Catalytic Role of Proton Transfers in the Formation of a Tetrahedral Adduct in a Serine Carboxyl Peptidase

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• 作者：Haobo Guo ; Alexander Wlodawer ; Toru Nakayama ; Qin Xu ; Hong Guo
• 刊名：Biochemistry
• 出版年：2006
• 出版时间：August 1, 2006
• 年：2006
• 卷：45
• 期：30
• 页码：9129 - 9137
• 全文大小：432K
• 年卷期：v.45,no.30(August 1, 2006)
• ISSN：1520-4995

文摘

Quantum mechanical/molecular mechanical molecular dynamics and 2D free energy simulationsare performed to study the formation of a tetrahedral adduct by an inhibitor N-acetyl-isoleucyl-prolyl-phenylalaninal (AcIPF) in a serine-carboxyl peptidase (kumamolisin-As) and elucidate the role of protontransfers during the nucleophilic attack by the Ser278 catalytic residue. It is shown that although theserine-carboxyl peptidases have a fold resembling that of subtilisin, the proton transfer processes duringthe nucleophilic attack by the Ser residue are likely to be more complex for these enzymes compared tothe case in classical serine proteases. The computer simulations demonstrate that both general base andacid catalysts are required for the formation and stabilization of the tetrahedral adduct. The 2D free energymaps further demonstrate that the proton transfer from Ser278 to Glu78 (the general base catalyst) issynchronous with the nucleophilic attack, whereas the proton transfer from Asp164 (the general acidcatalyst) to the inhibitor is not. The dynamics of the protons at the active site in different stages of thenucleophilic attack as well as the motions of the corresponding functional groups are also studied. It isfound that the side chain of Glu78 is generally rather flexible, consistent with its possible multifunctionalrole during catalysis. The effects of proton shuffling from Asp82 to Glu78 and from Glu32 to Asp82 areexamined, and the results indicate that such proton shuffling may not play an important role in thestabilization of the tetrahedral intermediate analogue.