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*

*Korrespondierende/r Autor/in für diese Arbeit

Publikation: Beitrag in einer FachzeitschriftArtikel

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. At a G1P concentration that excludes the substrate-bound E-Glc, the transfer/hydrolysis 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.

Originalspracheenglisch
Seiten (von - bis)2933-2943
Seitenumfang11
FachzeitschriftBiotechnology and Bioengineering
Jahrgang117
Ausgabenummer10
Frühes Online-Datum23 Jun 2020
DOIs
PublikationsstatusVeröffentlicht - 1 Okt 2020

ASJC Scopus subject areas

  • !!Applied Microbiology and Biotechnology
  • Bioengineering
  • Biotechnology

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