate constants were commonly measured from semilogarithmic plots of the activity versus time, which resolved into two straight lines, indicating that the inactivation process consisted of fast and slow phases as a first-order reaction. This result also implied that transient partially folded intermediate existed during tyrosinase unfolding pathway. Mushroom tyrosinase had different behaviors to denaturants in regard with: noncooperative binding manner by SDS while cooperative interactions by GdnHCl and urea; in equilibrium state, SDS-micelle never completely inactivated enzyme activity while GdnHCl has single step denaturation and urea induced a typical transition-like process. Various kinetic parameters for each denaturant were calculated and the possible unfolding pathway scheme was discussed.
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PMID:Kinetic inactivation study of mushroom tyrosinase: intermediate detection by denaturants. 1469 Feb 49

A novel copper-containing enzyme named COI (catechol oxidase I) has been isolated and purified from tobacco by extracting acetone-emerged powder with phosphate buffer, centrifugation at low temperature, ammonium sulfate fractional precipitation, and column chromatography on DEAE-sephadex (A-50), sephadex (G-75), and DEAE-celluse (DE-52). PAGE, SDS-PAGE were used to detect the enzyme purity, and to determine its molecular weight. Then the secondary structures of COI at different pH, different temperatures and different concentrations of guanidine hydrochloride (GdnHCl) were studied by the FT-IR, Fourier self-deconvolution spectra, and circular dichroism (CD). At pH 2.0, the contents of both alpha-helix and anti-parallel beta-sheet decrease, and that of random coil increases, while beta-turn is unchanged compared with the neutral condition (pH 7.0). At pH 11.0, the results indicate that the contents of alpha-helix, anti-parallel beta-sheet and beta-turn decrease, while random coil structure increases. According to the CD measurements, the relative average fractions of alpha-helix, anti-parallel beta-sheet, beta-turn/parallel beta-sheet, aromatic residues and disulfide bond, and random coil/gamma-turn are 41.7%, 16.7%, 23.5%, 11.3%, and 6.8% at pH 7.0, respectively, while 7.2%, 7.7%, 15.2%, 10.7%, 59.2% at pH 2.0, and 20.6%, 9.5%, 15.2%, 10.5%, 44.2% at pH 11.0. Both alpha-helix and random coil decrease with temperature increasing, and anti-parallel beta-sheet increases at the same time. After incubated in 6 mol/L guanidine hydrochloride for 30 min, the fraction of alpha-helix almost disappears (only 1.1% left), while random coil/gamma-turn increases to 81.8%, which coincides well with the results obtained through enzymatic activity experiment.
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PMID:The studies of FT-IR and CD spectroscopy on catechol oxidase I from tobacco. 1616 22

In the present investigation, the previous ultraviolet irradiated mutant strain of Aspergillus oryzae UV-7 was further improved in terms of 3,4 dihydroxy phenyl L-alanine (L-DOPA) activity after chemical mutagenesis through 1-methyl 3-nitro 1-nitroso guanidine (MNNG = 250-1500 microg/ml) treatment (0-30 min). Among several mutant variants, the one that produced a larger amount of L-DOPA from L-tyrosine was designated to as ME2 and it was made 2-deoxy-D-glucose-resistant by growing it at various concentrations of 2 dg (0.01-0.025 %, w/v) in Vogel's agar medium. Relatively better production of L-DOPA (> 0.60 mg/ml) was obtained when 2.0% (w/v) glucose was used as a carbon source in the mycelium production medium and the tyrosinase activity increased constitutively (1.08 mg/ml), which resulted in a greater production of L-DOPA. At optimum pH0 (pH 6.0) and reaction time (60 min), more than 65% sugar was utilized for cell mass formation. The maximum conversion of L-tyrosine to L-DOPA (0.428 mg/ml) was achieved 60 min after the biochemical reaction. Mould mycelium was used for microbiological conversion of L-tyrosine to L-DOPA because tyrosinases, beta-carboxylases, and tyrosine hydroxylases are intracellular enzymes. The effect of illite (1.0 x 10(6)-6.0 x 10(6) M) on biochemical conversion of L-tyrosine to L-DOPA by Aspergillus oryzae ME(2 )was also carried out. Best results of L-DOPA biosynthesis were observed when the concentration of illite was 3.5 x 10(-6) M (1.686 mg/ml L-DOPA produced with 1.525 mg/ml consumption of L-tyrosine). It was noted that the addition of illite not only increased enzyme activity but also enhanced the permeability of cell membrane to facilitate the secretion of enzymes into the reaction broth. The comparison of kinetic parameters showed the ability of mutant to yield L-DOPA (i.e., Yp/x 7.360 +/- 0.04 mg/mg). When the culture grown on various illite concentrations was monitored for Qp, Qs, and qp, there was significant enhancement (p < 0.025) in these variables over the control, which indicate that the study can be commercially applicable on stirred and magnetic rotary drums. Overall, there was up to 3-fold (Qp = 0.290 mg/L-DOPA produced/ml/h) enhancement in the product formation rate, which is highly encouraging (HS, LSD 0.456).
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PMID:Innovative effect of illite on improved microbiological conversion of L-tyrosine to 3,4 dihydroxy phenyl L-alanine (L-DOPA) by Aspergillus oryzae ME2 under acidic reaction conditions. 1703 88

Polyphenol oxidase (PPO; EC 1.14.18.1) catalyzes the hydroxylation of monophenols to o-diphenols (cresolase activity) and the oxidation of o-diphenols to o-quinones (catecholase activity), leading to browning in plants and produce. Further interest in the enzyme has been triggered by the active role that it plays in plant defense systems. PPO can be found in latent forms and is activated in vitro by various agents including urea, detergents, and proteases. The activation of PPO from several sources by sodium dodecyl sulfate (SDS) has been extensively investigated, but reports on the effect of other detergents or on the differential effect of detergents on each of PPO's activities are scarce. In addition, investigations on the enzyme in other plant parts besides fruits and vegetables are also scarce. Here, the effect of various detergents and chaotropic agents on PPO from dormant saffron (Crocus sativus L.) corm extract was investigated. SDS and sarkosyl activated the cresolase activity, while only SDS activated the catecholase activity. All other detergents tested, in milli- or micromolar concentrations, inhibited the cresolase activity but barely affected the catecholase activity. In contrast, urea and guanidine-HCl drastically inhibited the catecholase activity but moderately inhibited the cresolase activity. The same effects were obtained on the partially purified enzyme. Results identified a PPO, present in dormant corms, which was activated only by anionic detergents and was inhibited by other reputed activating agents such as urea. Results also emphasized the differences in structure and accessibility of the active sites for cresolase and catecholase activities.
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PMID:Effect of ionic detergents, nonionic detergents, and chaotropic agents on polyphenol oxidase activity from dormant saffron (Crocus sativus L.) corms. 1740 18

A new hybrid permethylated-amine-guanidine ligand based on a 1,3-propanediamine backbone (2L) and its Cu-O2 chemistry is reported. [(2L)CuI(MeCN)]1+ complex readily oxygenates at low temperatures in polar aprotic solvents to form a bis(mu-oxo)dicopper(III) (O) species (2b), similar to the parent bis-guanidine ligand complex (1b) and permethylated-diamine ligand complex (3b). UV-vis and X-ray absorption spectroscopy experiments confirm this assignment of 2b as an O species, and full formation of the 2:1 Cu-O2 complex is demonstrated by an optical titration with ferrocene-monocarboxylic acid (FcCOOH). The UV-vis spectra of 1b and 2b with guanidine ligation show low-intensity visible features assigned as guanidine pi --> Cu2O2 core transitions by time-dependent density functional theory (TD-DFT) calculations. Comparison of the reactivity among the three related complexes (1b-3b) with phenolate at 195 K is particularly insightful as only 2b hydroxylates 2,4-di-tert-butylphenolate to yield 3,5-di-tert-butylcatecholate (>95% yield) with the oxygen atom derived from O2, reminiscent of tyrosinase reactivity. 1b is unreactive, while 3b yields the C-C radical-coupled bis-phenol product. Attenuated outer-sphere oxidative strength of the O complexes and increased phenolate accessibility to the Cu2O2 core are attributes that correlate with phenolate hydroxylation reactivity observed in 2b. The comparative low-temperature reactivity of 1b-3b with FcCOOH (O-H BDE 71 kcal mol(-1)) to form the two-electron, two-proton reduced bis(mu-hydroxo)dicopper(II,II) complex is quantitative and presumably precedes through two sequential proton-coupled electron transfer (PCET) steps. Optical titrations along with DFT calculations support that the reduced complexes formed in the first step are more powerful oxidants than the parent O complexes. These mechanistic insights aid in understanding the phenol to bis-phenol reactivity exhibited by 2b and 3b.
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PMID:Phenolate hydroxylation in a bis(mu-oxo)dicopper(III) complex: lessons from the guanidine/amine series. 1911 46

New active sites can be introduced into naturally occurring enzymes by the chemical modification of specific amino acid residues in concert with genetic techniques. Chemical strategies have had a significant impact in the field of enzyme design such as modifying the selectivity and catalytic activity which is very different from those of the corresponding native enzymes. Thus, chemical modification has been exploited for the incorporation of active site binding analogs onto protein templates and for atom replacement in order to generate new functionality such as the conversion of a hydrolase into a peroxidase. The introduction of a coordination complex into a substrate binding pocket of trypsin could probably also be extended to various enzymes of significant therapeutic and biotechnological importance. The aim of this study is the conversion of trypsin into a copper enzyme: tyrosinase by chemical modification. Tyrosinase is a biocatalyst (EC.1.14.18.1) containing two atoms of copper per active site with monooxygenase activity. The active site of trypsin (EC 3.4.21.4), a serine protease was chemically modified by copper (Cu(+2)) introduced p-aminobenzamidine (pABA- Cu(+2): guanidine containing schiff base metal chelate) which exhibits affinity for the carboxylate group in the active site as trypsin-like inhibitor. Trypsin and the resultant semisynthetic enzyme preparation was analysed by means of its trypsin and catechol oxidase/tyrosinase activity. After chemical modification, trypsin-pABA-Cu(+2) preparation lost 63% of its trypsin activity and gained tyrosinase/catechol oxidase activity. The kinetic properties (K(cat), K(m), K(cat)/K(m)), optimum pH and temperature of the trypsin-pABA-Cu(+2) complex was also investigated.
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PMID:Conversion of trypsin to a copper enzyme: tyrosinase/catechol oxidase by chemical modification. 2002 99


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