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

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. ORCID:, Pudney, Christopher R. and Jones, D. Dafydd ORCID: 2023. Glycosylation increases active site rigidity leading to improved enzyme stability and turnover. The FEBS Journal 290 (15) , pp. 3812-3827. 10.1111/febs.16783

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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.

Item Type: Article
Date Type: Publication
Status: Published
Schools: Biosciences
Additional Information: License information from Publisher: LICENSE 1: URL:
Publisher: Wiley
ISSN: 1742-464X
Funders: BBSRC, Wellcome Trust
Date of First Compliant Deposit: 4 April 2023
Date of Acceptance: 27 March 2023
Last Modified: 30 Nov 2023 09:41

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