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-α-d-glucopyranosyloxy-1-[3H]butyl)-6-deoxy-4-thio-α-d-xylo-hex-5-enopyranosyl]-α-maltoside (12a, G1-G3*), methyl 4-O-[4-S-(3-azi-4-α-maltosyloxy-1-[3H]butyl)-6-deoxy-4-thio-α-d-xylo-hex-5-enopyranosyl]-α-d-glucopyranoside (15a, G2-G2*) and methyl 4-S-(3-azi-4-α-maltotriosyloxy-1-[3H]butyl)-6-deoxy-4-thio-α-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.