Seigner C, Prodanov E, Marchis-Mouren G
Eur J Biochem. 1985 Apr 1;148(1):161-8. doi: 10.1111/j.1432-1033.1985.tb08820.x.
Hydrolysis of small substrates (maltose, maltotriose and o-nitrophenylmaltoside) catalysed by porcine pancreatic alpha-amylase was studied from a kinetic viewpoint over a wide range of substrate concentrations. Non-linear double-reciprocal plots are obtained at high maltose, maltotriose and o-nitrophenylmaltoside concentrations indicating typical substrate inhibition. These results are consistent with the successive binding of two molecules of substrate per enzyme molecule with dissociation constants Ks1 and Ks2. The Hill plot, log [v/(V-v)] versus log [S], is clearly biphasic and allows the dissociation constants of the ES1 and ES2 complexes to be calculated. Maltose and maltotriose are inhibitors of the amylase-catalysed amylose and o-nitrophenylmaltoside hydrolysis. The inhibition is of the competitive type. The (apparent) inhibition constant Kiapp varies with the inhibitor concentration. These results are also consistent with the successive binding of at least two molecules of maltose or maltotriose per amylase molecule with the dissociation constants Ki1 and Ki2. These inhibition studies show that small substrates and large polymeric ones are hydrolysed at the same catalytic site(s). The values of the dissociation constants Ks1 and Ki1 of the maltose-amylase complexes are identical. According to the five-subsite energy profile previously determined, at low concentration, maltose (as substrate and as inhibitor) binds to the same two sites (4,5) or (3,4), maltotriose (as substrate and as inhibitor) and o-nitrophenyl-maltoside (as substrate) bind to the same three subsites (3,4,5). The dissociation constants Ks2 and Ki2 determined at high substrate and inhibitor concentration are consistent with the binding of the second ligand molecule at a single subsite. The binding mode of the second molecule of maltose (substrate) and o-nitrophenylmaltoside remains uncertain, very likely because of the inaccuracy due to simplifications in the calculations of the subsite binding energies. No binding site(s) outside the catalytic one has been taken into account in this model.
从动力学角度，在较宽的底物浓度范围内研究了猪胰α-淀粉酶催化的小底物（麦芽糖、麦芽三糖和邻硝基苯麦芽苷）的水解反应。在高麦芽糖、麦芽三糖和邻硝基苯麦芽苷浓度下获得了非线性双倒数图，表明存在典型的底物抑制作用。这些结果与每个酶分子连续结合两个底物分子且解离常数分别为Ks1和Ks2一致。希尔图，即log [v/(V - v)]对log [S]作图，明显呈双相，可计算出ES1和ES2复合物的解离常数。麦芽糖和麦芽三糖是淀粉酶催化的直链淀粉和邻硝基苯麦芽苷水解反应的抑制剂。这种抑制作用属于竞争性类型。（表观）抑制常数Kiapp随抑制剂浓度而变化。这些结果也与每个淀粉酶分子至少连续结合两个麦芽糖或麦芽三糖分子且解离常数分别为Ki1和Ki2一致。这些抑制研究表明，小底物和大聚合物底物在相同的催化位点水解。麦芽糖 - 淀粉酶复合物的解离常数Ks1和Ki1的值相同。根据先前确定的五亚位点能量分布，在低浓度下，麦芽糖（作为底物和抑制剂）结合到相同的两个位点（4,5）或（3,4），麦芽三糖（作为底物和抑制剂）和邻硝基苯麦芽苷（作为底物）结合到相同的三个亚位点（3,4,5）。在高底物和抑制剂浓度下测定的解离常数Ks2和Ki2与第二个配体分子在单个亚位点的结合一致。麦芽糖（底物）和邻硝基苯麦芽苷第二个分子的结合模式仍不确定，很可能是由于亚位点结合能计算简化导致的不准确。该模型未考虑催化位点之外的结合位点。
