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On the donor substrate dependence of group transfer reactions by hydrolytic enzymes: insight from kinetic analysis of sucrose phosphorylase-catalyzed transglycosylation
  • Mario Klimacek,
  • Alexander Sigg,
  • Bernd Nidetzky
Mario Klimacek
Graz University of Technology
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Alexander Sigg
Graz University of Technology
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Bernd Nidetzky
Graz University of Technology
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Peer review status:Published

08 May 2020Submitted to Biotechnology and Bioengineering
08 May 2020Submission Checks Completed
08 May 2020Assigned to Editor
10 May 2020Reviewer(s) Assigned
24 May 2020Review(s) Completed, Editorial Evaluation Pending
24 May 2020Editorial Decision: Revise Minor
15 Jun 20201st Revision Received
15 Jun 2020Submission Checks Completed
15 Jun 2020Assigned to Editor
21 Jun 2020Editorial Decision: Accept
21 Jun 2020Review(s) Completed, Editorial Evaluation Pending
Published in Biotechnology and Bioengineeringy. 23 Jun 2020. 10.1002/bit.27471

Abstract

Chemical group-transfer reactions by hydrolytic enzymes have considerable importance in biocatalytic synthesis and are exploited broadly in commercial-scale chemical production. Mechanistically, these reactions have in common the involvement of a covalent enzyme intermediate which is formed upon enzyme reaction with the donor substrate and is subsequently intercepted by a suitable acceptor. Here, we studied the glycosylation of glycerol from sucrose by sucrose phosphorylase (SucP) to clarify a peculiar, yet generally important characteristic of this reaction: partitioning between glycosylation of glycerol and hydrolysis depends on the type and the concentration of the donor substrate used (here: sucrose, α-D-glucose 1-phosphate (G1P)). We develop a kinetic framework to analyze the effect and provide evidence that, when G1P is used as donor substrate, hydrolysis occurs not only from the β-glucosyl-enzyme intermediate (E-Glc), but additionally from a noncovalent complex of E-Glc and substrate which unlike E-Glc is unreactive to glycerol. Depending on the relative rates of hydrolysis of free and substrate-bound E-Glc, inhibition (Leuconostoc mesenteroides SucP) or apparent activation (Bifidobacterium adolescentis SucP) is observed at high donor substrate concentration. Using G1P at a concentration excluding the substrate-bound E-Glc, the product ratio changes to a value consistent with reaction exclusively through E-Glc, independent of the donor substrate used. Collectively, these results give explanation for a kinetic behavior of SucP not previously accounted for, provide essential basis for design and optimization of the synthetic reaction, and establish a theoretical framework for the analysis of kinetically analogous group transfer reactions by hydrolytic enzymes.