Wang J, Xiang Y F, Lim C
Department of Molecular and Medical Genetics, University of Toronto, Ontario, Canada.
Protein Eng. 1994 Jan;7(1):75-82. doi: 10.1093/protein/7.1.75.
The 1.65 A X-ray structure of papain, which exhibits a Cys25-His159-Asn175 triad, does not correspond to the catalytically active ion pair state since Cys25 is oxidized to cysteic acid and His159 is predominantly neutral. Thus, stochastic boundary molecular dynamics simulations starting from the 1.65 A X-ray structure of papain have been performed for Cys25 and His159 in the SH-ImH+, SH-Im, S(-)-ImH+ and S(-)-Im states and for Asp158 mutated to Asn, Glu and Gly in the ion pair state. By comparing the resulting averaged structures and analyzing the trajectories of certain interatomic distances, important differences in the active-site geometry of papain have been found. In particular, the initial Cys25(S-)-His159(ImH+)-Asn175(C = O) triad found in the X-ray structure is retained in all the structures except the wild type and Asp158-->Asn ion pair states where there is a conformational transition to form the triad, Cys25(S-)-His159(ImH+)-Asp158(COO-). Both triads, Cys25(S-)-His159(ImH+)-Asp158(COO-) and Cys25(S-)-His159(ImH+)-Asn175(C = O) are postulated to participate in catalysis and their roles are discussed. Thus, catalysis does not take place from a single steric position but a two-state mechanism.
木瓜蛋白酶的1.65埃X射线结构显示出半胱氨酸25-组氨酸159-天冬酰胺175三联体，但它并不对应于催化活性离子对状态，因为半胱氨酸25被氧化成了半胱氨酸磺酸，且组氨酸159主要呈中性。因此，针对处于SH-ImH⁺、SH-Im、S(-)-ImH⁺和S(-)-Im状态的半胱氨酸25和组氨酸159，以及在离子对状态下突变为天冬酰胺、谷氨酸和甘氨酸的天冬氨酸158，从木瓜蛋白酶的1.65埃X射线结构出发进行了随机边界分子动力学模拟。通过比较所得的平均结构并分析某些原子间距离的轨迹，发现了木瓜蛋白酶活性位点几何结构的重要差异。特别是，在X射线结构中发现的初始半胱氨酸25(S⁻)-组氨酸159(ImH⁺)-天冬酰胺175(C=O)三联体在所有结构中都得以保留，除了野生型和天冬氨酸158→天冬酰胺离子对状态，在这两种状态下会发生构象转变以形成三联体半胱氨酸25(S⁻)-组氨酸159(ImH⁺)-天冬氨酸158(COO⁻)。假定半胱氨酸25(S⁻)-组氨酸159(ImH⁺)-天冬氨酸158(COO⁻)和半胱氨酸25(S⁻)-组氨酸159(ImH⁺)-天冬酰胺175(C=O)这两种三联体都参与催化，并对它们的作用进行了讨论。因此，催化并非从单一空间位置发生，而是通过一种双态机制。
