Skip to main content
SpringerLink
Log in

Estimating the energetic contribution of hydrogen bonding to the stability of Candida methylica formate dehydrogenase by using double mutant cycle

  • Published:
Biotechnology Letters Aims and scope Submit manuscript

Abstract

An homology model of Candida methylica formate dehydrogenase (cmFDH) was constructed based on the Pseudomonas sp. 101 formate dehydrogenase (psFDH) structure. In wild type  cmFDH, Thr169 and Thr226 can form hydrogen bonds with each other. We measured the interaction energy between the two threonines independent of other interactions in the proteins by using a so-called double mutant cycle and assessing the protein stability from the concentration of guanidine hydrochloride needed to denature 50% of the molecules. We conclude that the hydrogen bonds stabilize the wild type protein by −4 kcal mol−1.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Byrne MP, Manuel RL, LG Lowe, Stites WE (1995) Energetic contribution of side chain hydrogen bonding to the stability of staphylococcal nuclease. Biochemistry 34: 13949–13960.

    Google Scholar 

  • Carter P, Winter G, Wilkinson A, Fersht AR (1984) The use of double mutants to detect structural changes in the active site of the tyrosyl-tRNA synthetase (Bacillus stearothermophilus). Cell 38: 835–840. 1140

    Google Scholar 

  • Fersht AR (1999) Structure and Mechanism in Protein Science: A Guide to Enzyme Catalysis and Protein Folding. NewYork: W.H. Freeman and Company.

    Google Scholar 

  • Karaguler NG, Sessions RB, Clarke AR, Holbrook JJ (2001) A single mutation in the NAD-specific formate dehydrogenase from Candida methylicaallows the enzyme to use NADP. Biotechnol.Lett. 23: 283–287.

    Google Scholar 

  • Karaguler NG, Sessions RB, Holbrook JJ (2001) Role of glutamate-52 in the mechanism of L-lactate dehydrogenase from Bacillus stearothermophilus. Biotechnol. Lett. 23: 395–399.

    Google Scholar 

  • Lamzin V, Dauter Z, Popov V, Harutyunhan E, Wilson K (1994) High resolution structures of holo and apo formate dehydrogenase. J. Mol. Biol. 236: 759–785.

    Google Scholar 

  • Lazaridis T, Archontis G, Karplus M (1995) Enthalpic contribution to protein stability: insights from atom-based calculations and statistical mechanics. Adv. Protein Chem. 47: 231–306.

    Google Scholar 

  • Leatherbarrow RJ (1992) Grafit 3.0. Staines, UK: Erithacus Software.

    Google Scholar 

  • Pace CN (1995) Evaluating contribution of hydrogen bonding and hydrophobic bonding to protein folding. Meth. Enzymol. 259: 538–554.

    Google Scholar 

  • Serrano L, Horovitz A, Avron B, Bycroft M, Fersht AR (1990) Estimating the contribution of engineered surface electrostatic interactions to protein stability by using double mutant cycles. Biochemistry 29: 9343–9352.

    Google Scholar 

  • Sessions RB, Dewar V, Clarke AR, Holbrook JJ (1997) A model of plasmodium falciparumlactate dehydrogenase and its implications for design of improved antimalarials and the enhanced detection of parasitaemia. Protein Eng. 10: 301–306.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Biochemistry, School of Medical Sciences, University Walk, Bristol, BS8 1TD, UK

    Nevin Gül Karagüler, Richard B. Sessions, Kathleen M. Moreton, Anthony R. Clarke & J. John Holbrook

Authors
  1. Nevin Gül Karagüler
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Richard B. Sessions
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kathleen M. Moreton
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Anthony R. Clarke
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. J. John Holbrook
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Nevin Gül Karagüler.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Karagüler, N.G., Sessions, R.B., Moreton, K.M. et al. Estimating the energetic contribution of hydrogen bonding to the stability of Candida methylica formate dehydrogenase by using double mutant cycle. Biotechnology Letters 26, 1137–1140 (2004). https://doi.org/10.1023/B:BILE.0000035485.43826.03

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1023/B:BILE.0000035485.43826.03

Search

Navigation