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1.
J Fluoresc ; 24(6): 1849-59, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25345860

RESUMO

The plasma membrane H(+)-ATPase from Kluyveromyces lactis contains 14 tryptophan residues. Binding a nucleotide or unfolding with Gnd-HCl quenched intrinsic fluorescence by ≈60% suggesting that in the H(+)-ATPase-Nucleotide complex there is solvent-mediated collisional quenching of W505 fluorescence. N-bromosuccinimide (NBS) treatment of H(+)-ATPase modified a single W residue in both native and Gnd-HCl-unfolded H(+)-ATPase. Denaturing the H(+)-ATPase with 1% SDS led to expose six tryptophan residues while requiring 17 NBS/H(+)-ATPase. The remaining eight tryptophan residues kept buried indicating a highly stable TM domain. Acrylamide generated static quenching of fluorescence; partial in the native enzyme (V = 0.43 M(-1)) and complete in the Gnd-HCl-unfolded H(+)-ATPase (V = 0.81 M(-1)). Collisional quenching (K sv) increased from 3.12 to 7.45 M(-1) upon H(+)-ATPase unfolding. W505 fluorescence titration with NBS yielded a molar ratio of 6 NBS/H(+)-ATPase and quenched ≈ 60% fluorescence. In the recombinant N-domain, the distance between W505 and MantATP was estimated to be 21 Å by FRET. The amino acid residues involved in nucleotide binding were identified by N-domain molecular modelling and docking with ATP. In the N-domain/ATP complex model, the distance between W505 and ATP was 20.5 Å. ATP binding leads to a conformational change in the N-domain of H(+)-ATPase that exposes W505 to the environment.


Assuntos
Trifosfato de Adenosina/metabolismo , Membrana Celular/enzimologia , Kluyveromyces/enzimologia , ATPases Translocadoras de Prótons/metabolismo , Proteínas Recombinantes/metabolismo , Triptofano/química , Sequência de Aminoácidos , Sítios de Ligação , Bromosuccinimida/química , Bromosuccinimida/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , ATPases Translocadoras de Prótons/química , ATPases Translocadoras de Prótons/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Homologia de Sequência de Aminoácidos , Triptofano/metabolismo
2.
Biotechnol Lett ; 33(7): 1423-7, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21373922

RESUMO

Trametes cervina lignin peroxidase (LiP) lacks a catalytic tryptophan strictly conserved in other LiP and versatile peroxidases. It contains tyrosine(181) at the potential catalytic site. This protein and the well-characterized Phanerochaete chrysosporium LiP with the catalytic tryptophan(171) have been chemically modified: the tryptophan-specific modification with N-bromosuccinimide sufficiently disrupted oxidation of veratryl alcohol by P. chrysosporium LiP, whereas the activity of T. cervina LiP was not affected, suggesting no catalytic tryptophan in T. cervina LiP. On the other hand, the tyrosine-specific modification with tetranitromethane did not affect the activities of P. chrysosporium LiP lacking tyrosine but inactivated T. cervina LiP due to the nitration of tyrosine(181). These results strongly suggest that tyrosine(181) is at the catalytic site in T. cervina LiP.


Assuntos
Peroxidases/metabolismo , Trametes/enzimologia , Tirosina/metabolismo , Álcoois Benzílicos/metabolismo , Bromosuccinimida/metabolismo , Domínio Catalítico , Modelos Moleculares , Peroxidases/química , Phanerochaete/enzimologia , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Tetranitrometano/metabolismo , Tirosina/química
3.
Biochimie ; 93(3): 409-17, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21078359

RESUMO

Pea lectin (PSL) is a dimeric protein in which each subunit comprises two intertwined, post-translationally processed polypeptide chains--a long ß-fragment and a short α-fragment. Using guanidine hydrochloride-induced denaturation, we have investigated and characterized the species obtained in the unfolding equilibrium of PSL by steady-state and time-resolved fluorescence, phosphorescence, and selective chemical modification. During unfolding, the fragment chains become separated, and the unfolding pattern reveals a ß-fragment as intermediate that has the molten globule characteristics. As examined by 8-anilino-1-naphthalenesulfonate (ANS) binding, the fragment intermediate shows ~20 fold increase in ANS fluorescence, and a large increase in ANS lifetime (12.8 ns). The tryptophan environment of the molten globule ß-fragment has been probed by selective modification with N-bromosuccinimide (NBS), which shows that two tryptophans, possibly Trp 53 and Trp 152 are oxidized while the other Trp 128 remains resistant to oxidation. The different types of tryptophan environment for the intermediate are supported by phosphorescence studies at 77 K, which gives a (0,0) band at 410 nm. These results seem to indicate that the larger fragment chain of PSL can independently behave as a monomeric or single domain protein that undergoes unfolding through intermediate state(s), and may provide important insight into the folding problem of oligomeric proteins in general and lectins in particular.


Assuntos
Fragmentos de Peptídeos/química , Lectinas de Plantas/química , Desdobramento de Proteína , Acrilamida/química , Bromosuccinimida/metabolismo , Guanidina/farmacologia , Modelos Moleculares , Naftalenossulfonatos/metabolismo , Fragmentos de Peptídeos/metabolismo , Lectinas de Plantas/metabolismo , Dobramento de Proteína/efeitos dos fármacos , Estrutura Secundária de Proteína , Desdobramento de Proteína/efeitos dos fármacos , Espectrometria de Fluorescência , Temperatura
4.
Biophys Chem ; 151(3): 170-7, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20688426

RESUMO

Staphylococcal nuclease (SNase) has a single Trp residue at position 140. Circular dichroism, intrinsic and ANS-binding fluorescence, chemical titrations and enzymatic assays were used to measure the changes of its structure, stability and activities as the Trp was mutated or replaced to other positions. The results show that W140 is critical to SNase structure, stability, and function. Mutants such as W140A, F61W/W140A, and Y93W/W140A have unfolding, corrupted secondary and tertiary structures, diminished structural stability and attenuated catalytic activity as compared to the wild type. The deleterious effects of W140 substitution cannot be compensated by concurrent changes at topographical locations of position 61 or 93. Local hydrophobicity defined as a sum of hydrophobicity around a given residue within a distance is found to be a relevant property to SNase folding and stability.


Assuntos
Nuclease do Micrococo/química , Triptofano , Naftalenossulfonato de Anilina/metabolismo , Bromosuccinimida/metabolismo , Dicroísmo Circular , Estabilidade Enzimática , Guanidina/farmacologia , Nuclease do Micrococo/genética , Nuclease do Micrococo/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Conformação Proteica , Desdobramento de Proteína/efeitos dos fármacos , Espectrometria de Fluorescência , Trifluoretanol/metabolismo
5.
J Biomol Struct Dyn ; 26(6): 781-6, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19385706

RESUMO

Beta-N-acetyl-D-glucosaminidase (NAGase, EC3.2.1.52) plays important role in molting, digestion of chitinous foods, and defense systems against parasites in prawn (Litopenaeus vannamei). However, study on functional groups and catalytic mechanism of NAGase are yet limited. The modification of the active center of NAGase from prawn has been first studied. The results demonstrate that the disulfide bonds and the carbamidine groups of arginine residues are not essential to the enzyme's activity. The modification of indole group of tryptophan of the enzyme by N-bromosuccinimide (NBS) can lead to the complete inactivation, accompanying the absorption decreasing at 276 nm, indicating that tryptophan is essential residue to the enzyme. The modificatory kinetics of NAGase in the appropriate concentrations of NBS solution has been studied and the numbers of essential tryptophan residues have been determined using the kinetic method of the substrate reaction. The result shows that only one tryptophan residue is essential for enzyme activity. And the modifications of histidine, lysine residue, and the carboxyl groups also inactivate the enzyme completely or incompletely. The results showed that the carboxyl groups of acidic amino acid, imidazole groups of histidine residue, amino groups of lysine residue, and indole group of tryptophan were essential for the activity of enzyme.


Assuntos
Acetilglucosaminidase/metabolismo , Penaeidae/enzimologia , Acetilglucosaminidase/química , Aminoácidos/química , Aminoácidos/metabolismo , Animais , Arginina/química , Arginina/metabolismo , Bromosuccinimida/química , Bromosuccinimida/metabolismo , Catálise , Domínio Catalítico , Dissulfetos/química , Dissulfetos/metabolismo , Histidina/química , Histidina/metabolismo , Cinética , Espectrofotometria Ultravioleta , Especificidade por Substrato , Triptofano/química , Triptofano/metabolismo
6.
Biochim Biophys Acta ; 1780(9): 1017-22, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18570898

RESUMO

A mannose/glucose-specific lectin has been purified from Chinese evergreen chinkapin (Castanopsis chinensis) seeds, one of the most popular foods in East Asia. This lectin, designated as CCL, exhibited hemagglutinating activity in mouse and rabbit erythrocytes. It displayed a single band with a molecular mass of 29 kDa in SDS-PAGE and a 120-kDa peak in gel-filtration on Superdex-200. Its hemagglutinating activity was stable in the pH range 6-12 and at temperatures below 60 degrees C. The N-terminal amino acid sequence of CCL differed from those of other lectins in the same family. CCL inhibited the proliferation of HepG2 cells and adult emergence in fruitflies. CCL exhibited mitogenic activity toward mouse splenocytes, and induced nitric oxide production from mouse peritoneal macrophages but was devoid of inhibitory activity toward mycelial growth and HIV-1 reverse transcriptase.


Assuntos
Fagaceae/química , Glucose/metabolismo , Manose/metabolismo , Lectinas de Plantas/isolamento & purificação , Sequência de Aminoácidos , Animais , Bromosuccinimida/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Drosophila/efeitos dos fármacos , Eletroforese em Gel de Poliacrilamida , Humanos , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Mitógenos/farmacologia , Dados de Sequência Molecular , Óxido Nítrico/biossíntese , Lectinas de Plantas/química , Lectinas de Plantas/farmacologia , Espectrometria de Fluorescência
7.
Biochem J ; 386(Pt 2): 387-93, 2005 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-15461584

RESUMO

VPs (versatile peroxidases) sharing the functions of LiP (lignin peroxidase) and MnP (manganese peroxidase) have been described in basidiomycetous fungi Pleurotus and Bjerkandera. Despite the importance of this enzyme in polymer degradation, its reactivity with polymeric substrates remains poorly understood. In the present study, we first report that, unlike LiP, VP from Pleurotus ostreatus directly oxidized two polymeric substrates, bovine pancreatic RNase and Poly R-478, through a long-range electron pathway without redox mediators. P. ostreatus produces several MnP isoenzymes, including the multifunctional enzyme MnP2 (VP) and a typical MnP isoenzyme MnP3. MnP2 (VP) depolymerized a polymeric azo dye, Poly R-478, to complete its catalytic cycle. Reduction of the oxidized intermediates of MnP2 (VP) to its resting state was also observed for RNase. RNase inhibited the oxidation of VA (veratryl alcohol) in a competitive manner. Blocking of the exposed tryptophan by N-bromosuccinimide inhibited the oxidation of RNase and VA by MnP2 (VP), but its Mn2+-oxidizing activity was retained, suggesting that Trp-170 exposed on an enzyme surface is a substrate-binding site both for VA and the polymeric substrates. The direct oxidation of RNase and Poly R by MnP2 (VP) is in sharp contrast with redox mediator-dependent oxidation of these polymers by LiP from Phanerochaete chrysosporium. Molecular modelling of MnP2 (VP) revealed that the differences in the dependence on redox mediators in polymer oxidation by MnP2 (VP) and LiP were explained by the anionic microenvironment surrounding the exposed tryptophan.


Assuntos
Complexos Multienzimáticos/metabolismo , Peroxidases/metabolismo , Pleurotus/enzimologia , Polímeros/metabolismo , Antraquinonas/química , Antraquinonas/metabolismo , Álcoois Benzílicos/antagonistas & inibidores , Álcoois Benzílicos/metabolismo , Bromosuccinimida/química , Bromosuccinimida/metabolismo , Catálise , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Isoenzimas/metabolismo , Manganês/antagonistas & inibidores , Manganês/metabolismo , Modelos Moleculares , Peso Molecular , Oxirredução , Peroxidases/química , Pleurotus/crescimento & desenvolvimento , Polímeros/química , Ribonucleases/metabolismo , Especificidade por Substrato
8.
J Am Chem Soc ; 126(41): 13306-11, 2004 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-15479085

RESUMO

Peptidylglycine alpha-amidating monooxygenase catalyzes the biosynthesis of peptide hormones through radical cleavage of the C-terminal glycine residues of the corresponding prohormones. We have correlated ab initio calculations of radical stabilization energies and studies of free radical brominations with the extent of catalysis displayed by peptidylglycine alpha-amidating monooxygenase, to identify classes of inhibitors of the enzyme. In particular we find that, in closely related systems, the substitution of glycolate for glycine reduces the calculated radical stabilization energy by 34.7 kJ mol(-1), decreases the rate of bromination with N-bromosuccinimide at reflux in carbon tetrachloride by a factor of at least 2000, and stops catalysis by the monooxygenase, while maintaining binding to the enzyme.


Assuntos
Glicina/análogos & derivados , Glicina/metabolismo , Oxigenases de Função Mista/antagonistas & inibidores , Complexos Multienzimáticos/antagonistas & inibidores , Bromosuccinimida/química , Bromosuccinimida/metabolismo , Radicais Livres/química , Radicais Livres/metabolismo , Glicina/química , Hidrocarbonetos Bromados/química , Hidrocarbonetos Bromados/metabolismo , Cinética , Oxigenases de Função Mista/metabolismo , Complexos Multienzimáticos/metabolismo , Termodinâmica
9.
Biochemistry ; 40(7): 1945-55, 2001 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-11329261

RESUMO

Glucose oxidase (GOD) from Aspergillus niger is an acidic dimeric enzyme having a high degree of localization of negative charges on the enzyme surface and dimer interface. We have studied the effect of monovalent cations on the structure and stability of GOD using various optical spectroscopic techniques, limited proteolysis, size exclusion chromatography, differential scanning calorimetry, and enzymic activity measurements. The monovalent cations were found to influence the enzymic activity and tertiary structure of GOD, but no effect on the secondary structure of the enzyme was observed. The monovalent cation-stabilized GOD was found to have a more compact dimeric structure but lower enzymic activity than the native enzyme. The enzyme's K(m) for D-glucose was found to be slightly enhanced for the monovalent cation-stabilized enzyme (maximum enhancement of about 35% for LiCl) as compared to native GOD. Comparative denaturation studies on the native and monovalent cation-stabilized enzyme demonstrated a significant resistance of cation-stabilized GOD to urea (about 50% residual activity at 6.5 M urea) and thermal denaturation (Delta T(m) maximum of 10 degrees C compared to native enzyme). However, pH-induced denaturation showed a destabilization of monovalent cation-stabilized GOD as compared to the native enzyme. The effectiveness of monovalent cations in stabilizing GOD structure against urea and thermal denaturation was found to follow the Hofmeister series: K(+) > Na(+) > Li(+).


Assuntos
Cátions Monovalentes/química , Glucose Oxidase/química , Glucose Oxidase/metabolismo , Aspergillus niger/enzimologia , Sítios de Ligação , Bromosuccinimida/metabolismo , Varredura Diferencial de Calorimetria , Cromatografia em Gel , Dicroísmo Circular , Reagentes de Ligações Cruzadas/metabolismo , Endopeptidases/metabolismo , Ativação Enzimática , Estabilidade Enzimática , Glucose/metabolismo , Glutaral/metabolismo , Temperatura Alta , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Cloreto de Lítio/química , Cloreto de Potássio/química , Conformação Proteica , Desnaturação Proteica , Cloreto de Sódio/química , Espectrometria de Fluorescência , Relação Estrutura-Atividade , Triptofano/metabolismo , Ureia
10.
Biotechnol Prog ; 16(4): 577-82, 2000.
Artigo em Inglês | MEDLINE | ID: mdl-10933831

RESUMO

The rapid inactivation of 3-HBA-6-hydroxylase by 100 microM diethylpyrocarbonate or 40 microM N-bromosuccinimide and protection offered by the substrate, 3-hydroxybenzoate, against these chemical modifications implicate the involvement of histidine and tryptophan in the catalytic activity of the enzyme. Inactivation of the enzyme by diethylpyrocarbonate followed pseudo-first-order kinetics, and an "n" value of 1.3 was obtained. Inactivation of the enzyme by N-bromosuccinimide was instantaneous and failed to follow pseudo-first-order kinetics. Distinct and incremental changes in the UV absorption, emission fluorescence, and near UV-CD spectra of the enzyme upon its titration with increasing concentrations of diethylpyrocarbonate or N-bromosuccinimide may be ascribed to modification and/or changes in the microenvironment of aromatic amino acid residue(s) such as tryptophan in the enzyme.


Assuntos
Bromosuccinimida/metabolismo , Dietil Pirocarbonato/metabolismo , Oxigenases de Função Mista/metabolismo , Indicadores e Reagentes , Cinética , Análise Espectral
11.
Biochem J ; 346 Pt 1: 223-32, 2000 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-10657261

RESUMO

Equinatoxin II (EqtII) is a eukaryotic cytolytic toxin that avidly creates pores in natural and model lipid membranes. It contains five tryptophan residues in three different regions of the molecule. In order to study its interaction with the lipid membranes, three tryptophan mutants, EqtII Trp(45), EqtII Trp(116/117) and EqtII Trp(149), were prepared in an Escherichia coli expression system [here, the tryptophan mutants are classified according to the position of the remaining tryptophan residue(s) in each mutated protein]. They all possess a single intrinsic fluorescent centre. All mutants were less haemolytically active than the wild-type, although the mechanism of erythrocyte damage was the same. EqtII Trp(116/117) resembles the wild-type in terms of its secondary structure content, as determined from Fourier-transform infrared (FTIR) spectra and its fluorescent properties. Tryptophans at these two positions are buried within the hydrophobic interior of the protein, and are transferred to the lipid phase during the interaction with the lipid membrane. The secondary structure of the other two mutants, EqtII Trp(45) and EqtII Trp(149), was altered to a certain extent. EqtII Trp(149) was the most dissimilar from the wild-type, displaying a higher content of random-coil structure. It also retained the lowest number of nitrogen-bound protons after exchange with (2)H(2)O, which might indicate a reduced compactness of the molecule. Tryptophans in EqtII Trp(45) and EqtII Trp(149) were more exposed to water, and also remained as such in the membrane-bound form.


Assuntos
Venenos de Cnidários/química , Venenos de Cnidários/metabolismo , Citotoxinas/metabolismo , Mutação/genética , Anêmonas-do-Mar/química , Triptofano/metabolismo , Acrilamida/metabolismo , Sequência de Aminoácidos , Animais , Bromosuccinimida/metabolismo , Bovinos , Morte Celular/efeitos dos fármacos , Venenos de Cnidários/genética , Venenos de Cnidários/toxicidade , Citotoxinas/química , Citotoxinas/genética , Citotoxinas/toxicidade , Eritrócitos/citologia , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Fluorescência , Hemólise/efeitos dos fármacos , Lipossomos/química , Lipossomos/metabolismo , Dados de Sequência Molecular , Nitrogênio/metabolismo , Porinas/química , Porinas/genética , Porinas/metabolismo , Porinas/toxicidade , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/toxicidade , Espectroscopia de Infravermelho com Transformada de Fourier , Relação Estrutura-Atividade , Triptofano/genética , Água/metabolismo
12.
Mol Reprod Dev ; 54(4): 402-9, 1999 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-10542381

RESUMO

We have determined the presence of cysteine in the protein PL-I from the sperm of the surf clam Spisula solidissima. The existence of cysteine in this histone H1-related protein is responsible for its previously described aggregation behavior. The location of this residue, within the trypsin-resistant domain of the protein, has been established. We have also shown that cysteine is ubiquitously present in the PL-I proteins from the sperm of other bivalve mollusks but is absent from other PL of smaller molecular mass (PL-II, PL-III, PL-IV). We have also found cysteine to be present in the PL-I from a tunicate (Chelysoma productum) but absent in a PL-I from a fish (Mullus barbatus). The possible significance of the unusual occurrence of cysteine in these histone-H1-related proteins is discussed.


Assuntos
Cisteína/análise , Proteínas de Ligação a DNA/metabolismo , Histonas/metabolismo , Espermatozoides/metabolismo , Sequência de Aminoácidos , Animais , Bivalves/química , Bromosuccinimida/metabolismo , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Endopeptidases/metabolismo , Proteínas Homeobox A10 , Proteínas de Homeodomínio , Masculino , Metaloendopeptidases , Dados de Sequência Molecular , Oxirredução , Homologia de Sequência de Aminoácidos , Compostos de Sulfidrila/metabolismo
13.
Biochem Mol Biol Int ; 41(5): 951-9, 1997 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-9137826

RESUMO

The tryptophan residues in green crab (scylla serrata) alkaline phosphatase (EC 3.1.3.1) have been modified by N-bromosuccinimide (NBS). The modification of five tryptophan residues leads to complete loss of enzymatic activity. With the increase of NBS concentration, both the absorption at 278 nm and the fluorescence emission intensity at 335 nm of the modified enzyme decreased markedly indicating the modification of tryptophan residues. Quantitative treatment of the data (Tsou, Sci. Sinica 1962, 11, 1535-1558) shows that among the tryptophan residues modified, one is essential for its catalytic activity. The presence of the substrate markedly protects the modification of tryptophan residues as well as the inactivation, suggesting that the essential tryptophan residue is situated at the active site of this enzyme.


Assuntos
Fosfatase Alcalina/metabolismo , Braquiúros/enzimologia , Triptofano/metabolismo , Fosfatase Alcalina/análise , Fosfatase Alcalina/efeitos dos fármacos , Animais , Bromosuccinimida/metabolismo , Bromosuccinimida/farmacologia , Cinética , Espectrofotometria , Triptofano/análise
14.
Arch Biochem Biophys ; 320(2): 280-8, 1995 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-7625835

RESUMO

Treatment of spinach leaf ferredoxin:NADP+ oxidoreductase (FNR) with N-bromosuccinimide (NBS), under conditions where approximately one tryptophan residue per enzyme was modified, resulted in a loss of between 80 and 85% of the activity of the enzyme when electron transfer from NADPH to either ferredoxin or 2,6-dichlorophenol-indophenol was measured. Amino acid analysis revealed no detectable modification by NBS of any FNR amino acids other than tryptophan. Complex formation with ferredoxin, but not with NADP+, prevented both the inhibition of activity and the modification of tryptophan caused by the treatment with NBS. Modification of one FNR tryptophan residue had no significant effect on the Km values of the enzyme for either ferredoxin or NADPH or on the binding constants for the FNR complexes with either ferredoxin or NADP+. NBS treatment had only very small effects on the absorbance and circular dichroism spectra of FNR and did not significantly affect either the oxidation-reduction midpoint potential of the FAD prosthetic group of the enzyme or inhibit the reduction of the FAD group by NADPH. These results raise the possibility that a tryptophan residue may play a role in the electron transfer between the FAD of FNR and the enzyme substrate, ferredoxin.


Assuntos
Bromosuccinimida/metabolismo , Ferredoxina-NADP Redutase/metabolismo , Spinacia oleracea/enzimologia , Bromosuccinimida/farmacologia , Oxirredução , Triptofano/metabolismo
15.
J Biol Chem ; 270(19): 11383-90, 1995 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-7744774

RESUMO

Efficient fluorescence energy transfer from aromatic residues to the pyridine moiety of the bound coenzyme (NAD) of UDP-glucose 4-epimerase from Kluyveromyces fragilis had been reported earlier (Mukherji, S., and Bhaduri, A. (1992) J. Biol. Chem. 267, 11709-11713). We have employed N-bromosuccinimide (NBS) to identify tryptophan as the exclusive aromatic donor in the energy transfer. The characteristic UV absorption spectrum associated with Trp oxidation is observed during NBS modification of two of the four Trp residues of native epimerase along with concomitant inactivation of the enzyme. Excellent correlation between the observed inactivation and abolition of fluorescence energy transfer to coenzyme from Trp in epimerase upon treatment with NBS implicates the involvement of the same two tryptophans in both catalytic activity and fluorescence energy transfer. SDS-polyacrylamide gel electrophoresis and fluorescence data preclude gross structural/conformational changes in epimerase due to NBS oxidation. The susceptible tryptophans do not reside at the substrate binding site as substrates and UMP fail to protect against NBS modification. However, failure of sodium borohydride to reduce the bound NAD in the NBS-inactivated epimerase suggests that the reactive tryptophans are close to the coenzyme. Tryptophan fluorescence lifetime values of 1.9 and 3.9 ns for the native and 3.5 ns for the NBS-modified epimerase, complemented by a linear Stern-Volmer plot (effective Stern-Volmer constant = 2.85 M-1) of acrylamide quenching, suggest that the two key tryptophans are buried close to an intrinsic quencher, presumably NAD.


Assuntos
Kluyveromyces/enzimologia , Triptofano , UDPglucose 4-Epimerase/química , UDPglucose 4-Epimerase/metabolismo , Sítios de Ligação , Bromosuccinimida/metabolismo , Bromosuccinimida/farmacologia , Transferência de Energia , Cinética , NAD/análise , Espectrometria de Fluorescência , UDPglucose 4-Epimerase/antagonistas & inibidores
16.
Biochem Int ; 22(6): 977-82, 1990 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-1965282

RESUMO

Chemical modification of tryptophanyl residues of NADPH - adrenodoxin reductase by N - bromosuccinimide and trichloroethanol prevents the interaction of the enzyme with adrenodoxin. The modification does not touch other amino acid residues besides tryptophan (tyrosine, lysine and cysteine) or disturb the structure of protein. The presence of adrenodoxin suppresses the modification. The data obtained indicate the participation of adrenodoxin reductase tryptophan residues in the interaction with adrenodoxin.


Assuntos
Adrenodoxina/metabolismo , Ferredoxina-NADP Redutase/metabolismo , Triptofano/química , Córtex Suprarrenal/enzimologia , Bromosuccinimida/metabolismo , Grupo dos Citocromos c/metabolismo , Transporte de Elétrons , Etilenocloroidrina/análogos & derivados , Etilenocloroidrina/metabolismo , Ferredoxina-NADP Redutase/antagonistas & inibidores , Oxirredução , Espectrofotometria
17.
FEBS Lett ; 234(1): 31-4, 1988 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-3134256

RESUMO

The site-specific phosphorylation of bovine histone H1 by protein kinase C was investigated in order to further elucidate the substrate specificity of protein kinase C. Protein kinase C was found to phosphorylate histone H1 to 1 mol per mol. Using N-bromosuccinimide and thrombin digestions, the phosphorylation site was localized to the globular region of the protein, containing residues 71-122. A tryptic peptide containing the phosphorylation site was purified. Modification of the phosphoserine followed by amino acid sequence analysis demonstrated that protein kinase C phosphorylated histone H1 on serine 103. This sequence, Gly97-Thr-Gly-Ala-Ser-Gly-Ser(PO4)-Phe-Lys105, supports the contention that basic amino acid residues C-terminal to the phosphorylation site are sufficient determinants for phosphorylation by protein kinase C.


Assuntos
Histonas/metabolismo , Fosfosserina/metabolismo , Proteína Quinase C/metabolismo , Serina/análogos & derivados , Sequência de Aminoácidos , Animais , Bromosuccinimida/metabolismo , Bovinos , Cromatografia Líquida de Alta Pressão , Dados de Sequência Molecular , Fosforilação , Ratos , Especificidade por Substrato , Trombina/metabolismo
18.
Biochemistry ; 21(26): 6843-50, 1982 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-7159567

RESUMO

The environment of tryptophan residues of the gamma subunit derived from the 7S nerve growth factor has been studied by intrinsic fluorescence, solute quenching, and oxidation with N-bromosuccinimide (NBS). The native protein has a fluorescence emission maximum of 345 nm with a tryptophan quantum yield of 0.10. A red shift in the emission maximum occurs between 3 and 6 M urea; a 20% increase in quantum yield occurred at 7-8 M urea. NBS (20-24 mol of NBS/mol of protein) completely oxidized one tryptophan of the gamma subunit, causing a 70% quenching of the intrinsic fluorescence, a complete loss of esterolytic activity toward synthetic substrates, an inability to combine with alpha and beta subunits to re-form the 7S complex, and a shift of about 12% of the structure from beta strands to random coil as determined by circular dichroism. About 80% of the fluorescing tryptophans are accessible to quenching by acrylamide but not to potassium iodide. These results suggest the presence of an exposed tryptophan contributing greater than or equal to 70% of the native fluorescence, located near a negative charge, critical to the esterase activity and possibly to interaction with the beta subunit to re-form 7S NGF.


Assuntos
Bromosuccinimida/metabolismo , Fatores de Crescimento Neural/análise , Succinimidas/metabolismo , Triptofano/análise , Cromatografia em Gel , Dicroísmo Circular , Eletroforese em Gel de Poliacrilamida , Fluorescência , Substâncias Macromoleculares , Matemática
19.
J Biochem ; 83(5): 1503-10, 1978 May.
Artigo em Inglês | MEDLINE | ID: mdl-96111

RESUMO

Four tryptophan residues of saccharifying alpha-amylase from B. subtilis out of eleven in total are reactive towards N-bromosuccinimide (NBS), suggesting that they are on the surface of the enzyme. This is consistent with the results of solvent perturbation difference spectrophotometry with ethylene glycol. One of four tryptophan residues was clearly distinguished from the other three in reactivity with NBS by the stopped-flow method. This most reactive tryptophan residue was not protected from modification by substrates of analogs, indicating that the tryptophan is not located in the substrate binding site. One of the other three tryptophan residues, probably the second most reactive one, is considered to be related in some way to the glycosyl transfer in the reaction of the enzyme with maltose as a substrate.


Assuntos
Amilases/metabolismo , Bacillus subtilis/enzimologia , Triptofano/metabolismo , alfa-Amilases/metabolismo , Sítios de Ligação , Bromosuccinimida/metabolismo , Cinética , Maltose/metabolismo , Conformação Proteica , Especificidade por Substrato
20.
Tex Rep Biol Med ; 34(2-4): 315-29, 1976.
Artigo em Inglês | MEDLINE | ID: mdl-20674

RESUMO

A relationship between in vitro rate of oxidation by N-bromosuccinimide (NBS) and the pharmacologic activity (pA2) of different beta-adrenergic blockers for different blocking agent-tissue combinations has been studied. The rates of oxidation of the alcoholic group in the drugs by NBS, as well as their molecular conformations as represented by molecular models, were studied in order to determine requirements for selectivity and potency of action of beta-adrenergic blocking agents. Using data from all 7 drugs studied--both nonselective and selective blocking agents--no significant correlation between pA2 and -log k2 (k2 is the second order rate constant for the oxidative reaction) was found. If data from only the 4 nonselective agents were used (16 drug-tissue combinations), a correlation significant at p less than 0.01 was found. Hypotheses are presented to account for the selective action of some beta-adrenergic blocking agents.


Assuntos
Antagonistas Adrenérgicos beta/farmacologia , Bromosuccinimida/metabolismo , Succinimidas/metabolismo , Butoxamina/metabolismo , Relação Dose-Resposta a Droga , Etanolaminas/metabolismo , Cinética , Conformação Molecular , Pindolol/metabolismo , Practolol/metabolismo , Propranolol/metabolismo , Sotalol/metabolismo , Relação Estrutura-Atividade
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