Glycosylation increases active site rigidity leading to improved enzyme stability and turnover

Krithika Ramakrishnan, Rachel L. Johnson, Samuel D. Winter, Harley L. Worthy, Christopher Thomas, Diana C. Humer, Oliver Spadiut, Sarah H. Hindson, Stephen Wells, Andrew H. Barratt, Georgina E. Menzies, Christopher R. Pudney, D. Dafydd Jones

Research output: Contribution to journalArticlepeer-review


Glycosylation is the most prevalent protein post-translational modification, with a quarter of glycosylated proteins having enzymatic properties. Yet, the full impact of glycosylation on the protein structure–function relationship, especially in enzymes, is still limited. Here, we show that glycosylation rigidifies the important commercial enzyme horseradish peroxidase (HRP), which in turn increases its turnover and stability. Circular dichroism spectroscopy revealed that glycosylation increased holo-HRP's thermal stability and promoted significant helical structure in the absence of haem (apo-HRP). Glycosylation also resulted in a 10-fold increase in enzymatic turnover towards o-phenylenediamine dihydrochloride when compared to its nonglycosylated form. Utilising a naturally occurring site-specific probe of active site flexibility (Trp117) in combination with red-edge excitation shift fluorescence spectroscopy, we found that glycosylation significantly rigidified the enzyme. In silico simulations confirmed that glycosylation largely decreased protein backbone flexibility, especially in regions close to the active site and the substrate access channel. Thus, our data show that glycosylation does not just have a passive effect on HRP stability but can exert long-range effects that mediate the ‘native’ enzyme's activity and stability through changes in inherent dynamics.

Original languageEnglish
JournalFEBS Journal
Early online date2 Apr 2023
Publication statusE-pub ahead of print - 2 Apr 2023


  • Enzyme enhancment
  • Enzyme rigidity
  • glycosylation
  • molecular dynamics
  • post-translational modification

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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