Structural mutations that probe the interactions between the catalytic and dianion activation sites of triosephosphate isomerase

Abstract

Triosephosphate isomerase (TIM) catalyzes the isomerization of dihydroxyacetone phosphate to form d-glyceraldehyde 3-phosphate. The effects of two structural mutations in TIM on the kinetic parameters for catalysis of the reaction of the truncated substrate glycolaldehyde (GA) and the activation of this reaction by phosphite dianion are reported. The P168A mutation results in similar 50- and 80-fold decreases in (kcat/Km)E and (kcat/Km)E·HPi, respectively, for deprotonation of GA catalyzed by free TIM and by the TIM·HPO(3)(2-) complex. The mutation has little effect on the observed and intrinsic phosphite dianion binding energy or the magnitude of phosphite dianion activation of TIM for catalysis of deprotonation of GA. A loop 7 replacement mutant (L7RM) of TIM from chicken muscle was prepared by substitution of the archaeal sequence 208-TGAG with 208-YGGS. L7RM exhibits a 25-fold decrease in (kcat/Km)E and a larger 170-fold decrease in (kcat/Km)E·HPi for reactions of GA. The mutation has little effect on the observed and intrinsic phosphodianion binding energy and only a modest effect on phosphite dianion activation of TIM. The observation that both the P168A and loop 7 replacement mutations affect mainly the kinetic parameters for TIM-catalyzed deprotonation but result in much smaller changes in the parameters for enzyme activation by phosphite dianion provides support for the conclusion that catalysis of proton transfer and dianion activation of TIM take place at separate, weakly interacting, sites in the protein catalyst.

Figure 1

Dependence of the second-order rate constants k_cat/K_m for the TIM-catalyzed turnover of the free carbonyl form of [1-¹³C]-GA in D₂O on [HPO₃²⁻] at pD 7.0 and 25 °C at I = 0.1, NaCl. The data were fit to eq 5 derived for the model shown in Scheme 3. A. Wildtype cTIM. B. L7RM cTIM. C. P168A TbbTIM.

Figure 2

Superposition of models, from X-ray crystal structures, which show the active sites of unliganded wildtype TbbTIM (gold, PDB entry 5TIM), PGA-liganded TIM from Leishmania mexicana (cyan, PDB entry 1N55); and, PGA-liganded P168A mutant TbbTIM (green, PDB entry 2J27). The ligand induced enzyme conformational changes are observed for wildtype and P168A mutant TbbTIM are similar, except that the carboxylate side chain of Glu167 remains in open “swung-out” conformation at the P168A mutant.

Figure 3

Free energy profiles for turnover of GA by free TIM (E_O) and by TIM that is saturated with HPO₃²⁻ (E_C•HPO₃²⁻), constructed for Scheme 5 using the kinetic parameters reported in Table 3. The profiles show the activation free energy changes calculated using the Eyring equation at 298 K for reactions catalyzed by wildtype and P168A mutant TbbTIM. (A) Reactions catalyzed by wildtype cTIM. The difference between the total intrinsic phosphite dianion binding energy of −6.4 kcal/mol and ΔG^o = −2.4 kcal/mol for binding of HPO₃²⁻ to the inactive open enzyme E_O to give the active closed liganded enzyme E_C•HPO₃²⁻ is attributed to ΔG_C = 4.0 kcal/mol for the conformational change that converts E_O to E_C. (B) Reactions catalyzed by the P168A mutant. The observed barriers for conversion of E_O to the transition state for the unactivated and phosphite dianion activated reaction are 2.6 kcal/mol higher than for the wildtype cTIM catalyzed reaction (green bars, Figure 3B).

Figure 4

Reprinted from Ref. . A representation of the structure of the closed form of TIM from chicken muscle in the region of the active site. This Figure shows the important interactions between flexible loop 6 (Pro166 to Ala176) and loop 7 (Tyr208 to Ser211), which form upon binding of PGH, an analog of the enediolate reaction intermediate (PDB entry 1TPH).^, The 208-YGGS sequence was replaced by 208-TGAG at the loop 7 mutant of cTIM.

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Scheme 5

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