Studies on the biotin-binding sites of avidin and streptavidin. Tyrosine residues are involved in the binding site.
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.
Gitlin
G
G
Department of Biophysics, Weizmann Institute of Science, Rehovot, Israel.
Bayer
E A
EA
Wilchek
M
M
D000595:Amino Acid Sequence; D001360:Avidin / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D001665:Binding Sites; D001710:Biotin / Q000378:metabolism*; D055598:Chemical Phenomena; D002621:Chemistry; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin; D006868:Hydrolysis; D007202:Indicators and Reagents; D008969:Molecular Sequence Data; D009578:Nitrobenzenes; D010446:Peptide Fragments; D019809:Streptavidin; D014357:Trypsin; D014443:Tyrosine
D001426:Bacterial Proteins; D007202:Indicators and Reagents; D009578:Nitrobenzenes; D010446:Peptide Fragments; D001360:Avidin; C034442:4-nitrobenzenesulfonyl fluoride; D014443:Tyrosine; D001710:Biotin; D019809:Streptavidin; D002918:Chymotrypsin; D014357:Trypsin