Pasternak A, Liu X, Lin T Y, Hedstrom L
Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02454, USA.
Biochemistry. 1998 Nov 17;37(46):16201-10. doi: 10.1021/bi980951d.
The zymogen and mature enzyme forms of trypsin-like serine proteases exhibit a wide range of activities. The prototypical trypsinogen-trypsin system is an example of a minimally active zymogen and a maximally active mature protease. The present work identifies several features of trypsinogen which govern its activity. Our results indicate that rat trypsin is 10(8)-fold more active than rat trypsinogen. Rat trypsinogen appears to be less active than bovine trypsinogen. His40 is believed to be an important determinant of zymogen activity. We are unable to verify this role for His40 in trypsinogen since the mutation of His40 to Phe appears to change the trypsin-substrate interface. Deletion of the N-terminal Ile16 from trypsin is expected to produce a trypsinogen-like protein since the Ile16-Asp194 salt bridge cannot form. Such mutants have higher activity and BPTI affinity than trypsinogen, which indicates that the activation peptide stabilizes the inactive trypsinogen conformation. The mutation of Lys15 to Ala increases the BPTI affinity and activity of trypsinogen to an even greater extent; thus, removal of Lys15 can account for the effect of the loss of the activation peptide. These results suggest that Lys15 is an important determinant of zymogen activity. The mutation of Asp194 to Asn also increases the BPTI affinity and activity of trypsinogen. This result suggests that in addition to stabilizing the active conformation of trypsin via the Ile16-Asp194 salt bridge, Asp194 also maintains the inactive conformation of trypsinogen. A correlation exists between the values of kcat/Km and BPTI affinity of mutant trypsinogens and trypsins. However, the slope of this correlation is 0.64, which indicates that different "active" conformations are involved in BPTI binding and substrate hydrolysis. DeltaI16V17 trypsinogen is the lone outlier; its BPTI affinity is higher than would be expected based on the value of kcat/Km. We show that the rate of BPTI association is slower for DeltaI16V17 trypsinogen than for a mutant trypsinogen with a similar BPTI affinity. This observation suggests that BPTI binds to an "active" trypsinogen conformation that is not kinetically accessible to substrates.
类胰蛋白酶丝氨酸蛋白酶的酶原形式和成熟酶形式表现出广泛的活性。典型的胰蛋白酶原 - 胰蛋白酶系统就是一个活性最低的酶原和活性最高的成熟蛋白酶的例子。目前的研究确定了胰蛋白酶原中一些决定其活性的特征。我们的结果表明，大鼠胰蛋白酶的活性比大鼠胰蛋白酶原高10⁸倍。大鼠胰蛋白酶原的活性似乎低于牛胰蛋白酶原。人们认为His40是酶原活性的一个重要决定因素。由于His40突变为Phe似乎改变了胰蛋白酶 - 底物界面，我们无法验证His40在胰蛋白酶原中的这一作用。从胰蛋白酶中删除N端的Ile16预计会产生一种类似胰蛋白酶原的蛋白质，因为Ile16 - Asp194盐桥无法形成。这种突变体比胰蛋白酶原具有更高的活性和对枯草杆菌蛋白酶抑制剂（BPTI）的亲和力，这表明激活肽稳定了无活性的胰蛋白酶原构象。将Lys15突变为Ala会更大程度地增加胰蛋白酶原对BPTI的亲和力和活性；因此，去除Lys15可以解释激活肽缺失的影响。这些结果表明Lys15是酶原活性的一个重要决定因素。将Asp194突变为Asn也会增加胰蛋白酶原对BPTI的亲和力和活性。这一结果表明，除了通过Ile16 - Asp194盐桥稳定胰蛋白酶的活性构象外，Asp194还维持了胰蛋白酶原的无活性构象。突变型胰蛋白酶原和胰蛋白酶的kcat/Km值与BPTI亲和力之间存在相关性。然而，这种相关性的斜率为0.64，这表明在BPTI结合和底物水解过程中涉及不同的“活性”构象。DeltaI16V17胰蛋白酶原是唯一的异常值；其对BPTI的亲和力高于根据kcat/Km值预期的值。我们表明，DeltaI16V17胰蛋白酶原与BPTI结合的速率比具有相似BPTI亲和力的突变型胰蛋白酶原慢。这一观察结果表明，BPTI结合到一种底物在动力学上无法接近的“活性”胰蛋白酶原构象上。
