Brown Christopher M, Ray Manisha, Eroy-Reveles Aura A, Egea Pascal, Tajon Cheryl, Craik Charles S
Graduate Group in Biochemistry and Molecular Biology, University of California, San Francisco, CA 94158, USA.
Chem Biol. 2011 Jan 28;18(1):48-57. doi: 10.1016/j.chembiol.2010.11.007.
The ability to follow enzyme activity in a cellular context represents a challenging technological frontier that impacts fields ranging from disease pathogenesis to epigenetics. Activity-based probes (ABPs) label the active form of an enzyme via covalent modification of catalytic residues. Here we present an analysis of parameters influencing potency of peptide phosphonate ABPs for trypsin-fold S1A proteases, an abundant and important class of enzymes with similar substrate specificities. We find that peptide length and stability influence potency more than sequence composition and present structural evidence that steric interactions at the prime-side of the substrate-binding cleft affect potency in a protease-dependent manner. We introduce guidelines for the design of peptide phosphonate ABPs and demonstrate their utility in a live-cell labeling application that specifically targets active S1A proteases at the cell surface of cancer cells.
在细胞环境中追踪酶活性的能力代表了一个具有挑战性的技术前沿，其影响范围涵盖从疾病发病机制到表观遗传学等多个领域。基于活性的探针（ABP）通过对催化残基的共价修饰来标记酶的活性形式。在此，我们对影响肽膦酸酯ABP对胰蛋白酶折叠S1A蛋白酶效力的参数进行了分析，胰蛋白酶折叠S1A蛋白酶是一类丰富且重要的酶，具有相似的底物特异性。我们发现，肽的长度和稳定性对效力的影响大于序列组成，并提供了结构证据表明底物结合裂隙prime侧的空间相互作用以蛋白酶依赖性方式影响效力。我们引入了肽膦酸酯ABP的设计指南，并展示了它们在活细胞标记应用中的效用，该应用可特异性靶向癌细胞表面的活性S1A蛋白酶。
