title: Enzyme Architecture: Self-Assembly of Enzyme and Substrate Pieces of Glycerol-3-Phosphate Dehydrogenase into a Robust Catalyst of Hydride Transfer.
abstract: The stabilization of the transition state for hlGPDH-catalyzed reduction of DHAP due to the action of the phosphodianion of DHAP and the cationic side chain of R269 is between 12.4 and 17 kcal/mol. The R269A mutation of glycerol-3-phosphate dehydrogenase (hlGPDH) results in a 9.1 kcal/mol destabilization of the transition state for enzyme-catalyzed reduction of dihydroxyacetone phosphate (DHAP) by NADH, and there is a 6.7 kcal/mol stabilization of this transition state by 1.0 M guanidine cation (Gua+) [J. Am. Chem. Soc. 2015, 137, 5312-5315]. The R269A mutant shows no detectable activity toward reduction of glycolaldehyde (GA), or activation of this reaction by 30 mM HPO32-. We report the unprecedented self-assembly of R269A hlGPDH, dianions (X2-= FPO32-, HPO32-, or SO42-), Gua+and GA into a functioning catalyst of the reduction of GA, and fourth-order reaction rate constants kcat/KGAKXKGua. The linear logarithmic correlation (slope = 1.0) between values of kcat/KGAKXfor dianion activation of wildtype hlGPDH-catalyzed reduction of GA and kcat/KGAKXKGuashows that the electrostatic interaction between exogenous dianions and the side chain of R269 is not significantly perturbed by cutting hlGPDH into R269A and Gua+pieces. The advantage for connection of hlGPDH (R269A mutant + Gua+) and substrate pieces (GA + HPi) pieces, (ΔGS‡)HPi+E+Gua= 5.6 kcal/mol, is nearly equal to the sum of the advantage to connection of the substrate pieces, (ΔGS‡)GA+HPi= 3.3 kcal/mol, for wildtype hlGPDH-catalyzed reaction of GA + HPi, and for connection of the enzyme pieces, (ΔGS‡)E+Gua= 2.4 kcal/mol, for Gua+activation of the R269A hlGPDH-catalyzed reaction of DHAP.
authors: Department of Chemistry, University at Buffalo, SUNY , Buffalo, New York 14260-3000, United States.
authors: Archie C
authors: AC
authors: Reyes
authors: Department of Chemistry, University at Buffalo, SUNY , Buffalo, New York 14260-3000, United States.
authors: Tina L
authors: TL
authors: Amyes
authors: Department of Chemistry, University at Buffalo, SUNY , Buffalo, New York 14260-3000, United States.
authors: John P
authors: JP
authors: Richard
mesh_terms: D055162:Biocatalysis; D005993:Glycerolphosphate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D009154:Mutation; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D005993:Glycerolphosphate Dehydrogenase