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Studies of Glu-416 Variants of β-Galactosidase (E. coli) Show that the Active Site Mg2+ is Not Important for Structure and Indicate that the Main Role of Mg2+ is to Mediate Optimization of Active Site Chemistry

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Abstract

Variants of β-galactosidase with Valine and with Glutamine replacing Glutamate-416 did not have a Mg2+ bound at the active site even at high Mg2+ concentrations (200 mM). They had low catalytic activity and the pH profiles were very different from those of the native enzyme. In addition, substrates, substrate analogs, transition state analogs and galactose bound very poorly. However, the orientation and conformation of the Mg2+ ligands (residues 416, 418, and 461) as well as the B-factors of these three side chains did not change significantly. The structures, conformations and B-factors of other active site residues were also essentially unchanged. These studies show that the active site Mg2+ is not necessary for structure and is, therefore, mainly important for modulating the chemistry and mediating the interactions between the active site components.

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Notes

  1. Mn2+ can substitute for Mg2+ [3] but this results in only small changes in activity and binding.

  2. The Advanced Light Source synchrotron access program is supported by grants from the Alberta Science and Research Authority and the Alberta Heritage Foundation for Medical Research. The Advanced Light Source at Lawrence Berkeley Laboratory is operated by the Department of Energy (USA) and supported by the National Institute of Health (USA). The National Science Foundation, the University of California and Henry Wheeler fund Beamline 8.3.1.

  3. The average B-factors for each of the residues of the enzymes were 23.7 and 21.6 for E416Q- and E416V-β-galactosidase, respectively. Totally disordered residues have B-factors of 100. Thus the ratios of the B-factors of the residues to the overall B-factors of the enzymes would be between 4 and 5 if they were in total disorder.

  4. The [Mg2+] at which the native enzyme has half of its full activity is about 10−7 M. Thus the concentration at which these studies were done (10−4 M) were several orders of magnitude higher than the Mg2+ dissociation constant for the native enzyme.

Abbreviations

Amp:

Ampicillin

CNS:

Crystallographic and NMR system

COOT:

Crystallographic object oriented tool kit

DMSO:

Dimethyl sulfoxide

EDTA:

Ethylene diamine tetra acetate

IPTG:

Isopropyl-thio-β-d-galactopyranoside

oNP:

o-Nitrophenol

oNPG:

o-Nitrophenyl-β-d-galactopyranoside

SDS–PAGE:

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis

TES:

N-tris[hydroxymethylmethyl-2-aminoethanesulfonic acid

References

  1. Huber RE, Kurz G, Wallenfels K (1976) Biochemistry 15:1994–2001

    Article  CAS  Google Scholar 

  2. Juers DH, Heightman TD, Vasella A, McCarter JD, Mackenzie L, Withers SG, Matthews BW (2001) Biochemistry 40:14781–14794

    Article  CAS  Google Scholar 

  3. Huber RE, Parfett C, Woulfe-Flanagan H, Thompson DJ (1979) Biochemistry 18:4090–4095

    Article  CAS  Google Scholar 

  4. Jacobson RH, Zhang XJ, DuBose RF, Matthews BW (1994) Nature 369:761–766

    Article  CAS  Google Scholar 

  5. Sutendra G, Wong S, Fraser ME, Huber RE (2007) Biochem Biophys Res Commun 352:566–570

    Article  CAS  Google Scholar 

  6. Juers DH, Wigley RH, Zhang X, Huber RE, Tronrud DE, Matthews BW (2000) Prot Sci 9:1685–1699

    Article  CAS  Google Scholar 

  7. Roth NJ, Huber RE (1994) Biochem Biophys Res Commun 201:866–870

    Article  CAS  Google Scholar 

  8. Roth NJ, Huber RE (1996) Biochem Biophys Res Commun 219:111–115

    Article  CAS  Google Scholar 

  9. Selwood T, Sinnott ML (1990) Biochem J 268:317–323

    CAS  Google Scholar 

  10. Richard JP, Huber RE, Lin S, Heo C, Amyes TL (1996) Biochemistry 35:12377–12386

    Article  CAS  Google Scholar 

  11. Juers DH, Rob B, Dugdale ML, Rahimzadeh N, Giang C, Lee M, Matthews BW, Huber RE (2009) Prot Sci 18:1281–1292

    Article  CAS  Google Scholar 

  12. Evans PR (1993) Data reduction. Proceedings of CCP4 Study Weekend on Data collection and processing, pp 114–122

  13. Kabsch W (1988) J Appl Cryst 21:916–924

    Article  CAS  Google Scholar 

  14. Leslie AGW (1991) In: Moras D, Podjarny AD, Thierry JC (eds) Crystallographic computing 5, from chemistry to biology. Oxford University Press, Oxford

  15. Brünger AT, Adams PD, Clore GM, DeLano WL, Gros P, Grosse-Kunstleve RW, Jiang JS, Kuszewski J, Nilges M, Pannu NS, Read RJ, Rice LM, Simonson T, Warren GL (1998) Acta Crystallogr D Biol Crystallogr 54:905–921

    Article  Google Scholar 

  16. Emsley P, Cowtan K (2004) Acta Crystallogr D Biol Crystallogr 60:2126–2132

    Article  Google Scholar 

  17. Winn M, Isupov M, Murshudov GN (2001) Acta Crystallogr D Biol Crystallogr D57:122–133

    Article  CAS  Google Scholar 

  18. Deschavanne PJ, Viratelle OM, Yon JM (1978) J Biol Chem 253:833–837

    CAS  Google Scholar 

  19. Huber RE, Gaunt MT (1983) Arch Biochem Biophys 220:263–271

    Article  CAS  Google Scholar 

Download references

Acknowledgments

A grant from the Natural Sciences and Engineering Research Council of Canada to REH (#1891) is gratefully appreciated. We thank Dr. Marie Fraser (Biological Sciences, University of Calgary) for help in processing the diffraction data.

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Author notes

  1. Nathan J. Roth

    Present address: Talecris Biotherapeutics, Inc., 85 T.W. Alexander Dr., Research Triangle Park, NC, 27709, USA

Authors and Affiliations

  1. Department of Biological Sciences, University of Calgary, Calgary, AB, T2N 1N4, Canada

    Summie Lo, Megan L. Dugdale, Nisha Jeerh, Tabitha Ku, Nathan J. Roth & Reuben E. Huber

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  1. Summie Lo
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  2. Megan L. Dugdale
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  3. Nisha Jeerh
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Correspondence to Reuben E. Huber.

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Lo, S., Dugdale, M.L., Jeerh, N. et al. Studies of Glu-416 Variants of β-Galactosidase (E. coli) Show that the Active Site Mg2+ is Not Important for Structure and Indicate that the Main Role of Mg2+ is to Mediate Optimization of Active Site Chemistry. Protein J 29, 26–31 (2010). https://doi.org/10.1007/s10930-009-9216-x

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