Wildtype and engineered monomeric triosephosphate isomerase from Trypanosoma brucei: partitioning of reaction intermediates in D2O and activation by phosphite dianion

Abstract

Product yields for the reactions of (R)-glyceraldehyde 3-phosphate (GAP) in D2O at pD 7.9 catalyzed by wildtype triosephosphate isomerase from Trypanosoma brucei brucei (Tbb TIM) and a monomeric variant (monoTIM) of this wildtype enzyme were determined by (1)H NMR spectroscopy and were compared with the yields determined in earlier work for the reactions catalyzed by TIM from rabbit and chicken muscle [O'Donoghue, A. C., Amyes, T. L., and Richard, J. P. (2005), Biochemistry 44, 2610 - 2621]. Three products were observed from the reactions catalyzed by TIM: dihydroxyacetone phosphate (DHAP) from isomerization with intramolecular transfer of hydrogen, d-DHAP from isomerization with incorporation of deuterium from D2O into C-1 of DHAP, and d-GAP from incorporation of deuterium from D2O into C-2 of GAP. The yield of DHAP formed by intramolecular transfer of hydrogen decreases from 49% for the muscle enzymes to 40% for wildtype Tbb TIM to 34% for monoTIM. There is no significant difference in the ratio of the yields of d-DHAP and d-GAP for wildtype TIM from muscle sources and Trypanosoma brucei brucei, but partitioning of the enediolate intermediate of the monoTIM reaction to form d-DHAP is less favorable ((k(C1))(D)/(k(C2))(D) = 1.1) than for the wildtype enzyme ((k(C1))(D)/(k(C2))(D) = 1.7). Product yields for the wildtype Tbb TIM and monoTIM-catalyzed reactions of glycolaldehyde labeled with carbon-13 at the carbonyl carbon ([1-(13)C]-GA) at pD 7.0 in the presence of phosphite dianion and in its absence were determined by (1)H NMR spectroscopy [Go, M. K., Amyes, T. L., and Richard, J. P. (2009) Biochemistry 48, 5769-5778]. There is no detectable difference in the yields of the products of wildtype muscle and Tbb TIM-catalyzed reactions of [1-(13)C]-GA in D2O. The kinetic parameters for phosphite dianion activation of the reactions of [1-(13)C]-GA catalyzed by wildtype Tbb TIM are similar to those reported for the enzyme from rabbit muscle [Amyes, T. L. and Richard, J. P. (2007) Biochemistry 46, 5841-5854], but there is no detectable dianion activation of the reaction catalyzed by monoTIM. The engineered disruption of subunit contacts at monoTIM causes movement of the essential side chains of Lys-13 and His-95 away from the catalytic active positions. We suggest that this places an increased demand that the intrinsic binding energy of phosphite dianion be utilized to drive the change in the conformation of monoTIM back to the active structure for wildtype TIM.

Figure 1

Rate and product data for the reaction of GAP (10 mM) catalyzed by Tbb TIM (0.60 nM) in D₂O buffered by 14 mM imidazole at pD 7.9 and 25 °C (I = 0.1, NaCl), determined by ¹H NMR spectroscopy. (A) The decrease with time in the fraction of remaining GAP. (B) The change with time in the fractional yields of only the products of enzymatic reaction of GAP, normalized using the sum of the observed fraction of d-GAP, DHAP and d-DHAP according to eq 4–6. The initial product yields reported in Table 2 were obtained by making a short linear extrapolation of product yields to zero reaction time. Key: (▲) yield of DHAP; (■) yield of d-DHAP; (●) yield of d-GAP.

Figure 2

Rate and product data for the reaction of [1-¹³C]-GA catalyzed by Tbb TIM in the presence of 15 mM phosphite dianion in D₂O buffered by 20 mM imidazole at pD 7.0 and 25 °C (I = 0.1, NaCl), determined by ¹H NMR spectroscopy. (A) The decrease with time in the fraction of remaining [1-¹³C]-GA. (B) The change with time in the fractional yields of the products, f_P, of the phosphite-activated TIM-catalyzed reaction of [1-¹³C]-GA. The initial product yields reported in Table 3 were obtained by making a short linear extrapolation of product yields to zero reaction time. Key: (▼) yield of [2-¹³C]-GA; (◆) yield of [2-¹³C, 2-²H]-GA; (■) yield of [1-¹³C, 2-²H]-GA.

Figure 3

Dependence of the observed second-order rate constant (k_cat/K_m)_obs (M⁻¹ s⁻¹) for the turnover of the free carbonyl form of [1-¹³C]-GA by Tbb TIM and of unlabeled GA by rabbit muscle TIM in D₂O on the concentration of added phosphite dianion at pD 7 and 25 °C (I = 0.10). Key: (●) Reaction of 20 mM [1-¹³C]-GA catalyzed by Tbb TIM; (▲) Reaction of 20 mM unlabeled GA catalyzed by rabbit muscle TIM (5); (▼) Reaction of 10 mM unlabeled GA catalyzed by rabbit muscle TIM (5).

Figure 4

A comparison of the amino acid sequences for wildtype Tbb TIM and the engineered monoTIM. The first amino acid residue in loop 3 that follows β-strand 3 is Gln-65. Residues 69 – 79 (red) from loop 3 were replaced by four amino acids (N A D A) that extend the β-strand 3 by one turn. Additional amino acid substitutions for the wildtype enzyme are shown in blue.

Figure 5

Superimposed crystal structures of the loop-closed Tbb TIM-PGH complex, shown in gold ribbons and in atoms colored by element (PDB entry 1TRD) and chicken muscle TIM-PGH complex, shown in sea green atoms and ribbons (PDB entry 1TPH).

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