title: Electrostatic stabilization and general base catalysis in the active site of the human protein disulfide isomerase a domain monitored by hydrogen exchange.
abstract: The nucleophilic Cys36 thiol of the human protein disulfide isomerase a domain is positioned over the N terminus of the alpha(2) helix. Amides in the active site exhibit diffusion-limited, hydroxide-catalyzed exchange, indicating that the local positive electrostatic potential decreases the pK value for peptide anion formation by at least 2 units so as to equal or exceed the acidity of water. In stark contrast to the pH dependence of exchange for simple peptides, the His38 amide in the reduced enzyme exhibits a maximum rate of exchange at pH 5 due to efficient general base catalysis by the neutral imidazole of its own side chain and suppression of its exchange by the ionization of the Cys36 thiol. Ionization of this thiol and deprotonation of the His38 side chain suppress the Cys39 amide hydroxide-catalyzed exchange by a million-fold. The electrostatic potential within the active site monitored by these exchange experiments provides a means of stabilizing the two distinct transition states that lead to substrate reduction and oxidation. Molecular modeling offers a role for the conserved Arg103 in coordinating the oxidative transition-state complex, thus providing further support for mechanisms of disulfide isomerization that utilize enzymatic catalysis at each step of the overall reaction.
authors.affiliation: Wadsworth Center, New York State Department of Health and Department of Biomedical Sciences, School of Public Health, University at Albany-SUNY, Empire State Plaza, Albany, NY 12201, USA.
authors.forename: Griselda
authors.initials: G
authors.lastname: Hernández
authors.forename: Janet S
authors.initials: JS
authors.lastname: Anderson
authors.forename: David M
authors.initials: DM
authors.lastname: LeMaster
mesh_terms: D000577:Amides / Q000737:chemistry; D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D006801:Humans; D006859:Hydrogen / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D006878:Hydroxides / Q000737:chemistry*; D007093:Imidazoles / Q000737:chemistry; D008958:Models, Molecular; D010084:Oxidation-Reduction; D010455:Peptides / Q000737:chemistry; D019704:Protein Disulfide-Isomerases / Q000737:chemistry*; D055672:Static Electricity
chemical_list: D000577:Amides; D006878:Hydroxides; D007093:Imidazoles; D010455:Peptides; D006859:Hydrogen; D019704:Protein Disulfide-Isomerases