Enzyme Architecture: A Startling Role for Asn270 in Glycerol 3-Phosphate Dehydrogenase-Catalyzed Hydride Transfer

Abstract

The side chains of R269 and N270 interact with the phosphodianion of dihydroxyacetone phosphate (DHAP) bound to glycerol 3-phosphate dehydrogenase (GPDH). The R269A, N270A, and R269A/N270A mutations of GPDH result in 9.1, 5.6, and 11.5 kcal/mol destabilization, respectively, of the transition state for GPDH-catalyzed reduction of DHAP by the reduced form of nicotinamide adenine dinucleotide. The N270A mutation results in a 7.7 kcal/mol decrease in the intrinsic phosphodianion binding energy, which is larger than the 5.6 kcal/mol effect of the mutation on the stability of the transition state for reduction of DHAP; a 2.2 kcal/mol stabilization of the transition state for unactivated hydride transfer to the truncated substrate glycolaldehyde (GA); and a change in the effect of phosphite dianion on GPDH-catalyzed reduction of GA, from strongly activating to inhibiting. The N270A mutation breaks the network of hydrogen bonding side chains, Asn270, Thr264, Asn205, Lys204, Asp260, and Lys120, which connect the dianion activation and catalytic sites of GPDH. We propose that this disruption dramatically alters the performance of GPDH at these sites.

Figure 1

Reactions of whole substrates RCH₂OPO₃^2– (k_cat/K_m) and substrate pieces RH + HPO₃^2– (k_cat/K_HPiK_d) catalyzed by OMPDC, TIM, and GPDH. In each case, 6–8 kcal/mol of the dianion binding energy is used to activate the enzyme for catalysis of the respective reactions of whole and truncated substrates.

Figure 2

Representation of the X-ray crystal structure of the nonproductive ternary complex of hlGPDH, DHAP, and NAD⁺ (Protein Data Bank entry 1WPQ), which shows (a) loop residues 292–297 (green) that fold over DHAP and (b) Arg269 and Asn270 that interact with the substrate phosphodianion. (c) Network of hydrogen-bonded side chains (Asn270, Thr264, Asn205, Lys204, Asp260, and Lys120) that connect the catalytic and dianion activation sites.

Scheme 1

Figure 3

Effect of increasing [HPO₃^2–] on the observed second-order rate constant for N270A hlGPDH-catalyzed reduction of GA at 25 °C and pH 7.5 (10 mM triethanolamine buffer). The data from the inset show a good fit to eq 1 derived for Scheme 2.

Scheme 2