The stabilization of the transition state for hIGPDH-catalyzed reduction of DHAP due to the action of the phosphodianion of DHAP and the cationic side chain of 8269 is between 12.4 and 17 kcal/mol. The R269A mutation of glycerol 3-phosphate dehydrogenase (h1GPDH) 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 hIGPDH, dianions (X2- = FPO32-, HPO32-, or SO42-), Gua(+) and GA into a functioning catalyst of the reduction of GA, and fourth-order reaction rate constants k(cat)/K-GA-KxKGua. The linear logarithmic correlation (slope = 1.0) between values of k(cat)/K-GA/K-X for dianion activation of wildtype h1GPDH-catalyzed reduction of GA and k(cat)/K-GA KxKG a shows that the electrostatic interaction between exogenous dianions and the side chain of R269 is not significantly perturbed by cutting hIGPDH into R269A and Gua pieces. The advantage for connection of h1GPDH (R269A mutant + Gua(+) and substrate pieces (GA + HP,) pieces, (Delta G(S)(double dagger)) (HPi+E+Gua) = 5.6 kcal/mol, is nearly equal to the sum of the advantage to connection of the substrate pieces, (Delta G(S)(double dagger))(GA+HPI) = 3.3 kcal/mol, for wildtype h1GPDH-catalyzed reaction of GA + HP upsilon and for connection of the enzyme pieces, (Delta G(S)(double dagger))(E+Gua) = 2.4 kcal/mol, for Gua(+) activation of the R269A h1GPDH-catalyzed reaction of DHAP.