Lehmann J, Marchis-Mouren G, Schiltz E, Schmidt-Schuchardt M
Institut für Organische Chemie und Biochemie, Universität Freiburg, Germany.
Carbohydr Res. 1994 Dec 2;265(1):19-30. doi: 10.1016/0008-6215(94)00225-8.
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.
4'-O-[4-S-(3-叠氮基-4-α-D-吡喃葡萄糖氧基-1-[³H]丁基)-6-脱氧-4-硫代-α-D-木糖基己-5-烯吡喃糖基]-α-麦芽糖甲基酯(12a, G1-G3*)、4-O-[4-S-(3-叠氮基-4-α-麦芽糖氧基-1-[³H]丁基)-6-脱氧-4-硫代-α-D-木糖基己-5-烯吡喃糖基]-α-D-葡萄糖吡喃糖苷甲基酯(15a, G2-G2*)和4-S-(3-叠氮基-4-α-麦芽三糖氧基-1-[³H]丁基)-6-脱氧-4-硫代-α-D-木糖基己-5-烯吡喃糖苷甲基酯(16a, G3-G1*)，并将其用作光亲和探针用于猪胰腺α-淀粉酶(PPA)的化学修饰。共价连接放射性的掺入量达到化学计量值的25%至38%。对三种标记的蛋白质制剂PPA-G1-G3*、PPA-G2-G2和PPA-G3-G1进行胰蛋白酶消化，并通过分级HPLC纯化标记的肽，共得到六个纯组分。根据已发表的三维结构，肽G1-G3-II、G2-G2-II和G2-G2-III是催化位点的一部分。G1-G3-I和G2-G2-I是表面结合位点的一部分。由G3-G1*标记的源自PPA的主要组分对应于一个既不靠近活性位点也不靠近表面淀粉结合结构域的区域，这清楚地表明存在第三个迄今未检测到的底物结合位点。
