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

• Published in: The FEBS journal Vol. 290; no. 15; pp. 3812 - 3827
• Main Authors: Ramakrishnan, Krithika, Johnson, Rachel L., Winter, Samuel D., Worthy, Harley L., Thomas, Christopher, Humer, Diana C., Spadiut, Oliver, Hindson, Sarah H., Wells, Stephen, Barratt, Andrew H., Menzies, Georgina E., Pudney, Christopher R., Jones, D. Dafydd
• Format: Journal Article
• Language: English
• Published: England Blackwell Publishing Ltd 01.08.2023; John Wiley and Sons Inc
• Subjects: Catalytic Domain; Circular dichroism; Dichroism; Dynamic stability; Enzyme enhancment; Enzyme rigidity; Enzyme Stability; Enzymes; Excitation spectra; Flexibility; Fluorescence; Fluorescence spectroscopy; Glycosylation; Horseradish peroxidase; Molecular Dynamics; Original; Peroxidase; Phenylenediamine; Post-Translational Modification; Protein Processing, Post-Translational; Protein structure; Proteins; Rigidity; Spectrometry, Fluorescence; Spectroscopy; Spectrum analysis; Structure-function relationships; Substrates; Thermal stability; Enzyme enhancment; glycosylation; post-translational modification; molecular dynamics; Enzyme rigidity
• ISSN: 1742-464X; 1742-4658
• DOI: 10.1111/febs.16783

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. Glycosylation is one of the most common post‐translational modifications in nature. Yet, its impact on the enzyme structure–function relationship is limited. Using a combination of experimental and computational modelling, we found that the haem‐enzyme horseradish peroxidase becomes more rigid on glycosylation. Rather than increased rigidity impeding catalysis as per the classic ‘activity‐stability trade‐off’ idea, glycosylation increases turnover by 10‐fold and makes the enzyme more stable.