Primary Deuterium Kinetic Isotope Effects: A Probe for the Origin of the Rate Acceleration for Hydride Transfer Catalyzed by Glycerol-3-Phosphate Dehydrogenase

Abstract

Large primary deuterium kinetic isotope effects (1° DKIEs) on enzyme-catalyzed hydride transfer may be observed when the transferred hydride tunnels through the energy barrier. The following 1° DKIEs on k_cat/ K_m and relative reaction driving force are reported for wild-type and mutant glycerol-3-phosphate dehydrogenase (GPDH)-catalyzed reactions of NADL (L = H, D): wild-type GPDH, ΔΔ G^⧧ = 0 kcal/mol, 1° DKIE = 1.5; N270A, 5.6 kcal/mol, 3.1; R269A, 9.1 kcal/mol, 2.8; R269A + 1.0 M guanidine, 2.4 kcal/mol, 2.7; R269A/N270A, 11.5 kcal/mol, 2.4. Similar 1° DKIEs were observed on k_cat. The narrow range of 1° DKIEs (2.4-3.1) observed for a 9.1 kcal/mol change in reaction driving force provides strong evidence that these are intrinsic 1° DKIEs on rate-determining hydride transfer. Evidence is presented that the intrinsic DKIE on wild-type GPDH-catalyzed reduction of DHAP lies in this range. A similar range of 1° DKIEs (2.4-2.9) on ( k_cat/ K_GA, M^-1 s^-1) was reported for dianion-activated hydride transfer from NADL to glycolaldehyde (GA) [Reyes, A. C.; Amyes, T. L.; Richard, J. P. J. Am. Chem. Soc. 2016, 138, 14526-14529]. These 1° DKIEs are much smaller than those observed for enzyme-catalyzed hydrogen transfer that occurs mainly by quantum mechanical tunneling. These results support the conclusion that the rate acceleration for GPDH-catalyzed reactions is due to the stabilization of the transition state for hydride transfer by interactions with the protein catalyst. The small 1° DKIEs reported for mutant GPDH-catalyzed and for wild-type dianion-activated reactions are inconsistent with a model where the dianion binding energy is utilized in the stabilization of a tunneling ready state.

Figure 1

Free-energy reaction profiles that illustrate different strategies for enzymes to reduce the effective activation barrier. (A) The deprotonation of DHAP to form an enediolate phosphate intermediate catalyzed by triosephosphate isomerase. The activation barrier to the enzyme-catalyzed reaction is reduced by protein–ligand interactions that result in similar stabilization of the enediolate intermediate and the enediolate-like transition state relative to the bound substrate. (B) A reaction coordinate profile for hydride transfer from NADH to the carbonyl group catalyzed by a representative dehydrogenase. The apparent activation barrier may be reduced below that for the formation of the transition state by quantum mechanical tunneling through the energy surface.

Scheme 1. GPDH-Catalyzed Reduction of the Whole Substrate DHAP and the Dianion-Activated Reduction of Substrate Piece Glycolaldehyde

Scheme 2. Minimal Kinetic Mechanism for GPDH-Catalyzed Reactions

Figure 2

Dependence of v/[E] (s^–1) on [DHAP] for the mutant hlGPDH-catalyzed reduction of DHAP by NADH or NADD (0.2 mM) at pH 7.5, 25 °C, and I = 0.12 (NaCl): (A) R269A mutant, (B) N270A mutant, (C) R269A/N270A mutant.

Figure 3

Dependence of v/[E] (s^–1) on [GA] for the N270A mutant hlGPDH-catalyzed reduction of DHAP by NADH or NADD (0.2 mM) at pH 7.5, 25 °C, and I = 0.12 (NaCl).

Scheme 3. Activation of the R269A Mutant hlGPDH-Catalyzed Reduction of S = DHAP by NADL

Figure 4

Effect of the guanidine cation on the R269A mutant hlGPDH-catalyzed reduction of DHAP by NADH or NADD for reactions at pH 7.5 (20 mM TEA buffer), 25 °C, 0.2 mM NADL, and I = 0.12 (NaCl): (⧫) 2 mM Gua⁺, (▼) 5 mM Gua⁺, (▲) 10 mM Gua⁺, (●) 15 mM Gua⁺, and (○) 20 mM Gua⁺.

Figure 5

Effect of increasing [Gua⁺] on the values of (k_cat/K_m)_obs determined as the slopes of correlations from Figure 4 for the R269A mutant hlGPDH-catalyzed reduction of DHAP by NADH and NADD at pH 7.5 (20 mM TEA buffer), 25 °C, 0.2 mM NADL, and I = 0.12 (NaCl).

Scheme 4. Kinetic Mechanism for the hlGPDH-Catalyzed Reduction of GA at Saturating Concentrations of NADH or NADD

Figure 6

Representation of the X-ray crystal structure of the nonproductive ternary complex between hlGPDH, DHAP, and NAD⁺ (PDB entry 1WPQ) showing the side chains for Arg-269 and Asn-270 that interact with the substrate phosphodianion. Also shown are loop residues 292–297 (green) that fold over DHAP and the hydrogen-bonded side chains [Asn-270, Thr-264, Asn-205, Lys-204, Asp-260, and Lys-120] that connect the catalytic and dianion activation sites.

Scheme 5. Cycle That Shows the Effect of Consecutive R269A and N270A Mutations on the Activation Barrier ΔG_DHAP^⧧ for the Wild-Type hlGPDH-Catalyzed Reduction of DHAP by NADH

Figure 7

Hypothetical reaction coordinates which show the barriers for the microscopic rate constants that control the rate-determining step for the hlGPDH-catalyzed reduction of S_ox = GA or DHAP: (k_–d)_S for the release of substrate S_ox; k_chem for hydride transfer; and (k_–d)_P for the release of product P_red. Hydride transfer is only partially rate-determining for the reaction catalyzed by wild-type hlGPDH (black lines). The observation of intrinsic kinetic isotope effects on the hlGPDH-catalyzed reactions of the substrate in pieces or the mutant hlGPDH-catalyzed reactions reflects the decreasing barriers to (k_–d)_S and (k_–d)_P and the increasing barrier to k_chem (blue lines), as described in the text.