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.

Scheme 1

Figure 1

Representations, from X-ray crystal structures, of the surfaces of enzyme-ligand complexes: the loops that trap the ligand in a catalytic cage are shaded red, and the guanidine side chains at the protein surface are shaded black. (A) The complex between ScOMPDC and 6-hydroxyuridine 5′-monophosphate (BMP) (PDB entry 1DQX). (B) The nonproductive ternary complex of dihydroxyacetone phosphate (DHAP) and NAD⁺ with hsGPDH (PDB entry 1WPQ).

Figure 2

Effect of Gua⁺ on R269A mutant hsGPDH-catalyzed reduction of DHAP by NADH for reactions at pH 7.5 (20 mM triethanolamine buffer), 25 °C, saturating [NADH] = 0.2 mM, and I = 0.12 (NaCl). (A) The increase in v/[E] (s^–1), with increasing [DHAP], for reactions at different fixed [Gua⁺]: (◆) 2 mM Gua⁺; (▼) 5 mM Gua⁺; (▲) 10 mM Gua⁺; (●) 15 mM Gua⁺; (○) 20 mM Gua⁺. (B) The effect of increasing [Gua⁺] on the values of (k_cat/K_m)_obs from Figure 2A.

Scheme 2

Scheme 3

Figure 3

Representations of X-ray crystal structures of ScOMPDC and hsGPDH. (A) ScOMPDC in a complex with 6-hydroxyuridine 5′-monophosphate (PDB entry 1DQX). (B) The nonproductive ternary complex of human liver hsGPDH with DHAP and NAD⁺ (PDB entry 1WPQ).