Campus Units

Chemistry, Biochemistry, Biophysics and Molecular Biology, Roy J. Carver Department of

Document Type

Article

Publication Version

Published Version

Publication Date

2-16-2018

Journal or Book Title

Journal of Biological Chemistry

Volume

293

Issue

7

First Page

2631

Last Page

2639

DOI

10.1074/jbc.RA117.001466

Abstract

The bacterial phosphotransferase system (PTS) is a signal transduction pathway that couples phosphoryl transfer to active sugar transport across the cell membrane. The PTS is initiated by phosphorylation of enzyme I (EI) by phosphoenolpyruvate (PEP). The EI phosphorylation state determines the phosphorylation states of all other PTS components and is thought to play a central role in the regulation of several metabolic pathways and to control the biology of bacterial cells at multiple levels, for example, affecting virulence and biofilm formation. Given the pivotal role of EI in bacterial metabolism, an improved understanding of the mechanisms controlling its activity could inform future strategies for bioengineering and antimicrobial design. Here, we report an enzymatic assay, based on Selective Optimized Flip Angle Short Transient (SOFAST) NMR experiments, to investigate the effect of the small-molecule metabolite α-ketoglutarate (αKG) on the kinetics of the EI-catalyzed phosphoryl transfer reaction. We show that at experimental conditions favoring the monomeric form of EI, αKG promotes dimerization and acts as an allosteric stimulator of the enzyme. However, when the oligomerization state of EI is shifted toward the dimeric species, αKG functions as a competitive inhibitor of EI. We developed a kinetic model that fully accounted for the experimental data and indicated that bacterial cells might use the observed interplay between allosteric stimulation and competitive inhibition of EI by αKG to respond to physiological fluctuations in the intracellular environment. We expect that the mechanism for regulating EI activity revealed here is common to several other oligomeric enzymes.

Comments

This article is published as Nguyen, Trang T., Rodolfo Ghirlando, and Vincenzo Venditti. "The oligomerization state of bacterial enzyme I (EI) determines EI’s allosteric stimulation or competitive inhibition by α-ketoglutarate." Journal of Biological Chemistry 293, no. 7 (2018): 2631. DOI: 10.1074/jbc.RA117.001466.

Rights

Works produced by employees of the U.S. Government as part of their official duties are not copyrighted within the U.S. The content of this document is not copyrighted.

Language

en

File Format

application/pdf

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