U.S.-Hungary Biochemical Research on Structurally Related Serine Proteases

美国-匈牙利关于“结构相关丝氨酸蛋白酶”的生化研究项目是由加州大学旧金山分校荷尔蒙研究所的William J.Rutter博士和布达佩斯Eotvos大学的Gabor Pal博士共同完成的。丝氨酸蛋白酶，如胰酶和胰凝乳酶，具有相同的三维结构和相同的催化装置，但表现出显著不同的底物特异性。因此，它们是研究底物识别过程(基态稳定)与催化(过渡态稳定)之间关系的理想模型。通过定点突变，对胰蛋白酶底物结合口袋(残基189195、214-220、225-228)进行了一系列改变，以产生具有凝乳酶样特异性的突变体。其中一个胰酶突变体(D189S，YG217-219SGG，Q192M)特别令人感兴趣。它对胰凝乳酶(P1=Leu，Phe，Tyr，Trp)的各种多肽底物的亲和力与胰凝乳酶相当，而其催化效率(Kcat/Km)比凝乳酶或胰蛋白酶低3-4个数量级。因此，重新设计的和表面上紧密的基态酶-底物相互作用似乎不能适当地促进过渡态的稳定。对这些结果的一种解释是，胰蛋白酶和胰凝乳素与它们自己的底物相互作用可能会导致不同的构象变化，这对最佳过渡态的稳定至关重要。在这项提议中，研究人员认为，在底物结合时介导这种不同结构变化的结构单元可能与所谓的胰蛋白酶和糜蛋白酶的“激活结构域”相同。研究人员认为，这些结构域在胰蛋白酶和糜蛋白酶中的不同构象灵活性可能代表了这两种酶不同底物特异性的结构基础。因此，为了互换胰酶和胰凝乳酶的底物特异性(而不是活性的显著丧失)，激活区域而不仅仅是底物结合口袋必须互换。研究人员将对这一假设进行检验。这个生物化学项目通过招募美国和东欧的顶尖专家来继续互补人才，并在相互感兴趣和能力很强的领域汇集资源，从而实现了促进科学知识发展的计划目标。

This US-Hungary biochemical research project on "Structurally Related Serine Proteases" is between Dr. William J. Rutter of The Hormone Research Institute, University of California-San Francisco, and Dr. Gabor Pal of Eotvos University, Budapest. Serene proteases, such as trypsin and chymotrypsin, share a 3-dimensional structure and possess an identical catalytic apparatus but display strikingly different substrate specificities. As such, they are ideal models to study how the substrate recognition process (ground state stabilization) relates to catalysis (transition state stabilization). Via site-directed mutagenesis, a series of alterations of the substrate binding pocket of trypsin (residues 189195, 214-220, 225-228) were made to produce mutants with chymotrypsin-like specificity. One of the trypsin mutants (D189S, YG217-219SGG, Q192M) is of particular interest. Its affinity toward various peptide substrates of chymotrypsin (P1=Leu, Phe, Tyr, Trp) is comparable to that of chymotrypsin, while its catalytic efficiency (Kcat/Km) is 3-4 orders of magnitude less than that of chymotrypsin or trypsin. Thus the redesigned and apparently tight enzyme-substrate interactions in the ground state do not seem to properly contribute to transition state stabilization. One interpretation of these results is that the interaction of trypsin and chyumotrypsin with their own substrates might induce differential conformational changes crucial for optimal transition state stabilization. In this proposal, the researchers suggest that the structural unit that mediates such differential structural changes upon substrate binding may be identical with the so-called "activation domain" of trypsin and chymotryupsin. The researchers' view is that the different conformational flexibility of these domains in trypsin and chymotrypsin may represent the structural basis for different substrate specificities of these two proteases. Thus, to interchange the substrate specificities of trypsin and chymotrypsin (without a dramatic loss of activity), the activation domains rather than just the substrate binding pockets have to be interchanged. The researchers will test this hypothesis. This project in biochemistry fulfills the program objective of advancing scientific knowledge by enlisting leading experts in the United States and Eastern Europe to continue complementary talents and pool resources in areas of strong mutual interest and competence.