DIFFERENTIAL CHEMICAL MODIFICATION OF SUBSTRATE-BINDING AREAS IN PORCINE-PANCREATIC ALPHA-AMYLASE BY 3 REGIOISOMERIC PHOTOLABILE LIGANDS

Three regioisomeric radiolabelled spacer-modified oligosaccharides: me thyl o-alpha-D-xylo-hex-5-enopyranosyl]-alpha-maltoside (12a, G1-G3), methyl D-xylo-hex-5-enopyranosyl]-alpha-D-glucopyranoside (15a, G2-G2 ) and methyl l)-6-deoxy-4-thio-alpha-D-xylo-hex-5-enopyranoside (16a, G3-G1) were synthesised and used as photoaffinity probes for the che mical modification of porcine-pancreatic alpha-amylase (PPA). Incorpor ation of covalently attached radioactivity amounted to 25-38% of the s toichiometric value. Tryptic digestion of the three labelled protein p reparations PPA-G1-G3, PPA-G2-G2*, and PPA-G3-G1* and the purificatio n of the labelled peptides by fractional HPLC yielded altogether six p ure components. On the basis of the published three-dimensional struct ure peptides G1-G3-II, G2-G2-II, and G2-G2-III were part of the cataly tic site. G1-G3-I and G2-G2-I were part of the surface binding site. T he 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 thir d, hitherto undetected, substrate-binding site.