Cooperative Kinetics of the Glucan Phosphatase Starch Excess4

Claudia A. Mak; Kenyon Weis; Tiffany Henao; Andrea Kuchtova; Tiantian Chen; Savita Sharma; David A. Meekins; Matthias Thalmann; Craig W. Vander Kooi; Madushi Raththagala

doi:10.1021/acs.biochem.1c00307

Cooperative Kinetics of the Glucan Phosphatase Starch Excess4

Claudia A. Mak, Kenyon Weis, Tiffany Henao, Andrea Kuchtova, Tiantian Chen, Savita Sharma, David A. Meekins, Matthias Thalmann, Craig W. Vander Kooi, Madushi Raththagala

Research output: Contribution to journal › Article › peer-review

7 Scopus citations

Abstract

Glucan phosphatases are members of a functionally diverse family of dual-specificity phosphatase (DSP) enzymes. The plant glucan phosphatase Starch Excess4 (SEX4) binds and dephosphorylates glucans, contributing to processive starch degradation in the chloroplast at night. Little is known about the complex kinetics of SEX4 when acting on its complex physiologically relevant glucan substrate. Therefore, we explored the kinetics of SEX4 against both insoluble starch and soluble amylopectin glucan substrates. SEX4 displays robust activity and a unique sigmoidal kinetic response to amylopectin, characterized by a Hill coefficient of 2.77 ± 0.63, a signature feature of cooperativity. We investigated the basis for this positive kinetic cooperativity and determined that the SEX4 carbohydrate-binding module (CBM) dramatically influences the binding cooperativity and substrate transformation rates. These findings provide insights into a previously unknown but important regulatory role for SEX4 in reversible starch phosphorylation and further advances our understanding of atypical kinetic mechanisms.

Original language	English
Pages (from-to)	2425-2435
Number of pages	11
Journal	Biochemistry
Volume	60
Issue number	31
DOIs	https://doi.org/10.1021/acs.biochem.1c00307
State	Published - Aug 10 2021

Bibliographical note

Publisher Copyright:
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Funding

This study was supported by the National Science Foundation award MCB-2012074 to M.R., CHE-1808304 to C.W.V.K., and Skidmore College start-up funds provided to M.R. Support was also provided by the Schupf Scholar Program to C.A.M. and Skidmore Summer Collaborative Research Fellowship to T.H. This work also used the support provided by the Swiss National Science Foundation project number P2ZHP3_178037 to M.T.

Funders	Funder number
Skidmore College
Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung	178037, P2ZHP3_178037
U.S. Department of Energy Chinese Academy of Sciences Guangzhou Municipal Science and Technology Project Oak Ridge National Laboratory Extreme Science and Engineering Discovery Environment National Science Foundation National Energy Research Scientific Computing Center National Natural Science Foundation of China	CHE-1808304, MCB-2012074

ASJC Scopus subject areas

Biochemistry

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title = "Cooperative Kinetics of the Glucan Phosphatase Starch Excess4",

abstract = "Glucan phosphatases are members of a functionally diverse family of dual-specificity phosphatase (DSP) enzymes. The plant glucan phosphatase Starch Excess4 (SEX4) binds and dephosphorylates glucans, contributing to processive starch degradation in the chloroplast at night. Little is known about the complex kinetics of SEX4 when acting on its complex physiologically relevant glucan substrate. Therefore, we explored the kinetics of SEX4 against both insoluble starch and soluble amylopectin glucan substrates. SEX4 displays robust activity and a unique sigmoidal kinetic response to amylopectin, characterized by a Hill coefficient of 2.77 ± 0.63, a signature feature of cooperativity. We investigated the basis for this positive kinetic cooperativity and determined that the SEX4 carbohydrate-binding module (CBM) dramatically influences the binding cooperativity and substrate transformation rates. These findings provide insights into a previously unknown but important regulatory role for SEX4 in reversible starch phosphorylation and further advances our understanding of atypical kinetic mechanisms.",

author = "Mak, \{Claudia A.\} and Kenyon Weis and Tiffany Henao and Andrea Kuchtova and Tiantian Chen and Savita Sharma and Meekins, \{David A.\} and Matthias Thalmann and \{Vander Kooi\}, \{Craig W.\} and Madushi Raththagala",

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doi = "10.1021/acs.biochem.1c00307",

language = "English",

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T1 - Cooperative Kinetics of the Glucan Phosphatase Starch Excess4

AU - Mak, Claudia A.

AU - Weis, Kenyon

AU - Henao, Tiffany

AU - Kuchtova, Andrea

AU - Chen, Tiantian

AU - Sharma, Savita

AU - Meekins, David A.

AU - Thalmann, Matthias

AU - Vander Kooi, Craig W.

AU - Raththagala, Madushi

PY - 2021/8/10

Y1 - 2021/8/10

N2 - Glucan phosphatases are members of a functionally diverse family of dual-specificity phosphatase (DSP) enzymes. The plant glucan phosphatase Starch Excess4 (SEX4) binds and dephosphorylates glucans, contributing to processive starch degradation in the chloroplast at night. Little is known about the complex kinetics of SEX4 when acting on its complex physiologically relevant glucan substrate. Therefore, we explored the kinetics of SEX4 against both insoluble starch and soluble amylopectin glucan substrates. SEX4 displays robust activity and a unique sigmoidal kinetic response to amylopectin, characterized by a Hill coefficient of 2.77 ± 0.63, a signature feature of cooperativity. We investigated the basis for this positive kinetic cooperativity and determined that the SEX4 carbohydrate-binding module (CBM) dramatically influences the binding cooperativity and substrate transformation rates. These findings provide insights into a previously unknown but important regulatory role for SEX4 in reversible starch phosphorylation and further advances our understanding of atypical kinetic mechanisms.

AB - Glucan phosphatases are members of a functionally diverse family of dual-specificity phosphatase (DSP) enzymes. The plant glucan phosphatase Starch Excess4 (SEX4) binds and dephosphorylates glucans, contributing to processive starch degradation in the chloroplast at night. Little is known about the complex kinetics of SEX4 when acting on its complex physiologically relevant glucan substrate. Therefore, we explored the kinetics of SEX4 against both insoluble starch and soluble amylopectin glucan substrates. SEX4 displays robust activity and a unique sigmoidal kinetic response to amylopectin, characterized by a Hill coefficient of 2.77 ± 0.63, a signature feature of cooperativity. We investigated the basis for this positive kinetic cooperativity and determined that the SEX4 carbohydrate-binding module (CBM) dramatically influences the binding cooperativity and substrate transformation rates. These findings provide insights into a previously unknown but important regulatory role for SEX4 in reversible starch phosphorylation and further advances our understanding of atypical kinetic mechanisms.

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SN - 0006-2960

VL - 60

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EP - 2435

JO - Biochemistry

JF - Biochemistry

IS - 31

ER -

Cooperative Kinetics of the Glucan Phosphatase Starch Excess4

Abstract

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