Escherichia coli β-Galactosidase Recognizes a High-Energy Conformation of C-Lactose, a Nonhydrolizable Substrate Analogue. NMR and Modeling Studies of the Molecular Complex

Abstract

The enzyme-bound conformation of C-lactose, an Escherichia coli β-galactosidase inhibitor has been determined by NMR spectroscopy. It is demonstrated that the enzyme selects a high-energy conformation of this closely related structural analogue of the natural substrate, lactose. In addition, a molecular modeling protocol has been performed in order to obtain a detailed three-dimensional structure of the complex that can explain, in structural terms, the role that the key amino acid residues play in the catalytic mechanism. The implications of the recognition of a high-energy conformation of the analogue are also outlined.

The Crystal Structure of Galacto-N-biose/Lacto-N-biose I Phosphorylase

“…Alternatively, the conformational itinerary of ␤-galactosidases (or phosphorylases acting on ␤-galactosyl bonds) may be different from those of ␤-glucosidases because of potential crashes between the axial C4 hydroxyl and ␤-anomeric substituents. Espinosa et al (59) conducted an NMR study of a nonhydrolyzable C-glycoside analogue of lactose bound to E. coli GH2 ␤-galactosidase. They suggested that the galactopyranose ring of the bound analog is not in a distorted conformation (e.g.…”

α-d-Glcp-(1↔1)-β-d-Galp-containing oligosaccharides, novel products from lactose by the action of β-galactosidase

“…This can be explained by the way the enzyme binds to lactose [13]. According to [13] lactose can be positioned on the enzyme via several hydrophobic contacts and intermolecular hydrogen bonds (Fig.…”

“…These spectra were acquired with the proton offset at about 4.6 ppm and a sweepwidth of about 6 ppm. In the 13 C dimension the offset was placed around 80 ppm and a sweep width of 60 ppm was used. Typically, 300-350 free induction decays of 1024 data points were acquired using 128-256 scans per decay.…”

“…For the NMR analysis, use was made of the chemical shifts of linear b-(1 4)-linked D-galactobi-to pentaose (see Table 1). The 1 H and 13 C chemical shifts of the latter compounds, which have not been reported so far in detail, are shown in Tables 1 and 2, respectively. Additionally to the known oligogalactosylated glucoses, each Bio-Gel P-2 fraction contained at least one less retarded component (Fig.…”

“…Proposed hydrogen bonding between b-galactosidase and lactose [13]. During the transgalactosylation process (B), the terminal residue of the galactosyl acceptor will be located in the same part of the pocket as Glc in a galactosidase reaction.…”