Schellenberger V, Turck C W, Hedstrom L, Rutter W J
Hormone Research Institute, University of California, San Francisco 94143.
Biochemistry. 1993 Apr 27;32(16):4349-53. doi: 10.1021/bi00067a026.
We have developed a rapid and convenient procedure for the characterization of the S' subsite specificity of serine proteases. A mixture of peptide nucleophiles is incubated with the enzyme in the presence of excess of a specific ester substrate. The decrease in each nucleophile concentration is monitored by high-performance liquid chromatography analysis of the dansylated mixture. Relative kinetic parameters for each nucleophile in the mixture are then calculated using a new statistical algorithm that relates all pairs of nucleophiles. As a first application, we investigated the S'1 subsite specificity of chymotrypsin, trypsin, and a recently described trypsin mutant, Tr-->Ch[S1 + L1 + L2] with chymotrypsin-like primary specificity [Hedstrom, L., Szilagyi, L., & Rutter, W. J. (1992) Science 255, 1249-1253]. For this purpose 21 peptide nucleophiles of the general structure H-Xaa-Ala-Ala-Ala-Ala-NH2 were prepared by multiple solid-phase synthesis, where Xaa represents D-alanine, citrulline, and all natural amino acids except cysteine. Relative second-order rate constants for the enzyme-catalyzed acyl transfer to these nucleophiles were determined over a range of 10(2). Chymotrypsin and trypsin have markedly different S'1 specificities. The order of preference in chymotrypsin-catalyzed acyl transfer reactions is positively charged > aliphatic > aromatic >> negatively charged, D-Ala, Pro P'1 side chain. Trypsin prefers hydrophobic residues, but like chymotrypsin aliphatic residues are better than aromatic residues in P'1 position. The S'1 specificity of the mutant Tr-->Ch[S1 + L1 + L2] is similar to the specificity of trypsin; however, P'1 aromatic residues have low reactivity characteristic of chymotrypsin.
我们已经开发出一种快速便捷的方法来表征丝氨酸蛋白酶的S'亚位点特异性。将肽亲核试剂混合物与酶在过量特定酯底物存在的情况下孵育。通过对丹磺酰化混合物进行高效液相色谱分析来监测每种亲核试剂浓度的降低。然后使用一种新的统计算法计算混合物中每种亲核试剂的相对动力学参数，该算法关联了所有亲核试剂对。作为首次应用，我们研究了胰凝乳蛋白酶、胰蛋白酶以及最近描述的具有胰凝乳蛋白酶样一级特异性的胰蛋白酶突变体Tr-->Ch[S1 + L1 + L2]的S'1亚位点特异性[赫德斯特伦，L.，西拉吉，L.，& 鲁特，W. J.（1992年）《科学》255，1249 - 1253]。为此，通过多步固相合成制备了21种具有通用结构H-Xaa-Ala-Ala-Ala-Ala-NH2的肽亲核试剂，其中Xaa代表D-丙氨酸、瓜氨酸以及除半胱氨酸外的所有天然氨基酸。测定了在10(2)范围内酶催化酰基转移至这些亲核试剂的相对二级速率常数。胰凝乳蛋白酶和胰蛋白酶具有明显不同的S'1特异性。在胰凝乳蛋白酶催化的酰基转移反应中，偏好顺序为带正电荷的 > 脂肪族的 > 芳香族的 >> 带负电荷的、D-丙氨酸、脯氨酸P'1侧链。胰蛋白酶偏好疏水残基，但与胰凝乳蛋白酶一样，P'1位置的脂肪族残基比芳香族残基更优。突变体Tr-->Ch[S1 + L1 + L2]的S'1特异性与胰蛋白酶的特异性相似；然而，P'1芳香族残基具有胰凝乳蛋白酶特有的低反应性。
