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

1988 ◽  
Vol 250 (1) ◽  
pp. 291-294 ◽  
Author(s):  
G Gitlin ◽  
E A Bayer ◽  
M Wilchek
Keyword(s):  
Binding Site ◽  
Active Site ◽  
Egg White ◽  
Binding Activity ◽  
Complete Loss ◽  
Tryptophan Residue ◽  
Tryptophan Residues ◽  
Biotin Binding ◽  
Specific Reagent

Egg-white avidin was modified with the tryptophan-specific reagent 2-hydroxy-5-nitrobenzyl bromide. The complete loss of biotin-binding activity was achieved upon modification of an average of one tryptophan residue per avidin subunit. The identity of the modified residues was determined by isolating the relevant tryptic and chymotryptic peptides from CNBr-cleaved avidin fragments. The results demonstrate that Trp-70 and Trp-110 are modified in approximately equivalent proportions. It is believed that these residues are located in the active site of avidin and take part in the binding of biotin.

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.

scholarly journals Studies on the biotin-binding site of avidin. Lysine residues involved in the active site

1987 ◽  
Vol 242 (3) ◽  
pp. 923-926 ◽  
Author(s):  
G Gitlin ◽  
E A Bayer ◽  
M Wilchek
Keyword(s):  
Amino Acid ◽  
Binding Site ◽  
Active Site ◽  
Amino Acid Analysis ◽  
Egg White ◽  
Reversed Phase ◽  
Complete Loss ◽  
Tryptic Peptides ◽  
Lysine Residues ◽  
Biotin Binding

Egg-white avidin was treated with 1-fluoro-2,4-dinitrobenzene. Modification of an average of one lysine residue per avidin subunit caused the complete loss of biotin binding. Tryptic peptides obtained from the 2,4-dinitrophenylated avidin were fractionated by reversed-phase h.p.l.c. Three peptides contained the 2,4-dinitrophenyl group. Amino acid analysis revealed that lysine residues 45, 94 and 111 are modified and probably comprise part of the biotin-binding site.

scholarly journals Studies on the biotin-binding sites of avidin and streptavidin. Tyrosine residues are involved in the binding site

1990 ◽  
Vol 269 (2) ◽  
pp. 527-530 ◽  
Author(s):  
G Gitlin ◽  
E A Bayer ◽  
M Wilchek
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Egg White ◽  
Binding Activity ◽  
Tyrosine Residue ◽  
Tyrosine Residues ◽  
Egg White Protein ◽  
Biotin Binding ◽  
The Difference ◽  
Specific Reagent

The involvement of tyrosine in the biotin-binding sites of the egg-white glycoprotein avidin and the bacterial protein streptavidin was examined by using the tyrosine-specific reagent p-nitrobenzenesulphonyl fluoride (Nbs-F). Modification of an average of about 0.5 mol of tyrosine residue/mol of avidin subunit caused the complete loss of biotin binding. This indicates that the single tyrosine residue (Tyr-33) in the avidin subunit is directly involved in the biotin-binding site and that its modification by Nbs also abolishes the binding properties of a neighbouring subunit. This suggests that the tyrosine residues of the egg-white protein may also contribute to the stabilization of the native protein structure. In streptavidin, however, the modification of an average of 3 mol of tyrosine residue/mol of subunit was required to inactivate completely the biotin-binding activity of the protein, but only 1 mol (average) of tyrosine residue/mol of subunit was protected in the presence of biotin. The difference between the h.p.l.c. elution profiles of the enzymic digests of Nbs-modified streptavidin and the Nbs-modified streptavidin-biotin complex revealed two additional fractions in the unprotected protein that contain Nbs-modified tyrosine residues. These residues, Tyr-43 (major fraction) and Tyr-54 (minor fraction), appear to contribute to the biotin-binding site in streptavidin.

scholarly journals Studies on the biotin-binding site of avidin. Minimized fragments that bind biotin

1991 ◽  
Vol 278 (2) ◽  
pp. 573-585 ◽  
Author(s):  
Y Hiller ◽  
E A Bayer ◽  
M Wilchek
Keyword(s):  
Binding Site ◽  
Binding Capacity ◽  
Binding Activity ◽  
Cleavage Sites ◽  
Structural Elements ◽  
Peptide Bonds ◽  
Binding Function ◽  
Biotin Binding ◽  
Hydrolysis Of ◽  
Extreme Stability

The object of this study was to define minimized biotin-binding fragments, or ‘prorecognition sites’, of either the egg-white glycoprotein avidin or its bacterial analogue streptavidin. Because of the extreme stability to enzymic hydrolysis, fragments of avidin were prepared by chemical means and examined for their individual biotin-binding capacity. Treatment of avidin with hydroxylamine was shown to result in new cleavage sites in addition to the known Asn-Gly cleavage site (position 88-89 in avidin). Notably, the Asn-Glu and Asp-Lys peptide bonds (positions 42-43 and 57-58 respectively) were readily cleaved; in addition, lesser levels of hydrolysis of the Gln-Pro (61-62) and Asn-Asp (12-13 and 104-105) bonds could be detected. The smallest biotin-binding peptide fragment, derived from hydroxylamine cleavage of either native or non-glycosylated avidin, was identified to comprise residues 1-42. CNBr cleavage resulted in a 78-amino acid-residue fragment (residues 19-96) that still retained activity. The data ascribe an important biotin-binding function to the overlapping region (residues 19-42) of avidin, which bears the single tyrosine moiety. This contention was corroborated by synthesizing a tridecapeptide corresponding to residues 26-38 of avidin; this peptide was shown to recognize biotin. Streptavidin was not susceptible to either enzymic or chemical cleavage methods used in this work. The approach taken in this study enabled the experimental distinction between the chemical and structural elements of the binding site. The capacity to assign biotin-binding activity to the tyrosine-containing domain of avidin underscores its primary chemical contribution to the binding of biotin by avidin.

scholarly journals Structure—function relationship of xylanase: fluorimetric analysis of the tryptophan environment

1996 ◽  
Vol 315 (2) ◽  
pp. 583-587 ◽  
Author(s):  
Kavita R. BANDIVADEKAR ◽  
Vasanti V. DESHPANDE
Keyword(s):  
Structure Function ◽  
Active Site ◽  
Guanidine Hydrochloride ◽  
Fluorescence Emission ◽  
Tryptophan Residue ◽  
Emission Maximum ◽  
Complete Inactivation ◽  
Tryptophan Residues ◽  
Structure Function Relationship ◽  
Function Relationship

Involvement of one out of three tryptophan residues in the active site of the low-molecular-mass xylanase from Chainia has been demonstrated previously [Deshpande, Hinge and Rao (1990) Biochim. Biophys. Acta 1041, 172–177]. The work described here aims at: (i) deducing the structure–function relationship for the tryptophan residue involved at the active site (a) by correlating the effect of N-bromosuccinimide (NBS) on the fluorescence and activity, and (b) by assessing the ability of xylan to protect against decrease in fluorescence versus activity of NBS-treated enzyme; and (ii) probing into the environment of the tryptophan residues by studying the quenching of their fluorescence by various solute quenchers in the presence and absence of guanidine hydrochloride (Gdn.HCl). Complete inactivation of the NBS-treated enzyme occurs well before the loss of fluorescence. Full protection by xylan (0.5%) of the inactivation of enzyme by NBS compared with 30% protection for the decrease in fluorescence confirms the participation of a single tryptophan at the substrate-binding site of the xylanase. The xylanase exhibited a rather low fluorescence emission maximum at 310 nm. There was no shift in the emission maximum on treatment of the enzyme with Gdn.HCl (6.5 M), indicating the rigidity of the microenvironment around tryptophan residues. The quenching studies with acrylamide suggested the occurrence of both collisional as well as static quenching processes. The enzyme retained full activity as well as the characteristic emission maximum at 310 nm in the presence of acrylamide (100 mM), indicating that quenching of fluorescence by acrylamide is a physical process. Acrylamide was more efficient as a quencher than CsCl or KBr. Treatment of the enzyme with Gdn.HCl resulted in an increase in accessibility of the quenchers to the fluorophore as suggested by an increase in the Stern–Volmer quenching constants (Ksv) of the solute quenchers. The analysis of Ksv and V values of KBr and CsCl suggests that the overall tryptophan microenvironment in the xylanase from Chainia is slightly electronegative.

Studies on the tryptophan residues of soybean agglutinin. Involvement in saccharide binding

1989 ◽  
Vol 9 (2) ◽  
pp. 189-198 ◽  
Author(s):  
Musti Joginadha Swamy ◽  
Avadhesha Surolia
Keyword(s):  
Active Site ◽  
Blue Shift ◽  
Binding Activity ◽  
Carbohydrate Binding ◽  
Side Chains ◽  
Soybean Agglutinin ◽  
Protein Fluorescence ◽  
Saccharide Binding ◽  
Tryptophan Residues ◽  
Tryptophan Side Chains

Modification of tryptophan side chains of soybean agglutinin (SBA) with N-bromosuccinimide results in a loss of the hemagglutinating and carbohydrate binding activities of the protein. One residue/subunit is probably essential for the binding activity. Modification leads to a large decrease in the fluorescene of the protein accompained by a blue shift. Iodide ion quenching of the protein fluorescence shows that saccharide binding results in a decreased accessibility of some of the tryptophan side chains. These results strongly point towards the involvement of tryptophan residues in the active site of SBA.

scholarly journals Structure and Molecular Recognition Mechanism of IMP-13 Metallo-β-Lactamase

2020 ◽  
Vol 64 (6) ◽  
Author(s):  
Charlotte A. Softley ◽  
Krzysztof M. Zak ◽  
Mark J. Bostock ◽  
Roberto Fino ◽  
Richard Xu Zhou ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
Rational Drug Design ◽  
Tryptophan Residue ◽  
Resonance Spectroscopy ◽  
Global Public Health ◽  
Recognition Mechanism ◽  
Gram Negative ◽  
Content Type ◽  
Locking Mechanism

ABSTRACT Multidrug resistance among Gram-negative bacteria is a major global public health threat. Metallo-β-lactamases (MBLs) target the most widely used antibiotic class, the β-lactams, including the most recent generation of carbapenems. Interspecies spread renders these enzymes a serious clinical threat, and there are no clinically available inhibitors. We present the crystal structures of IMP-13, a structurally uncharacterized MBL from the Gram-negative bacterium Pseudomonas aeruginosa found in clinical outbreaks globally, and characterize the binding using solution nuclear magnetic resonance spectroscopy and molecular dynamics simulations. The crystal structures of apo IMP-13 and IMP-13 bound to four clinically relevant carbapenem antibiotics (doripenem, ertapenem, imipenem, and meropenem) are presented. Active-site plasticity and the active-site loop, where a tryptophan residue stabilizes the antibiotic core scaffold, are essential to the substrate-binding mechanism. The conserved carbapenem scaffold plays the most significant role in IMP-13 binding, explaining the broad substrate specificity. The observed plasticity and substrate-locking mechanism provide opportunities for rational drug design of novel metallo-β-lactamase inhibitors, essential in the fight against antibiotic resistance.

scholarly journals Functional homology between the yeast regulatory proteins GAL4 and LAC9: LAC9-mediated transcriptional activation in Kluyveromyces lactis involves protein binding to a regulatory sequence homologous to the GAL4 protein-binding site.

1987 ◽  
Vol 7 (12) ◽  
pp. 4400-4406 ◽  
Author(s):  
K D Breunig ◽  
P Kuger
Keyword(s):  
Protein Binding ◽  
Binding Site ◽  
Transcriptional Activation ◽  
Kluyveromyces Lactis ◽  
Regulatory Protein ◽  
Binding Activity ◽  
Protein Binding Site ◽  
Regulatory Sequence ◽  
Functional Homology ◽  
Beta Galactosidase

As shown previously, the beta-galactosidase gene of Kluyveromyces lactis is transcriptionally regulated via an upstream activation site (UASL) which contains a sequence homologous to the GAL4 protein-binding site in Saccharomyces cerevisiae (M. Ruzzi, K.D. Breunig, A.G. Ficca, and C.P. Hollenberg, Mol. Cell. Biol. 7:991-997, 1987). Here we demonstrate that the region of homology specifically binds a K. lactis regulatory protein. The binding activity was detectable in protein extracts from wild-type cells enriched for DNA-binding proteins by heparin affinity chromatography. These extracts could be used directly for DNase I and exonuclease III protection experiments. A lac9 deletion strain, which fails to induce the beta-galactosidase gene, did not contain the binding factor. The homology of LAC9 protein with GAL4 (J.M. Salmeron and S. A. Johnston, Nucleic Acids Res. 14:7767-7781, 1986) strongly suggests that LAC9 protein binds directly to UASL and plays a role similar to that of GAL4 in regulating transcription.

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