Characterizing lysine acetylation of glucokinase

Glucokinase (GK) catalyzes the phosphorylation of glucose to form glucose‐6‐phosphate as the substrate of glycolysis for energy production. Acetylation of lysine residues in Escherichia coli GK has been identified at multiple sites by a series of proteomic studies, but the impact of acetylation on G...

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Bibliographic Details
Published in:Protein science 2024-01, Vol.33 (1), p.e4845-n/a
Main Authors: Fatema, Nour, Li, Xinyu, Gan, Qinglei, Fan, Chenguang
Format: Article
Language:English
Subjects:
Acetylation
Acetyllysine
deacetylase
Deacetylation
E coli
Escherichia coli - metabolism
Genetic code
genetic code expansion
Glucokinase
Glucokinase - genetics
Glucokinase - metabolism
Glucose
Glutamine
Glutamine - genetics
Glutamine - metabolism
Glycolysis
Lysine
Lysine - genetics
lysine acetylation
Phosphorylation
Protein Processing, Post-Translational
Proteomics
Substitutes
Substrates
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Summary:Glucokinase (GK) catalyzes the phosphorylation of glucose to form glucose‐6‐phosphate as the substrate of glycolysis for energy production. Acetylation of lysine residues in Escherichia coli GK has been identified at multiple sites by a series of proteomic studies, but the impact of acetylation on GK functions remains largely unknown. In this study, we applied the genetic code expansion strategy to produce site‐specifically acetylated GK variants which naturally exist in cells. Enzyme assays and kinetic analyses showed that lysine acetylation decreases the GK activity, mostly resulting from acetylation of K214 and K216 at the entrance of the active site, which impairs the binding of substrates. We also compared results obtained from the glutamine substitution method and the genetic acetyllysine incorporation approach, showing that glutamine substitution is not always effective for mimicking acetylated lysine. Further genetic studies as well as in vitro acetylation and deacetylation assays were performed to determine acetylation and deacetylation mechanisms, which showed that E. coli GK could be acetylated by acetyl‐phosphate without enzymes and deacetylated by CobB deacetylase.
ISSN:0961-8368
1469-896X
DOI:10.1002/pro.4845