Leuconostoc mesenteroides
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.
Keywords: Hydrolase; Transfer reaction; Glycoside Phosphorylase;
Glycoside hydrolase; Kinetic mechanism