Kinetic mechanism of indole-3-glycerol phosphate synthase

Sandra Schlee, Susanne Dietrich, Tomasz Kurćon, Pamela Delaney, Nina Goodey, Reinhard Sterner

Research output: Contribution to journalArticleResearchpeer-review

7 Citations (Scopus)

Abstract

The (βα)8-barrel enzyme indole-3-glycerol phosphate synthase (IGPS) catalyzes the multistep transformation of 1-(o- carboxyphenylamino)-1-deoxyribulose 5-phosphate (CdRP) into indole-3-glycerol phosphate (IGP) in tryptophan biosynthesis. Mutagenesis data and crystal structure analysis of IGPS from Sulfolobus solfataricus (sIGPS) allowed for the formulation of a plausible chemical mechanism of the reaction, and molecular dynamics simulations suggested that flexibility of active site loops might be important for catalysis. Here we developed a method that uses extrinsic fluorophores attached to active site loops to connect the kinetic mechanism of sIGPS to structure and conformational motions. Specifically, we elucidated the kinetic mechanism of sIGPS and correlated individual steps in the mechanism to conformational motions of flexible loops. Pre-steady-state kinetic measurements of CdRP to IGP conversion monitoring changes in intrinsic tryptophan and IGP fluorescence provided a minimal three-step kinetic model in which fast substrate binding and chemical transformation are followed by slow product release. The role of sIGPS loop conformational motion during substrate binding and catalysis was examined via variants that were covalently labeled with fluorescent dyes at the N-terminal extension of the enzyme and mobile active site loop β1α1. Analysis of kinetic data monitoring dye fluorescence revealed a conformational change that follows substrate binding, suggesting an induced-fit-type binding mechanism for the substrate CdRP. Global fitting of all kinetic results obtained with wild-type sIGPS and the labeled variants was best accommodated by a four-step kinetic model. In this model, both the binding of CdRP and its on-enzyme conversion to IGP are accompanied by conformational transitions. The liberation of the product from the active site is the rate-limiting step of the overall reaction. Our results confirm the importance of flexible active loops for substrate binding and catalysis by sIGPS.

Original languageEnglish
Pages (from-to)132-142
Number of pages11
JournalBiochemistry
Volume52
Issue number1
DOIs
StatePublished - 8 Jan 2013

Fingerprint

Indole-3-Glycerol-Phosphate Synthase
Kinetics
Catalytic Domain
Catalysis
Phosphates
Substrates
Tryptophan
Enzymes
Fluorescence
Sulfolobus solfataricus
Mutagenesis
Fluorophores
Monitoring
Biosynthesis
Molecular Dynamics Simulation
Fluorescent Dyes
Data structures
Molecular dynamics
Coloring Agents
Crystal structure

Cite this

Schlee, S., Dietrich, S., Kurćon, T., Delaney, P., Goodey, N., & Sterner, R. (2013). Kinetic mechanism of indole-3-glycerol phosphate synthase. Biochemistry, 52(1), 132-142. https://doi.org/10.1021/bi301342j
Schlee, Sandra ; Dietrich, Susanne ; Kurćon, Tomasz ; Delaney, Pamela ; Goodey, Nina ; Sterner, Reinhard. / Kinetic mechanism of indole-3-glycerol phosphate synthase. In: Biochemistry. 2013 ; Vol. 52, No. 1. pp. 132-142.
@article{dc9d3cd0270642f18838ba82264fca4d,
title = "Kinetic mechanism of indole-3-glycerol phosphate synthase",
abstract = "The (βα)8-barrel enzyme indole-3-glycerol phosphate synthase (IGPS) catalyzes the multistep transformation of 1-(o- carboxyphenylamino)-1-deoxyribulose 5-phosphate (CdRP) into indole-3-glycerol phosphate (IGP) in tryptophan biosynthesis. Mutagenesis data and crystal structure analysis of IGPS from Sulfolobus solfataricus (sIGPS) allowed for the formulation of a plausible chemical mechanism of the reaction, and molecular dynamics simulations suggested that flexibility of active site loops might be important for catalysis. Here we developed a method that uses extrinsic fluorophores attached to active site loops to connect the kinetic mechanism of sIGPS to structure and conformational motions. Specifically, we elucidated the kinetic mechanism of sIGPS and correlated individual steps in the mechanism to conformational motions of flexible loops. Pre-steady-state kinetic measurements of CdRP to IGP conversion monitoring changes in intrinsic tryptophan and IGP fluorescence provided a minimal three-step kinetic model in which fast substrate binding and chemical transformation are followed by slow product release. The role of sIGPS loop conformational motion during substrate binding and catalysis was examined via variants that were covalently labeled with fluorescent dyes at the N-terminal extension of the enzyme and mobile active site loop β1α1. Analysis of kinetic data monitoring dye fluorescence revealed a conformational change that follows substrate binding, suggesting an induced-fit-type binding mechanism for the substrate CdRP. Global fitting of all kinetic results obtained with wild-type sIGPS and the labeled variants was best accommodated by a four-step kinetic model. In this model, both the binding of CdRP and its on-enzyme conversion to IGP are accompanied by conformational transitions. The liberation of the product from the active site is the rate-limiting step of the overall reaction. Our results confirm the importance of flexible active loops for substrate binding and catalysis by sIGPS.",
author = "Sandra Schlee and Susanne Dietrich and Tomasz Kurćon and Pamela Delaney and Nina Goodey and Reinhard Sterner",
year = "2013",
month = "1",
day = "8",
doi = "10.1021/bi301342j",
language = "English",
volume = "52",
pages = "132--142",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "1",

}

Schlee, S, Dietrich, S, Kurćon, T, Delaney, P, Goodey, N & Sterner, R 2013, 'Kinetic mechanism of indole-3-glycerol phosphate synthase', Biochemistry, vol. 52, no. 1, pp. 132-142. https://doi.org/10.1021/bi301342j

Kinetic mechanism of indole-3-glycerol phosphate synthase. / Schlee, Sandra; Dietrich, Susanne; Kurćon, Tomasz; Delaney, Pamela; Goodey, Nina; Sterner, Reinhard.

In: Biochemistry, Vol. 52, No. 1, 08.01.2013, p. 132-142.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Kinetic mechanism of indole-3-glycerol phosphate synthase

AU - Schlee, Sandra

AU - Dietrich, Susanne

AU - Kurćon, Tomasz

AU - Delaney, Pamela

AU - Goodey, Nina

AU - Sterner, Reinhard

PY - 2013/1/8

Y1 - 2013/1/8

N2 - The (βα)8-barrel enzyme indole-3-glycerol phosphate synthase (IGPS) catalyzes the multistep transformation of 1-(o- carboxyphenylamino)-1-deoxyribulose 5-phosphate (CdRP) into indole-3-glycerol phosphate (IGP) in tryptophan biosynthesis. Mutagenesis data and crystal structure analysis of IGPS from Sulfolobus solfataricus (sIGPS) allowed for the formulation of a plausible chemical mechanism of the reaction, and molecular dynamics simulations suggested that flexibility of active site loops might be important for catalysis. Here we developed a method that uses extrinsic fluorophores attached to active site loops to connect the kinetic mechanism of sIGPS to structure and conformational motions. Specifically, we elucidated the kinetic mechanism of sIGPS and correlated individual steps in the mechanism to conformational motions of flexible loops. Pre-steady-state kinetic measurements of CdRP to IGP conversion monitoring changes in intrinsic tryptophan and IGP fluorescence provided a minimal three-step kinetic model in which fast substrate binding and chemical transformation are followed by slow product release. The role of sIGPS loop conformational motion during substrate binding and catalysis was examined via variants that were covalently labeled with fluorescent dyes at the N-terminal extension of the enzyme and mobile active site loop β1α1. Analysis of kinetic data monitoring dye fluorescence revealed a conformational change that follows substrate binding, suggesting an induced-fit-type binding mechanism for the substrate CdRP. Global fitting of all kinetic results obtained with wild-type sIGPS and the labeled variants was best accommodated by a four-step kinetic model. In this model, both the binding of CdRP and its on-enzyme conversion to IGP are accompanied by conformational transitions. The liberation of the product from the active site is the rate-limiting step of the overall reaction. Our results confirm the importance of flexible active loops for substrate binding and catalysis by sIGPS.

AB - The (βα)8-barrel enzyme indole-3-glycerol phosphate synthase (IGPS) catalyzes the multistep transformation of 1-(o- carboxyphenylamino)-1-deoxyribulose 5-phosphate (CdRP) into indole-3-glycerol phosphate (IGP) in tryptophan biosynthesis. Mutagenesis data and crystal structure analysis of IGPS from Sulfolobus solfataricus (sIGPS) allowed for the formulation of a plausible chemical mechanism of the reaction, and molecular dynamics simulations suggested that flexibility of active site loops might be important for catalysis. Here we developed a method that uses extrinsic fluorophores attached to active site loops to connect the kinetic mechanism of sIGPS to structure and conformational motions. Specifically, we elucidated the kinetic mechanism of sIGPS and correlated individual steps in the mechanism to conformational motions of flexible loops. Pre-steady-state kinetic measurements of CdRP to IGP conversion monitoring changes in intrinsic tryptophan and IGP fluorescence provided a minimal three-step kinetic model in which fast substrate binding and chemical transformation are followed by slow product release. The role of sIGPS loop conformational motion during substrate binding and catalysis was examined via variants that were covalently labeled with fluorescent dyes at the N-terminal extension of the enzyme and mobile active site loop β1α1. Analysis of kinetic data monitoring dye fluorescence revealed a conformational change that follows substrate binding, suggesting an induced-fit-type binding mechanism for the substrate CdRP. Global fitting of all kinetic results obtained with wild-type sIGPS and the labeled variants was best accommodated by a four-step kinetic model. In this model, both the binding of CdRP and its on-enzyme conversion to IGP are accompanied by conformational transitions. The liberation of the product from the active site is the rate-limiting step of the overall reaction. Our results confirm the importance of flexible active loops for substrate binding and catalysis by sIGPS.

UR - http://www.scopus.com/inward/record.url?scp=84872111702&partnerID=8YFLogxK

U2 - 10.1021/bi301342j

DO - 10.1021/bi301342j

M3 - Article

VL - 52

SP - 132

EP - 142

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 1

ER -

Schlee S, Dietrich S, Kurćon T, Delaney P, Goodey N, Sterner R. Kinetic mechanism of indole-3-glycerol phosphate synthase. Biochemistry. 2013 Jan 8;52(1):132-142. https://doi.org/10.1021/bi301342j