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 HPO₃^2-. We report the unprecedented self-assembly of R269A hlGPDH, dianions (X^2- = FPO₃^2-, HPO₃^2-, or SO₄^2-), Gua⁺ and GA into a functioning catalyst of the reduction of GA, and fourth-order reaction rate constants k_cat/K_GAK_XK_Gua. The linear logarithmic correlation (slope = 1.0) between values of k_cat/K_GAK_X for dianion activation of wildtype hlGPDH-catalyzed reduction of GA and k_cat/K_GAK_XK_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.

Figure 1

A comparison of the protein surfaces from X-ray crystal structures of the following: (A) The complex between OMPDC from yeast and 6-hydroxyuridine 5′-monophosphate (PDB entry 1DQX). (B) The nonproductive ternary complex of dihydroxyacetone phosphate (DHAP) and NAD⁺ with hlGPDH (PDP entry 1WPQ). These structures show the loops that trap the ligand in a protein cage shaded red, and the guanidine side chains at the protein surface shaded black. The respective enzyme-bound ligands at the structures represented by A and B are buried in the protein, with the phosphodianion projecting toward the surface, and in a stable ion pair with the guanidine side chains of R235 (OMPDC) or R269 (hlGPDH). Reproduced from ref (5a). Copyright 2015 American Chemical Society.

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Figure 2

Effect of inorganic dianions and Gua⁺ on R269A mutant hlGPDH-catalyzed reduction of GA by NADH, determined for reactions at pH 7.5, 25 °C, saturating [NADH] = 0.2 mM and I = 0.12 (NaCl). (A) The increase in v/[E][GA] (M^–1 s^–1), with increasing [HPO₃^2–], for reactions in the presence of 30 mM (open symbols) or 60 mM (closed symbols) total GA [carbonyl + hydrate] and at different fixed concentrations of Gua⁺. The equilibrium constant for hydration of GA is K_eq = [carbonyl]/[hydrate] = (6/94). Key: ([GA]_carbonyl = 3.6 mM) (▼) 30 mM Gua⁺; (⧫), 25 mM Gua⁺; (■), 20 mM Gua⁺; (▲), 15 mM Gua⁺; (●), 10 mM Gua⁺; ([GA]_carbonyl = 1.8 mM) (□), 30 mM Gua⁺; (◊), 15 mM Gua⁺. (B) The increase in v/[E][GA] (M^–1 s^–1), with increasing [FPO₃^2–], for reactions at [GA]_carbonyl = 3.6 mM and at different fixed concentrations of Gua⁺. Key: (▼) 30 mM Gua⁺; (⧫), 25 mM Gua⁺; (■), 20 mM Gua⁺; (▲), 15 mM Gua⁺; (●), 10 mM Gua⁺. (C) The increase in v/[E][GA] (M^–1 s^–1), with increasing [SO₄^2–], for reactions at 3.6 mM GA and at different fixed concentrations of Gua⁺. Key: (▼) 30 mM Gua⁺; (⧫), 25 mM Gua⁺; (■), 20 mM Gua⁺; (▲), 15 mM Gua⁺; (●), 10 mM Gua⁺. Figure 3 shows the linear plots of the slopes of these linear correlations, (k_cat/K_GAK_X)_obs, against [Gua⁺].

Figure 3

Effect of increasing concentrations of Gua⁺ on the observed third-order rate constants (k_cat/K_GAK_X)_obs (M^–2 s^–1, Figure 2) for dianion activation of R269A mutant hlGPDH-catalyzed reduction of GA: solid symbols, [GA]_carbonyl = 3.6 mM; open symbols, [GA]_carbonyl = 1.8 mM. The slopes of these linear correlations are the fourth order rate constants k_cat/K_GAK_XK_Gua reported in Table 1.

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Figure 4

(A) Representations of the X-ray crystal structure (PDB entry 1WPQ) of the nonproductive ternary Michaelis complex between wildtype hlGPDH, DHAP and NAD⁺. (B–D) Representations, generated in silico from Figure 4A by deletion of the relevant covalent linkage(s) and maintaining a fixed position for the remaining atoms, of the following hypothetical Michaelis complexes: (B) wildtype hlGPDH, GA and HP_i, (C) R269A hlGPDH, DHAP and Gua⁺, (D) R269A hlGPDH, GA, HP_i and Gua⁺.

Figure 5

Linear logarithmic correlation, with slope of 1.0, between the fourth order rate constants k_cat/K_GAK_XK_Gua for activation of R269A hlGPDH-catalyzed reduction of GA by the combined action of dianions X^2– and Gua⁺ and the third order rate constants k_cat/K_GAK_X for activation of wildtype hlGPDH-catalyzed reduction of GA by the same dianions.