title: Enzyme architecture: optimization of transition state stabilization from a cation-phosphodianion pair.
abstract: The side chain cation of R269 lies at the surface of l-glycerol 3-phosphate dehydrogenase (GPDH) and forms an ion pair to the phosphodianion of substrate dihydroxyacetone phosphate (DHAP), which is buried at the nonpolar protein interior. The R269A mutation of GPDH results in a 110-fold increase in K(m) (2.8 kcal/mol effect) and a 41,000-fold decrease in k(cat) (6.3 kcal/mol effect), which corresponds to a 9.1 kcal/mol destabilization of the transition state for GPDH-catalyzed reduction of DHAP by NADH. There is a 6.7 kcal/mol stabilization of the transition state for the R269A mutant GPDH-catalyzed reaction by 1.0 M guanidinium ion, and the transition state for the reaction of the substrate pieces is stabilized by an additional 2.4 kcal/mol by their covalent attachment at wildtype GPDH. These results provide strong support for the proposal that GPDH invests the 11 kcal/mol intrinsic phosphodianion binding energy of DHAP in trapping the substrate at a nonpolar active site, where strong electrostatic interactions are favored, and obtains a 9 kcal/mol return from stabilizing interactions between the side chain cation and transition state trianion. We propose a wide propagation for the catalytic motif examined in this work, which enables strong transition state stabilization from enzyme-phosphodianion pairs.
authors.affiliation: Department of Chemistry, University at Buffalo, SUNY, Buffalo, New York 14260-3000, United States.
authors.forename: Archie C
authors.initials: AC
authors.lastname: Reyes
authors.affiliation: Department of Chemistry, University at Buffalo, SUNY, Buffalo, New York 14260-3000, United States.
authors.forename: Astrid P
authors.initials: AP
authors.lastname: Koudelka
authors.affiliation: Department of Chemistry, University at Buffalo, SUNY, Buffalo, New York 14260-3000, United States.
authors.forename: Tina L
authors.initials: TL
authors.lastname: Amyes
authors.affiliation: Department of Chemistry, University at Buffalo, SUNY, Buffalo, New York 14260-3000, United States.
authors.forename: John P
authors.initials: JP
authors.lastname: Richard
mesh_terms: D004099:Dihydroxyacetone Phosphate / Q000378:metabolism*; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology; D005993:Glycerolphosphate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007477:Ions / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D009243:NAD / Q000378:metabolism; D009964:Orotidine-5'-Phosphate Decarboxylase / Q000737:chemistry; D017354:Point Mutation; D011487:Protein Conformation; D013816:Thermodynamics
chemical_list: D007477:Ions; D009243:NAD; D004099:Dihydroxyacetone Phosphate; D005993:Glycerolphosphate Dehydrogenase; D009964:Orotidine-5'-Phosphate Decarboxylase