Synthesis of (1, 2‐<sup>13</sup>C<sub>2</sub>) 2‐phosphoglycolic acid

O'Connor, Edward J.; Tomita, York; McDermott, Ann E.

doi:10.1002/jlcr.2580340806

Cited by 6 publications

(9 citation statements)

References 7 publications

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“…Sodium 2-phosphoglycolate was synthesized by following a published procedure. 42 Imidazole was recrystallized from benzene. All other chemicals were reagent grade or better and were used without further purification.…”

Section: Methodsmentioning

confidence: 99%

Structural Mutations That Probe the Interactions between the Catalytic and Dianion Activation Sites of Triosephosphate Isomerase

et al. 2013

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Triosephosphate isomerase (TIM) catalyzes the isomerization of dihydroxyacetone phosphate to form D-glyceraldehyde 3-phosphate. The effects of two structural mutations at 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-fold and 80-fold decreases, respectively, in (kcat/Km)E and (kcat/Km)E•HPi for deprotonation of GA catalyzed by free TIM and by the TIM•HPO32− complex. The mutation has little effect on the observed and intrinsic phosphite dianion binding energy, or on 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 for 208-YGGS. The 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 provide 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.

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Section: Methodsmentioning

confidence: 99%

Structural Mutations That Probe the Interactions between the Catalytic and Dianion Activation Sites of Triosephosphate Isomerase

et al. 2013

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“…2-Phosphoglycolic acid was prepared by a literature procedure. 51 The disodium salt of d -glyceraldehyde 3-phosphate diethyl acetal was prepared by a literature procedure 52 and purified by column chromatography over DEAE-Sephadex. Imidazole was recrystallized from benzene.…”

Section: Methodsmentioning

confidence: 99%

“…Deuterium oxide (99% D), deuterium chloride (35 wt %, 99.9% D), 15 NH 4 Cl, and [ 13 C 6 ]glucose were from Cambridge Isotope Laboratories. 2-Phosphoglycolic acid was prepared by a literature procedure . The disodium salt of d -glyceraldehyde 3-phosphate diethyl acetal was prepared by a literature procedure and purified by column chromatography over DEAE-Sephadex.…”

Section: Methodsmentioning

confidence: 99%

Enzyme Architecture: The Effect of Replacement and Deletion Mutations of Loop 6 on Catalysis by Triosephosphate Isomerase

Zhai

O’Donoghue

et al. 2014

Biochemistry

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Two mutations of the phosphodianion gripper loop in chicken muscle triosephosphate isomerase (cTIM) were examined: (1) the loop deletion mutant (LDM) formed by removal of residues 170–173 [Pompliano, D. L., et al. (1990) Biochemistry 29, 3186–3194] and (2) the loop 6 replacement mutant (L6RM), in which the N-terminal hinge sequence of TIM from eukaryotes, 166-PXW-168 (X = L or V), is replaced by the sequence from archaea, 166-PPE-168. The X-ray crystal structure of the L6RM shows a large displacement of the side chain of E168 from that for W168 in wild-type cTIM. Solution nuclear magnetic resonance data show that the L6RM results in significant chemical shift changes in loop 6 and surrounding regions, and that the binding of glycerol 3-phosphate (G3P) results in chemical shift changes for nuclei at the active site of the L6RM that are smaller than those of wild-type cTIM. Interactions with loop 6 of the L6RM stabilize the enediolate intermediate toward the elimination reaction catalyzed by the LDM. The LDM and L6RM result in 800000- and 23000-fold decreases, respectively, in kcat/Km for isomerization of GAP. Saturation of the LDM, but not the L6RM, by substrate and inhibitor phosphoglycolate is detected by steady-state kinetic analyses. We propose, on the basis of a comparison of X-ray crystal structures for wild-type TIM and the L6RM, that ligands bind weakly to the L6RM because a large fraction of the ligand binding energy is utilized to overcome destabilizing electrostatic interactions between the side chains of E168 and E129 that are predicted to develop in the loop-closed enzyme. Similar normalized yields of DHAP, d-DHAP, and d-GAP are formed in LDM- and L6RM-catalyzed reactions of GAP in D2O. The smaller normalized 12–13% yield of DHAP and d-DHAP observed for the mutant cTIM-catalyzed reactions compared with the 79% yield of these products for wild-type cTIM suggests that these mutations impair the transfer of a proton from O-2 to O-1 at the initial enediolate phosphate intermediate. No products are detected for the LDM-catalyzed isomerization reactions in D2O of [1-13C]GA and HPi, but the L6RM-catalyzed reaction in the presence of 0.020 M dianion gives a 2% yield of the isomerization product [2-13C,2-2H]GA.

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“…2-Phosphoglycolic acid was prepared according to a literature procedure (23). The barium salt of D-glyceraldehyde 3-phosphate diethyl acetal was prepared by Dr. James Tait according to a literature procedure (24).…”

Section: Methodsmentioning

confidence: 99%

Role of Lys-12 in Catalysis by Triosephosphate Isomerase: A Two-Part Substrate Approach

et al. 2010

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We report that the K12G mutation at triosephosphate isomerase (TIM) from Saccharomyces cerevisiae results in: (1) A ca. 50-fold increase in K m for the substrate glyceraldehyde 3-phosphate (GAP) and a 60-fold increase in K i for competitive inhibition by the intermediate analog 2-phosphoglycolate, resulting from the loss of stabilizing ground state interactions between the alkylammonium side chain of Lys-12 and the ligand phosphodianion group. (2) A 12,000-fold decrease in k cat for isomerization of GAP, suggesting a tightening of interactions between the side chain of Lys-12 and the substrate on proceeding from the Michaelis complex to the transition state. (3) A 6 × 10 5 -fold decrease in k cat /K m , corresponding to a total 7.8 kcal/mol stabilization of the transition state by the cationic side chain of Lys-12. The yields of the four products of the K12G TIM-catalyzed isomerization of GAP in D 2 O were quantified as: dihydroxyacetone phosphate (DHAP), 27%; [1(R)-2 H]-DHAP, 23%; [2(R)-2 H]-GAP, 31%; and 18% methylglyoxal from an enzyme-catalyzed elimination reaction. The K12G mutation has only a small effect on the relative yields of the three products of proton transfer to the TIM-bound enediol(ate) intermediate in D 2 O, but it strongly favors catalysis of the elimination reaction to give methylglyoxal. The K12G mutation also results in a ≥ 14-fold decrease in k cat /K m for isomerization of bound glycolaldehyde (GA), although the dominant observed product of the mutant enzyme-catalyzed reaction of [1-13 C]-GA in D 2 O is [1-13 C, 2,2-di-2 H]-GA from a nonspecific protein-catalyzed reaction. The observation that the K12G mutation results in a large decrease in k cat /K m for the reactions of both GAP and the neutral truncated substrate [1-13 C]-GA provides evidence for a stabilizing interaction between the cationic side chain of Lys-12 and negative charge that develops at the enolate-like oxygen in the transition state for deprotonation of the sugar substrate "piece".Triosephosphate isomerase (TIM) 1 catalyzes the stereospecific, reversible, 1,2-hydrogen shift at dihydroxyacetone phosphate (DHAP) to give (R)-glyceraldehyde 3-phosphate (GAP) by a single-base (Glu-165) proton transfer mechanism through an enzyme-bound cisenediol(ate) intermediate (Scheme 1) (1,2). The enzyme's low molecular weight (dimer, 26 kDa/subunit), high cellular abundance (3), and the centrality of proton transfer at carbon in metabolic processes (4-6) have made TIM a prominent target for studies on the mechanism of enzyme action (1,7-10).

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Synthesis of (1, 2‐¹³C₂) 2‐phosphoglycolic acid

Abstract: The synthesis of (1,2‐ 13C2) 2‐phosphoglycelic acid, 5, from commercially‐available (1,2‐ 13C2) bromoacetic acid is described along with some of its solution NMR characteristics.

Cited by 6 publications

References 7 publications

Structural Mutations That Probe the Interactions between the Catalytic and Dianion Activation Sites of Triosephosphate Isomerase

Structural Mutations That Probe the Interactions between the Catalytic and Dianion Activation Sites of Triosephosphate Isomerase

Enzyme Architecture: The Effect of Replacement and Deletion Mutations of Loop 6 on Catalysis by Triosephosphate Isomerase

Role of Lys-12 in Catalysis by Triosephosphate Isomerase: A Two-Part Substrate Approach

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Synthesis of (1, 2‐13C2) 2‐phosphoglycolic acid

Abstract: The synthesis of (1,2‐ 13C2) 2‐phosphoglycelic acid, 5, from commercially‐available (1,2‐ 13C2) bromoacetic acid is described along with some of its solution NMR characteristics.

Cited by 6 publications

References 7 publications

Structural Mutations That Probe the Interactions between the Catalytic and Dianion Activation Sites of Triosephosphate Isomerase

Structural Mutations That Probe the Interactions between the Catalytic and Dianion Activation Sites of Triosephosphate Isomerase

Enzyme Architecture: The Effect of Replacement and Deletion Mutations of Loop 6 on Catalysis by Triosephosphate Isomerase

Role of Lys-12 in Catalysis by Triosephosphate Isomerase: A Two-Part Substrate Approach

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Synthesis of (1, 2‐¹³C₂) 2‐phosphoglycolic acid