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

Autor: Ramakrishnan K; Molecular Biosciences Division, School of Biosciences, Cardiff University, UK., Johnson RL; Molecular Biosciences Division, School of Biosciences, Cardiff University, UK., Winter SD; Department of Biology and Biochemistry, University of Bath, UK., Worthy HL; Molecular Biosciences Division, School of Biosciences, Cardiff University, UK.; Biosciences, Faculty of Health and Life Sciences, University of Exeter, UK., Thomas C; Ortho Clinical Diagnostics, Pencoed, UK., Humer DC; Institute of Chemical, Environmental and Bioscience Engineering, Research Area Biochemical Engineering, TU Wien, Austria., Spadiut O; Institute of Chemical, Environmental and Bioscience Engineering, Research Area Biochemical Engineering, TU Wien, Austria., Hindson SH; Department of Biology and Biochemistry, University of Bath, UK., Wells S; Department of Physics, University of Bath, UK., Barratt AH; Molecular Biosciences Division, School of Biosciences, Cardiff University, UK., Menzies GE; Molecular Biosciences Division, School of Biosciences, Cardiff University, UK., Pudney CR; Department of Biology and Biochemistry, University of Bath, UK.; Centre for Therapeutic Innovation, University of Bath, UK., Jones DD; Molecular Biosciences Division, School of Biosciences, Cardiff University, UK.
Jazyk: angličtina
Zdroj: The FEBS journal [FEBS J] 2023 Aug; Vol. 290 (15), pp. 3812-3827. Date of Electronic Publication: 2023 Apr 03.
DOI: 10.1111/febs.16783
Abstrakt: 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.
(© 2023 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)
Databáze: MEDLINE
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