Differential chemical modification of substrate binding areas in porcine-pancreatic alpha-amylase by three regioisomeric photolabile ligands.
Three regioisomeric radiolabelled spacer-modified oligosaccharides: methyl 4'-O-[4-S-(3-azi-4-alpha-D-glucopyranosyloxy-1-[3H]butyl)-6- deoxy- 4-thio-alpha-D-xylo-hex-5-enopyranosyl]-alpha-maltoside (12a, G1-G3*), methyl 4-O-[4-S-(3-azi-4-alpha-maltosyloxy-1-[3H]butyl)-6-deoxy-4-t hio- alpha-D-xylo-hex-5-enopyranosyl]-alpha-D-glucopyranoside (15a, G2-G2*) and methyl 4-S-(3-azi-4-alpha-maltotriosyloxy-1-[3H]butyl)-6-deoxy-4-th io-alpha- D-xylo-hex-5-enopyranoside (16a, G3-G1*) were synthesised and used as photoaffinity probes for the chemical modification of porcine-pancreatic alpha-amylase (PPA). Incorporation of covalently attached radioactivity amounted to 25-38% of the stoichiometric value. Tryptic digestion of the three labelled protein preparations PPA-G1-G3*, PPA-G2-G2*, and PPA-G3-G1* and the purification of the labelled peptides by fractional HPLC yielded altogether six pure components. On the basis of the published three-dimensional structure peptides G1-G3-II, G2-G2-II, and G2-G2-III were part of the catalytic site. G1-G3-I and G2-G2-I were part of the surface binding site. The major component derived from PPA, labelled by G3-G1*, corresponded to an area that is neither close to the active site nor to the surface starch-binding domain, which clearly indicates the presence of a third, hitherto undetected, substrate-binding site.
Institut für Organische Chemie und Biochemie, Universität Freiburg, Germany.
J
J
Lehmann
G
G
Marchis-Mouren
E
E
Schiltz
M
M
Schmidt-Schuchardt
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002240:Carbohydrate Sequence; D008024:Ligands; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000138:chemical synthesis*; D010179:Pancreas / Q000201:enzymology; D010446:Peptide Fragments / Q000737:chemistry; D010777:Photochemistry; D013379:Substrate Specificity; D013552:Swine; D014357:Trypsin; D000516:alpha-Amylases / Q000737:chemistry*
D008024:Ligands; D009844:Oligosaccharides; D010446:Peptide Fragments; D000516:alpha-Amylases; D014357:Trypsin