Lesner A, Brzozowski K, Kupryszewski G, Rolka K
Faculty of Chemistry, University of Gdańsk, Gdańsk, PL-80-502, Poland.
Biochem Biophys Res Commun. 2000 Mar 5;269(1):81-4. doi: 10.1006/bbrc.2000.2257.
A series of trypsin chromogenic substrates with formula: Y-Ala-X-Abu-Pro-Lys-pNA, where X = Gly, Ala, Abu, Val, Leu, Phe, Ser, Glu and Y = Ac, H; pNA = p-nitroanilide was synthesized. The Cucurbita maxima trypsin inhibitor CMTI-III molecule was used as a vehicle to design the trypsin substrates. To evaluate the influence of position P(4) on the substrate-enzyme interaction, kinetic parameters of newly synthesized substrates with bovine beta-trypsin were determined. The increasing hydrophobicity of the amino acid residue (Gly, Ala, Abu, Val) introduced in position P(4) significantly enhanced the substrate specificity (k(cat)/K(m)) which was over 8 times higher for the last residue than that for the first one. The introduction of residues with more hydrophilic side chain (Glu, Ser) in this position reduced the value of this parameter. These results correspond well with those obtained using molecular dynamics of bovine beta-trypsin with monosubstituted CMTI-I analogues, indicating that in both trypsin substrate and inhibitor position 4 plays an important role in the interaction with the enzyme.
合成了一系列通式为Y-Ala-X-Abu-Pro-Lys-pNA的胰蛋白酶显色底物，其中X = Gly、Ala、Abu、Val、Leu、Phe、Ser、Glu，Y = Ac、H；pNA = 对硝基苯胺。以南瓜胰蛋白酶抑制剂CMTI-III分子为载体设计胰蛋白酶底物。为评估P(4)位对底物-酶相互作用的影响，测定了新合成底物与牛β-胰蛋白酶的动力学参数。引入P(4)位的氨基酸残基（Gly、Ala、Abu、Val）疏水性增加，显著增强了底物特异性（k(cat)/K(m)），最后一个残基的该参数比第一个残基高出8倍以上。在此位置引入具有更多亲水性侧链的残基（Glu、Ser）会降低该参数的值。这些结果与使用牛β-胰蛋白酶与单取代CMTI-I类似物的分子动力学得到的结果非常吻合，表明在胰蛋白酶底物和抑制剂中，4位在与酶的相互作用中都起着重要作用。
