scholarly journals Chemical modification of cellulase from Aspergillus niger

1977 ◽  
Vol 167 (3) ◽  
pp. 549-556 ◽  
Author(s):  
P L Hurst ◽  
P A Sullivan ◽  
M G Shepherd
Keyword(s):  
Ph Dependence ◽  
Cellulase Activity ◽  
Tryptophan Residue ◽  
Carboxyl Group ◽  
Enzyme Molecule ◽  
Thiol Groups ◽  
Original Activity ◽  
Activity Inhibition ◽  
Tryptophan Residues ◽  
Benzyl Halides

N-Bromosuccinimide completely inactivated the cellulase, and titration experiments showed that oxidation of one tryptophan residue per cellulase molecule coincided with 100% inactivation. CM-cellulose protected the enzyme from inactivation by N-bromosuccinimide. The cellulase was inhibited by active benzyl halides, and reaction with 2-hydroxy-5-nitrobenzyl bromide resulted in the incorporation of 2.3 hydroxy-5-nitrobenzyl groups per enzyme molecule; one tryptophan residue was shown to be essential for activity. Diazocarbonyl compounds in the presence of Cu2+ ions inhibited the enzyme. The pH-dependence of inactivation was consistent with the reaction occurring with a protonated carboxyl group. Carbodi-imide inhibited the cellulase, and kinetic analysis indicated that there was an average of 1 mol of carbodi-imide binding to the cellulase during inactivation. Treatment of the cellulase with diethyl pyrocarbonate resulted in the modification of two out of the four histidine residues present in the cellulase. The modified enzyme retained 40% of its original activity. Inhibition of cellulase activity by the metal ions Ag+ and Hg2+ was ascribed to interaction with tryptophan residues, rather than with thiol groups.

scholarly journals Probing the active site of glyoxalase I from human erythrocytes by use of the strong reversible inhibitor S-p-bromobenzylglutathione and metal substitutions

1981 ◽  
Vol 197 (1) ◽  
pp. 67-75 ◽  
Author(s):  
Anne-Charlotte Aronsson ◽  
Siv Sellin ◽  
Gudrun Tibbelin ◽  
Bengt Mannervik
Keyword(s):  
Catalytic Activity ◽  
Linear Regression Analysis ◽  
Strong Support ◽  
Human Erythrocytes ◽  
Reversible Inhibitor ◽  
Tryptophan Residue ◽  
Glyoxalase I ◽  
Enzyme Molecule ◽  
Binding Parameters ◽  
Thiol Groups

Glyoxalase I from human erythrocytes was studied by use of the strong reversible competitive inhibitor S-p-bromobenzylglutathione. Replacements of cobalt, manganese and magnesium for the essential zinc in the enzyme were made by a new procedure involving 10% methanol as a stabilizer of the enzyme. The Km value for the adduct of methylglyoxal and glutathione was essentially unchanged by the metal substitutions, whereas the inhibition constant for S-p-bromobenzylglutathione increased from 0.08μm for the Zn-containing enzyme to 1.3, 1.7 and 2.4μm for Co-, Mn- and Mg-glyoxalase I respectively. Binding of the inhibitor to the enzyme caused quenching of the tryptophan fluorescence of the protein, from which the binding parameters could be determined by the use of non-linear regression analysis. The highest dissociation constant was obtained for apoenzyme (6.9μm). The identity of the corresponding kinetic and binding parameters of the native enzyme and the Zn2+-re-activated apoenzyme and the clear differences from the parameters of the other metal-substituted enzyme forms give strong support to the previous identification of zinc as the natural metal cofactor of glyoxalase I. Binding to apoenzyme was also shown by the use of S-p-bromobenzylglutathione as a ligand in affinity chromatography and as a protector in chemical modification experiments. The tryptophan-modifying reagent 2-hydroxy-5-nitrobenzyl bromide caused up to 85% inactivation of the enzyme. After blocking of the thiol groups (about 8 per enzyme molecule) 6.1 2-hydroxy-5-nitrobenzyl groups were incorporated. Inclusion of S-p-bromobenzylglutathione with the modifying reagent preserved the catalytic activity of the enzyme completely and decreased the number of modified residues to 4.4 per enzyme molecule. The findings indicate the presence of one tryptophan residue in the active centre of each of the two subunits of the enzyme. Thiol groups appear not to be essential for catalytic activity. The presence of at least two categories of tryptophan residues in the protein was also shown by quenching of the fluorescence by KI.

scholarly journals Posttranslational isoprenylation of tryptophan in bacteria

2017 ◽  
Vol 13 ◽  
pp. 338-346 ◽  
Author(s):  
Masahiro Okada ◽  
Tomotoshi Sugita ◽  
Ikuro Abe
Keyword(s):  
Quorum Sensing ◽  
Cyclic Peptide ◽  
Cysteine Residues ◽  
Tryptophan Residue ◽  
Biosynthetic Pathways ◽  
Thiol Groups ◽  
Tryptophan Residues ◽  
Peptide Pheromone

Posttranslational isoprenylation is generally recognized as a universal modification of the cysteine residues in peptides and the thiol groups of proteins in eukaryotes. In contrast, the Bacillus quorum sensing peptide pheromone, the ComX pheromone, possesses a posttranslationally modified tryptophan residue, and the tryptophan residue is isoprenylated with either a geranyl or farnesyl group at the gamma position to form a tricyclic skeleton that bears a newly formed pyrrolidine, similar to proline. The post-translational dimethylallylation of two tryptophan residues of a cyclic peptide, kawaguchipeptin A, from cyanobacteria has also been reported. Interestingly, the modified tryptophan residues of kawaguchipeptin A have the same scaffold as that of the ComX pheromones, but with the opposite stereochemistry. This review highlights the biosynthetic pathways and posttranslational isoprenylation of tryptophan. In particular, recent studies on peptide modifying enzymes are discussed.

scholarly journals The reactivities and ionization properties of the active-site dithiol groups of mammalian protein disulphide-isomerase

1991 ◽  
Vol 275 (2) ◽  
pp. 335-339 ◽  
Author(s):  
H C Hawkins ◽  
R B Freedman
Keyword(s):  
Rate Constant ◽  
Ph Dependence ◽  
Order Rate Constant ◽  
Second Order ◽  
Native Enzyme ◽  
Liver Protein ◽  
Thiol Groups ◽  
Protein Disulphide Isomerase ◽  
Order Rate ◽  
Reactive Groups

1. The number of reactive thiol groups in mammalian liver protein disulphide-isomerase (PDI) in various conditions was investigated by alkylation with iodo[14C]acetate. 2. Both the native enzyme, as isolated, and the urea-denatured enzyme contained negligible reactive thiol groups; the enzyme reduced with dithiothreitol contained two groups reactive towards iodoacetic acid at pH 7.5, and up to five reactive groups were detectable in the reduced denatured enzyme. 3. Modification of the two reactive groups in the reduced native enzyme led to complete inactivation, and the relationship between the loss of activity and the extent of modification was approximately linear. 4. Inactivation of PDI by alkylation of the reduced enzyme followed pseudo-first-order kinetics; a plot of the pH-dependence of the second-order rate constant for inactivation indicated that the essential reactive groups had a pK of 6.7 and a limiting second-order rate constant at high pH of 11 M-1.s-1. 5. Since sequence data on PDI show the presence within the polypeptide of two regions closely similar to thioredoxin, the data strongly indicate that these regions are chemically and functionally equivalent to thioredoxin. 6. The activity of PDI in thiol/disulphide interchange derives from the presence of vicinal dithiol groups in which one thiol group of each pair has an unusually low pK and high nucleophilic reactivity at physiological pH.

Fluorescent Properties of Firefly Luciferases and Their Complexes with Luciferin

2000 ◽  
Vol 20 (1) ◽  
pp. 21-30 ◽  
Author(s):  
Ekaterina I. Dementieva ◽  
Elena A. Fedorchuk ◽  
Lubov Yu. Brovko ◽  
Alexander P. Savitskii ◽  
Natalya N. Ugarova
Keyword(s):  
Energy Transfer ◽  
Tryptophan Fluorescence ◽  
Tryptophan Residue ◽  
Amino Acid Residues ◽  
Photinus Pyralis ◽  
Fluorescent Properties ◽  
Effective Energy Transfer ◽  
Tryptophan Residues ◽  
Luciola Mingrelica

Fluorescence of luciferases from Luciola mingrelica (single tryptophanresidue, Trp-419) and Photinus pyralis (two tryptophan residues, Trp-417,Trp-426) was studied. Analysis of quenching of tryptophan fluorescenceshowed that the tryptophan residue conserved in all luciferases is notaccessible for charged quenchers, which is explained by the presence ofpositively and negatively charged amino acid residues in the close vicinityto it. An effective energy transfer from tryptophan to luciferin wasobserved during quenching of tryptophan fluorescence of both luciferaseswith luciferin. From the data on the energy transfer, the distance betweenthe luciferin molecule and Trp-417 (419) in the luciferin–luciferasecomplex was calculated: 11–15 Å for P. pyralis and 12–17Å for L. mingrelica luciferases. The role of the conserved Trp residuein the catalysis is discussed.

scholarly journals Membrane insertion of the N-terminal α-helix of equinatoxin II, a sea anemone cytolytic toxin

2004 ◽  
Vol 384 (2) ◽  
pp. 421-428 ◽  
Author(s):  
Ion GUTIÉRREZ-AGUIRRE ◽  
Ariana BARLIČ ◽  
Zdravko PODLESEK ◽  
Peter MAČEK ◽  
Gregor ANDERLUH ◽  
...  
Keyword(s):  
Lipid Membranes ◽  
Lipid Membrane ◽  
Model Systems ◽  
Tryptophan Residue ◽  
Sea Anemones ◽  
Fluorescence Measurements ◽  
Tryptophan Residues ◽  
Equinatoxin Ii ◽  
Α Helix ◽  
Pore Forming Proteins

Equinatoxin II (Eqt-II) is a member of the actinoporins, a unique family of cytotoxins comprising 20 kDa pore-forming proteins isolated from sea anemones. Actinoporins bind preferentially to lipid membranes containing sphingomyelin, and create cation-selective pores by oligomerization of three to four monomers. Previous studies have shown that regions of Eqt-II crucial for its cytolytic mechanism are an exposed aromatic cluster and the N-terminal region containing an amphipathic α-helix. In the present study, we have investigated the transfer of the N-terminal α-helix into the lipid membrane by the use of three mutants containing an additional tryptophan residue in different positions within the amphipathic α-helix (Ile18→Trp, Val22→Trp and Ala25→Trp). The interaction of the mutants with different model systems, such as lipid monolayers, erythrocytes and ghost membranes, was extensively characterized. Intrinsic fluorescence measurements and the use of vesicles containing brominated phospholipids indicated a deep localization of the N-terminal amphipathic helix in the lipid bilayer, except for the case of Val22→Trp. This mutant is stabilized in a state immediately prior to final pore formation. The introduction of additional tryptophan residues in the sequence of Eqt-II has proved to be a suitable approach to monitor the new environments that surround defined regions of the molecule upon membrane interaction.

scholarly journals Chemical modification of a xylanase from a thermotolerant Streptomyces. Evidence for essential tryptophan and cysteine residues at the active site

1989 ◽  
Vol 261 (1) ◽  
pp. 49-55 ◽  
Author(s):  
S S Keskar ◽  
M C Srinivasan ◽  
V V Deshpande
Keyword(s):  
Chemical Modification ◽  
Active Site ◽  
Submerged Culture ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Cellulase Activity ◽  
Original Activity ◽  
Larch Wood ◽  
End Products ◽  
Temperature Optima

Extracellular xylanase produced in submerged culture by a thermotolerant Streptomyces T7 growing at 37-50 degrees C was purified to homogeneity by chromatography on DEAE-cellulose and gel filtration on Sephadex G-50. The purified enzyme has an Mr of 20,463 and a pI of 7.8. The pH and temperature optima for the activity were 4.5-5.5 and 60 degrees C respectively. The enzyme retained 100% of its original activity on incubation at pH 5.0 for 6 days at 50 degrees C and for 11 days at 37 degrees C. The Km and Vmax. values, as determined with soluble larch-wood xylan, were 10 mg/ml and 7.6 x 10(3) mumol/min per mg of enzyme respectively. The xylanase was devoid of cellulase activity. It was completely inhibited by Hg2+ (2 x 10(-6) M). The enzyme degraded xylan, producing xylobiose, xylo-oligosaccharides and a small amount of xylose as end products, indicating that it is an endoxylanase. Chemical modification of xylanase with N-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide and p-hydroxymercuribenzoate (PHMB) revealed that 1 mol each of tryptophan and cysteine per mol of enzyme were essential for the activity. Xylan completely protected the enzyme from inactivation by the above reagents, suggesting the presence of tryptophan and cysteine at the substrate-binding site. Inactivation of xylanase by PHMB could be restored by cysteine.

scholarly journals Interactions between apo-(d-β-hydroxybutyrate dehydrogenase) and phospholipids studied by intrinsic and extrinsic fluorescence

1986 ◽  
Vol 237 (2) ◽  
pp. 359-364 ◽  
Author(s):  
M S el Kebbaj ◽  
N Latruffe ◽  
M Monsigny ◽  
A Obrenovitch
Keyword(s):  
Conformational Changes ◽  
Negative Charge ◽  
Positive Charge ◽  
Tryptophan Residue ◽  
Fluorescence Energy Transfer ◽  
Hydrophobic Region ◽  
Phospholipid Liposomes ◽  
Tryptophan Residues ◽  
Beta Hydroxybutyrate ◽  
Fluorescence Energy

Interactions of D-beta-hydroxybutyrate dehydrogenase with phospholipids were investigated by both intrinsic- and extrinsic-fluorescence approaches. The intrinsic fluorescence, mainly caused by tryptophan residues, increased upon re-activation in the presence of phospholipids bearing a positive charge, i.e. phosphatidylcholine, but decreased in the presence of non-re-activating phospholipids with a negative charge. This indicates either that the environment of tryptophan residues is affected by charges rather than by hydrophobic chains of phospholipids, or that the enzyme undergoes different conformational changes depending on the nature of the phospholipids. On the other hand, the graph of the temperature-dependence of the fluorescence intensities of the enzyme embedded in dimyristoylphosphatidylcholine liposomes exhibits a break around 21 degrees C. This indicates either that at least one tryptophan residue is closely in contact with the hydrophobic chains of phospholipids or that there is a change in the environment of tryptophan residues owing to the physical state of the phospholipids. The addition of D-beta-hydroxybutyrate apo-dehydrogenase to phospholipid liposomes containing diphenylhexatriene (a fluorescent probe) increased the diphenylhexatriene fluorescence polarization. Moreover, there was a partial fluorescence energy transfer from tryptophan to diphenylhexatriene. These results strongly favour the possibility that there is a portion of the enzyme polypeptide chain inserted into the phospholipid hydrophobic region. All these results demonstrate that D-beta-hydroxybutyrate apo-dehydrogenase interacts with both polar and hydrophobic parts of phospholipids and leads to small, but essential, conformational changes of the enzyme.

scholarly journals Studies on the biotin-binding site of streptavidin. Tryptophan residues involved in the active site

1988 ◽  
Vol 256 (1) ◽  
pp. 279-282 ◽  
Author(s):  
G Gitlin ◽  
E A Bayer ◽  
M Wilchek
Keyword(s):  
Binding Site ◽  
Reversed Phase ◽  
Binding Activity ◽  
Complete Loss ◽  
Tryptophan Residue ◽  
Tryptophan Residues ◽  
Lysine Residues ◽  
Biotin Binding ◽  
Peptide Fractions ◽  
Specific Reagent

Streptavidin, the non-glycosylated bacterial analogue of the egg-white glycoprotein avidin, was modified with the tryptophan-specific reagent 2-hydroxy-5-nitrobenzyl (Hnb) bromide. As with avidin, complete loss of biotin-binding activity was achieved upon modification of an average of one tryptophan residue per streptavidin subunit. Tryptic peptides obtained from an Hnb-modified streptavidin preparation were fractionated by reversed-phase h.p.l.c., and three major Hnb-containing peptide fractions were isolated. Amino acid and N-terminal sequence analysis revealed that tryptophan residues 92, 108 and 120 are modified and probably comprise part of the biotin-binding site of the streptavidin molecule. Unlike avidin, the modification of lysine residues in streptavidin failed to result in complete loss of biotin-binding activity. The data imply subtle differences in the fine structure of the respective biotin-binding sites of the two proteins.

Binding Studies of Bile Acids using the Native Fluorescence of the Tryptophan Residue Of Bax Protein

2006 ◽  
Vol 26 (3) ◽  
pp. 245-250 ◽  
Author(s):  
Wei Zhang ◽  
Clifford J. Steer ◽  
Kenneth T. Douglas ◽  
Cecilia M. P. Rodrigues
Keyword(s):  
Bile Acids ◽  
Direct Interaction ◽  
Tryptophan Fluorescence ◽  
Potential Interaction ◽  
Tryptophan Residue ◽  
Binding Studies ◽  
Native Fluorescence ◽  
Bax Protein ◽  
Tryptophan Residues ◽  
Direct Binding

Ursodeoxycholic acid (UDCA) and its taurine-conjugate, tauroursodeoxycholic acid (TUDCA), play a unique role in modulating the apoptotic threshold in cells. The mechanism is thought to involve, in part, inhibition of translocation for Bax from the cytosol to mitochondria. Here, we attempted to use the native fluorescence of the tryptophan residues of Bax to determine whether bile acids bind directly to recombinant Bax protein. The results showed that UDCA had no effect on the tryptophan fluorescence of Bax. Similarly, there was no evidence of direct binding between Bax protein and the more hydrophobic bile acid, deoxycholic acid (DCA). In contrast, the fluorescence change detected for Bax solution titrated against TUDCA in dimethylsulfoxide was greater than that observed with solvent alone. In conclusion, data from fluorescence spectroscopy does not support a direct interaction of UDCA or DCA with Bax protein, whereas it suggests that there may be some potential interaction with TUDCA.

scholarly journals A study of the subunit structure and the thiol reactivity of bovine liver uridine diphosphate glucose dehydrogenase

1972 ◽  
Vol 129 (4) ◽  
pp. 821-830 ◽  
Author(s):  
P. A. Gainey ◽  
T. C. Pestell ◽  
C. F. Phelps
Keyword(s):  
Amino Acid ◽  
Amino Acid Analysis ◽  
Peptide Mapping ◽  
Ph Dependence ◽  
Glucose Dehydrogenase ◽  
Group Analysis ◽  
Subunit Structure ◽  
Uridine Diphosphate ◽  
Thiol Groups ◽  
Thiol Reactivity

1. The amino acid analysis of UDP-glucose dehydrogenase is reported. 2. N-Terminal-group analysis indicates only one type of N-terminal amino acid, methionine, to be present. 3. Peptide ‘mapping’ in conjunction with the amino acid analysis indicates that the subunits of the enzyme are similar if not identical. 4. The various kinetic classes of thiol group were investigated by reaction with 5,5′-dithiobis-(2-nitrobenzoate). 5. NAD+, UDP-glucose and UDP-xylose protect the two rapidly reacting thiol groups of the hexameric enzyme. 6. Inactivation of the enzyme with 5,5′-dithiobis-(2-nitrobenzoate) indicates the involvement of six thiol groups in the maintenance of enzymic activity. 7. The pH-dependence of UDP-xylose inhibition of the enzyme was investigated. 8. The group involved in the binding of UDP-xylose to the protein has a heat of ionization of about 33kJ/mol and a pK of 8.4–8.6. 9. It is suggested that UDP-xylose has a cooperative homotropic effect on the enzyme.

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