The oligomerization state of bacterial enzyme I (EI) determines EI's allosteric stimulation or competitive inhibition by α-ketoglutarate.
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.
['Nguyen|Trang T|TT|', 'Ghirlando|Rodolfo|R|', 'Venditti|Vincenzo|V|']
D000494:Allosteric Regulation D001426:Bacterial Proteins D004791:Enzyme Inhibitors D004926:Escherichia coli D007656:Ketoglutaric Acids D007700:Kinetics D010731:Phosphoenolpyruvate Sugar Phosphotransferase System D017852:Phosphotransferases (Nitrogenous Group Acceptor)
signaling SOFAST-TROSY allosteric regulation enzyme stimulation inhibition mechanism nuclear magnetic resonance (NMR) protein assembly protein oligomerization