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

这个关于“结构相关丝氨酸蛋白酶”的美国-匈牙利生化研究项目是由加州大学旧金山分校激素研究所的William J. Rutter博士和布达佩斯Eotvos大学的Gabor Pal博士共同完成的。宁静的蛋白酶，如胰蛋白酶和凝乳胰蛋白酶，共享一个三维结构，具有相同的催化装置，但显示出明显不同的底物特异性。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袋（残基189195,214 - 220,225 -228）进行一系列改变，产生具有凝乳胰蛋白酶样特异性的突变体。其中一种胰蛋白酶突变体（D189S, YG217-219SGG, Q192M）特别令人感兴趣。它对凝乳胰蛋白酶的各种肽底物（P1=Leu, Phe, Tyr, Trp）的亲和力与凝乳胰蛋白酶相当，而其催化效率（Kcat/Km）比凝乳胰蛋白酶或胰蛋白酶低3-4个数量级。因此，在基态中重新设计的和明显紧密的酶-底物相互作用似乎并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凝乳胰蛋白酶与它们自身底物的相互作用可能会引起对最佳过渡态稳定至关重要的不同构象变化。在这项提议中，研究人员提出，在底物结合后介导这种差异结构变化的结构单元可能与胰蛋白酶和凝乳蛋白酶的所谓“激活域”相同。研究人员认为，胰蛋白酶和凝乳胰蛋白酶中这些结构域的不同构象灵活性可能代表了这两种蛋白酶不同底物特异性的结构基础。因此，为了交换胰蛋白酶和凝乳胰蛋白酶的底物特异性（而不显著失去活性），必须交换激活域而不仅仅是底物结合袋。研究人员将验证这一假设。该生物化学项目通过招募美国和东欧的顶尖专家，在共同感兴趣和能力强的领域继续补充人才和汇集资源，实现了推进科学知识的计划目标。

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.