Ruggles Sandra Waugh, Fletterick Robert J, Craik Charles S
Graduate Group in Biophysics, University of California, San Francisco, California 94143-2280, USA.
J Biol Chem. 2004 Jul 16;279(29):30751-9. doi: 10.1074/jbc.M400949200. Epub 2004 May 3.
Granzymes are trypsin-like serine proteases mediating apoptotic cell death that are composed of two genetically distinct subfamilies: granzyme A-like proteases resemble trypsin in their active site architecture, while granzyme B-like proteases are quite distinct. Granzyme B prefers substrates containing P4 to P1 amino acids Ile/Val, Glu/Met/Gln, Pro/Xaa, and aspartic acid N-terminal to the proteolytic cleavage. By investigating the narrow extended specificity of the granzyme B-like proteases the mediators of their unique specificity are being defined. The foci of this study were the structural determinants Ile99, Tyr174, Arg192, and Asn218. Even modest mutations of these residues resulted in unique extended specificity profiles as determined using combinatorial substrate libraries and individual fluorogenic substrates. As with other serine proteases, Ile99 completely defines and predicts P2 specificity, primarily through the binding constant Km. Asn218 variants have minor effects alone but in combination with mutations at Arg192 and Ile99 alter P2 through P4 extended specificity. For each variant, the activity on its cognate substrate was equal to that of granzyme B for the same substrate. Thus, mutations at these determinants change extended selectivity preferentially over catalytic power. Additionally Asn218 variants result in increased activity on the wild type substrate, while the N218A/I99A variant disrupts the additivity between P2 and P4 specificity. This defines Asn218 not only as a determinant of specificity but also as a structural component required for P2 and P4 independence. This study confirms four determinants of granzyme B extended substrate specificity that constitute a canon applicable to the study of the remaining family members.
颗粒酶是介导凋亡性细胞死亡的胰蛋白酶样丝氨酸蛋白酶，由两个基因上不同的亚家族组成：颗粒酶A样蛋白酶在其活性位点结构上类似于胰蛋白酶，而颗粒酶B样蛋白酶则截然不同。颗粒酶B更喜欢含有P4至P1氨基酸Ile/Val、Glu/Met/Gln、Pro/Xaa以及蛋白水解切割N端天冬氨酸的底物。通过研究颗粒酶B样蛋白酶的狭窄扩展特异性，其独特特异性的介导因子正在被确定。本研究的重点是结构决定因素Ile99、Tyr174、Arg192和Asn218。使用组合底物文库和单个荧光底物测定，这些残基即使是适度的突变也会导致独特的扩展特异性谱。与其他丝氨酸蛋白酶一样，Ile99主要通过结合常数Km完全定义并预测P2特异性。Asn218变体单独作用时影响较小，但与Arg192和Ile99处的突变结合会改变P2至P4的扩展特异性。对于每个变体，其对同源底物的活性与颗粒酶B对相同底物的活性相同。因此，这些决定因素处的突变优先改变扩展选择性而非催化能力。此外，Asn218变体导致对野生型底物的活性增加，而N218A/I99A变体破坏了P2和P4特异性之间的加和性。这不仅将Asn218定义为特异性的决定因素，还定义为P2和P4独立性所需的结构成分。本研究证实了颗粒酶B扩展底物特异性的四个决定因素，这些因素构成了适用于研究其余家族成员的准则。
