title: Catalytic role of proton transfers in the formation of a tetrahedral adduct in a serine carboxyl peptidase.
abstract: Quantum mechanical/molecular mechanical molecular dynamics and 2D free energy simulations are 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 proton transfers during the nucleophilic attack by the Ser278 catalytic residue. It is shown that although the serine-carboxyl peptidases have a fold resembling that of subtilisin, the proton transfer processes during the nucleophilic attack by the Ser residue are likely to be more complex for these enzymes compared to the case in classical serine proteases. The computer simulations demonstrate that both general base and acid catalysts are required for the formation and stabilization of the tetrahedral adduct. The 2D free energy maps further demonstrate that the proton transfer from Ser278 to Glu78 (the general base catalyst) is synchronous with the nucleophilic attack, whereas the proton transfer from Asp164 (the general acid catalyst) to the inhibitor is not. The dynamics of the protons at the active site in different stages of the nucleophilic attack as well as the motions of the corresponding functional groups are also studied. It is found that the side chain of Glu78 is generally rather flexible, consistent with its possible multifunctional role during catalysis. The effects of proton shuffling from Asp82 to Glu78 and from Glu32 to Asp82 are examined, and the results indicate that such proton shuffling may not play an important role in the stabilization of the tetrahedral intermediate analogue.
authors.affiliation: Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville, Tennessee 37996, USA.
authors.forename: Haobo
authors.initials: H
authors.lastname: Guo
authors.forename: Alexander
authors.initials: A
authors.lastname: Wlodawer
authors.forename: Toru
authors.initials: T
authors.lastname: Nakayama
authors.forename: Qin
authors.initials: Q
authors.lastname: Xu
authors.forename: Hong
authors.initials: H
authors.lastname: Guo
mesh_terms: D001224:Aspartic Acid / Q000737:chemistry; D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002268:Carboxypeptidases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D002384:Catalysis; D003198:Computer Simulation; D004735:Energy Transfer / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000737:chemistry; D018698:Glutamic Acid / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D011522:Protons; D012694:Serine / Q000737:chemistry; D013816:Thermodynamics
chemical_list: D004791:Enzyme Inhibitors; D011522:Protons; D001224:Aspartic Acid; D018698:Glutamic Acid; D012694:Serine; D002268:Carboxypeptidases; C022633:serine carboxypeptidase; D016282:Aspartic Acid Endopeptidases; C078894:kumamolysin