Biochemical and Biophysical Research Communications
β-Galactosidase (Escherichia coli) has a second catalytically important Mg2+ site
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 kcat 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 kcat value than the wild-type enzyme at all Mg2+ concentrations. The kcat 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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