Enzyme Architecture: Self-Assembly of Enzyme and Substrate Pieces of Glycerol-3-Phosphate Dehydrogenase into a Robust Catalyst of Hydride Transfer

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 HPO 3 2- . We report the unprecedented self-assembly of R269A hlGPDH, dianions (X 2- = FPO 3 2- , HPO 3 2- , or SO 4 2- ), Gua + and GA into a functioning catalyst of the reduction of GA, and fourth-order reaction rate constants k cat /K GA K X K Gua . The linear logarithmic correlation (slope = 1.0) between values of k cat /K GA K X for dianion activation of wildtype hlGPDH-catalyzed reduction of GA and k cat /K GA K X K Gua shows 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 + HP i ) pieces, (ΔG S ‡ ) HPi+E+Gua = 5.6 kcal/mol, is nearly equal to the sum of the advantage to connection of the substrate pieces, (ΔG S ‡ ) GA+HPi = 3.3 kcal/mol, for wildtype hlGPDH-catalyzed reaction of GA + HP i , and for connection of the enzyme pieces, (ΔG S ‡ ) E+Gua = 2.4 kcal/mol, for Gua + activation of the R269A hlGPDH-catalyzed reaction of DHAP.