β-Galactosidase (Escherichia coli) has a second catalytically important Mg2+ site

doi:10.1016/j.bbrc.2006.11.061

Biochemical and Biophysical Research Communications

Volume 352, Issue 2, 12 January 2007, Pages 566-570

https://doi.org/10.1016/j.bbrc.2006.11.061 Get rights and content

Abstract

It is shown here that Escherichia coli β-galactosidase has a second Mg²⁺ binding site that is important for activity. Binding of Mg²⁺ to the second site caused the k_cat (with oNPG as the substrate) to increase about 100 s⁻¹; the K_m was not affected. The K_d for binding the second Mg²⁺ is about 10⁻⁴ M. Since the concentration of free Mg²⁺ in E. coli is about 1–2 mM, the second site is physiologically significant. Non-polar substitutions (Ala or Leu) for Glu-797, a residue in an active site loop, eliminated the k_cat increase. This indicates that the second Mg²⁺ site is near to Glu-797. The K_i values of transition state analogs were decreased by small but statistically significant amounts when the second Mg²⁺ site was occupied and Arrhenius plots showed that less entropic activation energy is required when the second site is occupied. These inhibitor and temperature results suggest that binding of the second Mg²⁺ helps to order the active site for stabilization of the transition state.

Section snippets

Materials and methods

β-Galactosidase expression. Escherichia coli strain LMG 194 (Invitrogen, California, USA) containing a lacZ deletion (F-ΔlacX74 galE thi rpsL ΔphoA (PvuII) Δara714 leu:Tn10) was used for β-galactosidase expression. The expression plasmid was pBAD/His/lacZ (Invitrogen) containing the lacZ gene and an ampicillin resistance gene. Mutants of pBAD/His/lacZ were made by Bio T&T Inc. (Quebec, Canada). The codon for Glu-797 was substituted with codons for Ala, Leu, Asp, and Gln. The mutated plasmids

Purity

The expressed and purified enzymes were >97% pure when analyzed by SDS–PAGE (data not shown).

Mg²⁺ titration

Plots of the k_cat values (with oNPG) of wild-type and the Glu-797 β-galactosidase variants are shown as functions of pMg (pMg = −log [Mg²⁺]_free) in Fig. 2. Each β-galactosidase variant had a lower k_cat value than the wild-type enzyme at all Mg²⁺ concentrations. The k_cat of wild-type β-galactosidase at very low Mg²⁺ concentrations was about 120 s⁻¹. This value increased to 498 s⁻¹, with a mid-point of about

Discussion

This study shows that there is a second Mg²⁺ site that affects the activity of β-galactosidase of E. coli, that Glu-797 is an essential component of this second site and that the effect is due to more favorable entropic stabilization of the transition state. A loop region (residues 794–803) within the active site of β-galactosidase [9], [11], [12], [13], [14] is “open” for parts of the enzyme reaction and “closed” for others. The role of this loop is not entirely known. When Glu-797 (a residue

Acknowledgments

This work was supported in part by NSERC (Canada) Grant #1896 to R.E.H. and an equipment grant to M.E.F. M.E.F. is a Biomedical Scholar supported by the Alberta Heritage Foundation for Medical Research. We thank Genesis Juat for excellent technical help.

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Biochemical and Biophysical Research Communications

Abstract

Section snippets

Materials and methods

Purity

Mg²⁺ titration

Discussion

Acknowledgments

References (20)

Site directed substitutions suggest that His-418 of β-galactosidase (E. coli) is a ligand to Mg²⁺

Biochem. Biophys. Res. Comm.

Site directed mutants of β-galactosidase show that Tyr-503 is unimportant in Mg²⁺ binding but that Glu-461 is very important and may be a ligand of Mg²⁺

Biochem. Biophys. Res. Comm.

Glu-416 of β-galactosidase (Escherichia coli) is a Mg²⁺ ligand and β-galactosidases with substitutions for Glu-416 are inactivated rather than activated by Mg²⁺

Biochem. Biophys. Res. Comm.

The role of magnesium ions in β-galactosidase-catalysed hydrolyses

Biochem. J.

Kinetic study of the activation process of β-galactosidase from Escherichia coli by Mg²⁺

Eur. J. Biochem.

pH dependence of the activity of β-galactosidase from Escherichia coli

Eur. J. Biochem.

Interaction of the lac Z β-galactosidase of Escherichia coli with some β-d-galactopyranoside competitive inhibitors

Biochem. J.

Interaction of divalent cations with β-galactosidase (Escherichia coli)

Biochemistry

Three-dimensionsal structure of β-galactosidase from E. coli

Nature

High resolution refinement of β-galactosidase in a new crystal form reveals multiple metal binding sites and provides a structural basis for complementation

Protein Sci.

Cited by (33)

The functional mutational landscape of the lacZ gene

Structural and functional insights of β galactosidase and its potential applications

Downstream processing technologies in the biocatalytic production of oligosaccharides

Hydrogen production by Escherichia coli during anaerobic utilization of mixture of lactose and glycerol: Enhanced rate and yield, prolonged production

Design and Synthesis of 2-Acetamido-2,3-dideoxythiodisaccharides via Diastereoselective Conjugate Addition to Sugar Enone O-Acetyl Oximes. Galactosidase Inhibition Studies

Purification and characterization of β-galactosidase from probiotic Pediococcus acidilactici and its use in milk lactose hydrolysis and galactooligosaccharide synthesis

β-Galactosidase (Escherichia coli) has a second catalytically important Mg2+ site

Abstract

Section snippets

Materials and methods

Purity

Mg2+ titration

Discussion

Acknowledgments

Biochem. Biophys. Res. Comm.

Biochem. Biophys. Res. Comm.

Biochem. Biophys. Res. Comm.

The role of magnesium ions in β-galactosidase-catalysed hydrolyses

Biochem. J.

Kinetic study of the activation process of β-galactosidase from Escherichia coli by Mg2+

Eur. J. Biochem.

pH dependence of the activity of β-galactosidase from Escherichia coli

Eur. J. Biochem.

Interaction of the lac Z β-galactosidase of Escherichia coli with some β-d-galactopyranoside competitive inhibitors

Biochem. J.

Interaction of divalent cations with β-galactosidase (Escherichia coli)

Biochemistry

Three-dimensionsal structure of β-galactosidase from E. coli

Nature

High resolution refinement of β-galactosidase in a new crystal form reveals multiple metal binding sites and provides a structural basis for complementation

Protein Sci.

β-Galactosidase (Escherichia coli) has a second catalytically important Mg²⁺ site

Mg²⁺ titration

Kinetic study of the activation process of β-galactosidase from Escherichia coli by Mg²⁺