Skip to main content
SpringerLink
Log in

Practical Considerations When Using Temperature to Obtain Rate Constants and Activation Thermodynamics of Enzymes with Two Catalytic Steps: Native and N460T-β-Galactosidase (E. coli) as Examples

  • Published:
The Protein Journal Aims and scope Submit manuscript

Abstract

The values of the rate constants and the associated enthalpies and entropies of enzymes with two catalytic steps can be measured by determining the effects of temperature on the k cat values. Practical considerations that should be taken into account when doing this are presented. The narrow temperature range available with enzymes and the sensitivity of pH to temperature mean that special attention to detail must be taken and this study highlights the assiduousness needed. The necessity of conversion of apparent k cat to true k cat values when assays are done with products having pKa values near to the assay pH is shown and the importance of obtaining sufficient data is emphasized. Reasons that non-linear regression should be used to obtain the estimates of rate constants and activation thermodynamic parameters are given. Other precautions and recommendations are also presented. Results obtained by this method for native β-galactosidase (E. coli) and for a β-galactosidase in which a Thr was substituted for Asn-460 were analyzed to demonstrate the valuable mechanistic details of enzymes that can be obtained from studies of this type.

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Scheme 1

Similar content being viewed by others

Notes

  1. If there were a large (~100-fold) difference in ∆H ‡2 and ∆H ‡3 , the transition from k 3 to k 2 being rate limiting would be distinct with a definite break in an Arrhenius plot rather than a curve. The lines would be linear on each side of the break. Thus, linear regression could be applied at the two sides of the break where k 2 and k 3 are definitely rate limiting. Such big differences in activation enthalpy between two catalytic steps are, however, unlikely with enzymes. Note that distinct breaks would also be seen for an enzyme with only one catalytic step if that enzyme undergoes a clear-cut temperature dependent conformation change that affects activity [14] and one must be careful in interpreting such graphs.

  2. A study of the temperature effects on β-galactosidase in the presence and absence of monovalent cations was included in an earlier study done in our laboratory [15]. It was not done as rigorously as the present study and the enzyme has low heat stability in the absence of monovalent cation but it showed that when no monovalent cation was present the entropy of k 2 for oNPG was decreased while the enthalpy was not changed significantly.

  3. The B-factors of d-galactonolactone (relative to the average B factors for the whole enzyme) are larger for N460T-ß-galactosidase than for native. In particular, the average B factors for C2 and O2 of the d-galactonolactone bound to N460T-β-galactosidase are 32 and 38% greater, respectively, than for native (relative to the average B-factor values of the whole proteins).

Abbreviations

Amp:

Ampicillin

BSA:

Bovine serum albumin

EDTA:

Ethylene diamine tetraacetic acid

LB:

Luria–Bertini

Onp:

o-Nitrophenol

oNPG:

o-Nitrophenyl-β-d-galactopyranoside

Pnp:

p-Nitrophenol

pNPG:

p-Nitrophenyl-β-d-galactopyranoside

TES:

N-Tris(hydroxymethyl) methyl-2-aminoethane-sulfonic acid

References

  1. Ayala YM, Di Cera E (2000) Prot Sci 9:1589–1593

    Article  CAS  Google Scholar 

  2. Cornish-Bowden A (2002) J Biosci 27:121–126

    Article  Google Scholar 

  3. Gebler JC, Aebersold R, Withers SG (1992) J Biol Chem 267:11126–11130

    CAS  Google Scholar 

  4. Huber RE, Brockbank RL (1987) Biochemistry 26:1526–1531

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  6. Stokes TM, Wilson IB (1972) Biochemistry 11:1061–1064

    Article  CAS  Google Scholar 

  7. Motulsky HJ, Christopoulos A (2003) www.graphpad.com, GraphPad Software Inc.

  8. Allan GF, Robinson RA, Bower VE (1962) J Phys Chem 66:171–172

    Article  Google Scholar 

  9. Robinson RA, Peiperl A (1963) J Phys Chem 67:1723–1724

    Article  CAS  Google Scholar 

  10. Motulsky HJ, Ransnas LA (1987) FASEB J 1:365–374

    CAS  Google Scholar 

  11. Huber RE, Gaunt MT, Hurlburt KL (1984) Arch Biochem Biophys 234:151

    Article  CAS  Google Scholar 

  12. McCarter JD, Adam MJ, Withers SG (1992) Biochem J 286:721–727

    CAS  Google Scholar 

  13. Huber RE, Hurlburt KL (1986) Arch Biochem Biophys 246:411–418

    Article  CAS  Google Scholar 

  14. Weingand-Ziade A, Renault F, Masson P (1999) Eur J Biochem 264:327–335

    Article  CAS  Google Scholar 

  15. Xu J, McRae MA, Harron S, Rob B, Huber RE (2004) Biochem Cell Biol 82:275–284

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

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

    John C. Kappelhoff, Su Yi Judy Liu, Megan L. Dugdale, Dayna L. Dymianiw, Larry R. Linton & Reuben E. Huber

Authors
  1. John C. Kappelhoff
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Su Yi Judy Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Megan L. Dugdale
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Dayna L. Dymianiw
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Larry R. Linton
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Reuben E. Huber
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Reuben E. Huber.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kappelhoff, J.C., Liu, S.Y.J., Dugdale, M.L. et al. Practical Considerations When Using Temperature to Obtain Rate Constants and Activation Thermodynamics of Enzymes with Two Catalytic Steps: Native and N460T-β-Galactosidase (E. coli) as Examples. Protein J 28, 96–103 (2009). https://doi.org/10.1007/s10930-009-9168-1

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10930-009-9168-1

Keywords

Search

Navigation