The role of phosphate in a multistep enzymatic reaction: reactions of the substrate and intermediate in pieces

Abstract

Several mechanistically unrelated enzymes utilize the binding energy of their substrate's nonreacting phosphoryl group to accelerate catalysis. Evidence for the involvement of the phosphodianion in transition state formation has come from reactions of the substrate in pieces, in which reaction of a truncated substrate lacking its phosphorylmethyl group is activated by inorganic phosphite. What has remained unknown until now is how the phosphodianion group influences the reaction energetics at different points along the reaction coordinate. 1-Deoxy-D-xylulose-5-phosphate (DXP) reductoisomerase (DXR), which catalyzes the isomerization of DXP to 2-C-methyl-D-erythrose 4-phosphate (MEsP) and subsequent NADPH-dependent reduction, presents a unique opportunity to address this concern. Previously, we have reported the effect of covalently linked phosphate on the energetics of DXP turnover. Through the use of chemically synthesized MEsP and its phosphate-truncated analogue, 2-C-methyl-D-glyceraldehyde, the current study revealed a loss of 6.1 kcal/mol of kinetic barrier stabilization upon truncation, of which 4.4 kcal/mol was regained in the presence of phosphite dianion. The activating effect of phosphite was accompanied by apparent tightening of its interactions within the active site at the intermediate stage of the reaction, suggesting a role of the phosphodianion in disfavoring intermediate release and in modulation of the on-enzyme isomerization equilibrium. The results of kinetic isotope effect and structural studies indicate rate limitation by physical steps when the covalent linkage is severed. These striking differences in the energetics of the natural reaction and the reactions in pieces provide a deeper insight into the contribution of enzyme-phosphodianion interactions to the reaction coordinate.

Scheme 1. Mechanism for DXR-Catalyzed Conversion of DXP to MEP and DE to 2MG

Scheme 2. Synthesis of 2-C-Methyl-d-glyceraldehyde (2MGA)

Reaction conditions: (a) (i) SOCl₂, CH₂Cl₂; (ii) CH₃ONHCH₃·HCl, pyridine; (b) modified α-AD mix;^, (c) Me₂C(OMe)₂, p-MeC₆H₄SO₃H; (d) LiAlH₄, THF; (e) Amberlite (H⁺ form), H₂O.

Scheme 3. Synthesis of 2-C-Methyl-d-erythrose 4-Phosphate (MEsP)

Reaction conditions: (a) From refs (1, 2); (b) H₂, Pd/C, MeOH; (c) 37 °C, H₂O (9 h); (d) 1 M NaOH.

Scheme 4. Non-essential Activation Model for Phosphite-Activated 2MGA Turnover by MtDXR

E = MtDXR·NADPH.

Figure 1

Dependence of the rate of MtDXR-catalyzed 2MGA turnover on the concentrations of (A) 2MGA ([HPO₃^2–], from bottom to top: 0, 4.7, 14.1, 23.5, 32.9, and 47.0 mM) and (B) phosphite dianion ([2MGA], from bottom to top: 0, 0.5, 0.8, 1.2, 1.7, 2.5, 4.2, and 5.9 mM) at pH 7.5, 25 °C (I = 0.2 M, NaCl). Curves are the result of nonlinear regression performed globally using eq 2.

Scheme 5. Simplified Kinetic Model for Turnover of DXP and MEsP

The isotope (H or D) and isotope-sensitive step are in red.

Scheme 6. Thermodynamic Box Describing Conversion of DE to 2MGA in the Absence and Presence of Phosphite Dianion

E = MtDXR·NADPH.

Figure 2

Superposition of the crystal structures of MtDXR with inhibitor FR-900098, Mn²⁺, and NADPH (not shown) (PDB entry 4A03, purple) and MtDXR with DE, Mn²⁺, NADPH (not shown), and HPO₃^2– (PDB entry 4RCV, green). Residues of the flexible loop, phosphodianion binding pocket, and metal-binding pocket are shown. Loop 189–206 is absent in the structure with the substrate in pieces (green).