Active-Site Glu165 Activation in Triosephosphate Isomerase and Its Deprotonation Kinetics

Hua Deng, R. Brian Dyer, Robert Callender

Research output: Contribution to journalArticle

Abstract

Triosephosphate isomerase (TIM) catalyzes the interconversion between dihydroxyacetone phosphate (DHAP) and d-glyceraldehyde 3-phosphate (GAP) via an enediol(ate) intermediate. The active-site residue Glu165 serves as the catalytic base during catalysis. It abstracts a proton from C1 carbon of DHAP to form the reaction intermediate and donates a proton to C2 carbon of the intermediate to form product GAP. Our difference Fourier transform infrared spectroscopy studies on the yeast TIM (YeTIM)/phosphate complex revealed a C=O stretch band at 1706 cm -1 from the protonated Glu165 carboxyl group at pH 7.5, indicating that the pK a of the catalytic base is increased by >3.0 pH units upon phosphate binding, and that the Glu165 carboxyl environment in the complex is still hydrophilic in spite of the increased pK a . Hence, the results show that the binding of the phosphodianion group is part of the activation mechanism which involves the pK a elevation of the catalytic base Glu165. The deprotonation kinetics of Glu165 in the μs to ms time range were determined via infrared (IR) T-jump studies on the YeTIM/phosphate and ("heavy enzyme") [U- 13 C,- 15 N]YeTIM/phosphate complexes. The slower deprotonation kinetics in the ms time scale is due to phosphate dissociation modulated by the loop motion, which slows down by enzyme mass increase to show a normal heavy enzyme kinetic isotope effect (KIE) ∼1.2 (i.e., slower rate in the heavy enzyme). The faster deprotonation kinetics in the tens of μs time scale is assigned to temperature-induced pK a decrease, while phosphate is still bound, and it shows an inverse heavy enzyme KIE ∼0.89 (faster rate in the heavy enzyme). The IR static and T-jump spectroscopy provides atomic-level resolution of the catalytic mechanism because of its ability to directly observe the bond breaking/forming process.

Original languageEnglish (US)
Pages (from-to)4230-4241
Number of pages12
JournalJournal of Physical Chemistry B
Volume123
Issue number19
DOIs
StatePublished - May 16 2019

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Triose-Phosphate Isomerase
Deprotonation
Catalytic Domain
phosphates
Phosphates
Chemical activation
activation
kinetics
Enzymes
enzymes
Glyceraldehyde 3-Phosphate
Dihydroxyacetone Phosphate
Yeast
Enzyme kinetics
Yeasts
yeast
Isotopes
Protons
Carbon
Infrared radiation

ASJC Scopus subject areas

  • Physical and Theoretical Chemistry
  • Surfaces, Coatings and Films
  • Materials Chemistry

Cite this

Active-Site Glu165 Activation in Triosephosphate Isomerase and Its Deprotonation Kinetics. / Deng, Hua; Dyer, R. Brian; Callender, Robert.

In: Journal of Physical Chemistry B, Vol. 123, No. 19, 16.05.2019, p. 4230-4241.

Research output: Contribution to journalArticle

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title = "Active-Site Glu165 Activation in Triosephosphate Isomerase and Its Deprotonation Kinetics",
abstract = "Triosephosphate isomerase (TIM) catalyzes the interconversion between dihydroxyacetone phosphate (DHAP) and d-glyceraldehyde 3-phosphate (GAP) via an enediol(ate) intermediate. The active-site residue Glu165 serves as the catalytic base during catalysis. It abstracts a proton from C1 carbon of DHAP to form the reaction intermediate and donates a proton to C2 carbon of the intermediate to form product GAP. Our difference Fourier transform infrared spectroscopy studies on the yeast TIM (YeTIM)/phosphate complex revealed a C=O stretch band at 1706 cm -1 from the protonated Glu165 carboxyl group at pH 7.5, indicating that the pK a of the catalytic base is increased by >3.0 pH units upon phosphate binding, and that the Glu165 carboxyl environment in the complex is still hydrophilic in spite of the increased pK a . Hence, the results show that the binding of the phosphodianion group is part of the activation mechanism which involves the pK a elevation of the catalytic base Glu165. The deprotonation kinetics of Glu165 in the μs to ms time range were determined via infrared (IR) T-jump studies on the YeTIM/phosphate and ({"}heavy enzyme{"}) [U- 13 C,- 15 N]YeTIM/phosphate complexes. The slower deprotonation kinetics in the ms time scale is due to phosphate dissociation modulated by the loop motion, which slows down by enzyme mass increase to show a normal heavy enzyme kinetic isotope effect (KIE) ∼1.2 (i.e., slower rate in the heavy enzyme). The faster deprotonation kinetics in the tens of μs time scale is assigned to temperature-induced pK a decrease, while phosphate is still bound, and it shows an inverse heavy enzyme KIE ∼0.89 (faster rate in the heavy enzyme). The IR static and T-jump spectroscopy provides atomic-level resolution of the catalytic mechanism because of its ability to directly observe the bond breaking/forming process.",
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N2 - Triosephosphate isomerase (TIM) catalyzes the interconversion between dihydroxyacetone phosphate (DHAP) and d-glyceraldehyde 3-phosphate (GAP) via an enediol(ate) intermediate. The active-site residue Glu165 serves as the catalytic base during catalysis. It abstracts a proton from C1 carbon of DHAP to form the reaction intermediate and donates a proton to C2 carbon of the intermediate to form product GAP. Our difference Fourier transform infrared spectroscopy studies on the yeast TIM (YeTIM)/phosphate complex revealed a C=O stretch band at 1706 cm -1 from the protonated Glu165 carboxyl group at pH 7.5, indicating that the pK a of the catalytic base is increased by >3.0 pH units upon phosphate binding, and that the Glu165 carboxyl environment in the complex is still hydrophilic in spite of the increased pK a . Hence, the results show that the binding of the phosphodianion group is part of the activation mechanism which involves the pK a elevation of the catalytic base Glu165. The deprotonation kinetics of Glu165 in the μs to ms time range were determined via infrared (IR) T-jump studies on the YeTIM/phosphate and ("heavy enzyme") [U- 13 C,- 15 N]YeTIM/phosphate complexes. The slower deprotonation kinetics in the ms time scale is due to phosphate dissociation modulated by the loop motion, which slows down by enzyme mass increase to show a normal heavy enzyme kinetic isotope effect (KIE) ∼1.2 (i.e., slower rate in the heavy enzyme). The faster deprotonation kinetics in the tens of μs time scale is assigned to temperature-induced pK a decrease, while phosphate is still bound, and it shows an inverse heavy enzyme KIE ∼0.89 (faster rate in the heavy enzyme). The IR static and T-jump spectroscopy provides atomic-level resolution of the catalytic mechanism because of its ability to directly observe the bond breaking/forming process.

AB - Triosephosphate isomerase (TIM) catalyzes the interconversion between dihydroxyacetone phosphate (DHAP) and d-glyceraldehyde 3-phosphate (GAP) via an enediol(ate) intermediate. The active-site residue Glu165 serves as the catalytic base during catalysis. It abstracts a proton from C1 carbon of DHAP to form the reaction intermediate and donates a proton to C2 carbon of the intermediate to form product GAP. Our difference Fourier transform infrared spectroscopy studies on the yeast TIM (YeTIM)/phosphate complex revealed a C=O stretch band at 1706 cm -1 from the protonated Glu165 carboxyl group at pH 7.5, indicating that the pK a of the catalytic base is increased by >3.0 pH units upon phosphate binding, and that the Glu165 carboxyl environment in the complex is still hydrophilic in spite of the increased pK a . Hence, the results show that the binding of the phosphodianion group is part of the activation mechanism which involves the pK a elevation of the catalytic base Glu165. The deprotonation kinetics of Glu165 in the μs to ms time range were determined via infrared (IR) T-jump studies on the YeTIM/phosphate and ("heavy enzyme") [U- 13 C,- 15 N]YeTIM/phosphate complexes. The slower deprotonation kinetics in the ms time scale is due to phosphate dissociation modulated by the loop motion, which slows down by enzyme mass increase to show a normal heavy enzyme kinetic isotope effect (KIE) ∼1.2 (i.e., slower rate in the heavy enzyme). The faster deprotonation kinetics in the tens of μs time scale is assigned to temperature-induced pK a decrease, while phosphate is still bound, and it shows an inverse heavy enzyme KIE ∼0.89 (faster rate in the heavy enzyme). The IR static and T-jump spectroscopy provides atomic-level resolution of the catalytic mechanism because of its ability to directly observe the bond breaking/forming process.

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