Skip to main content
SpringerLink
Log in

Enzyme Kinetics: Theory and Practice

  • Chapter
  • First Online:
Plant Metabolic Networks

Enzymes, like all positive catalysts, dramatically increase the rate of a given reaction. Enzyme kinetics is principally concerned with the measurement and mathematical description of this reaction rate and its associated constants. For many steps in metabolism, enzyme kinetic properties have been determined, and this information has been collected and organized in publicly available online databases (www.brenda.uni-koeln.de). In the first section of this chapter, we review the fundamentals of enzyme kinetics and provide an overview of the concepts that will help the metabolic modeler make the best use of this resource. The techniques and methods required to determine kinetic constants from purified enzymes have been covered in detail elsewhere [4, 12] and are not discussed here. In the second section, we will describe recent advances in the high throughput, high sensitivity measurement of enzyme activity, detail the methodology, and discuss the use of high throughput techniques for profiling large numbers of samples and providing a first step in the process of identifying potential regulatory candidates.

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

Access this chapter

Log in via an institution
Chapter
USD 29.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Notes

  1. 1.

    This format was invented in the early 1950s by the Hungarian G. Takatsky and became popular during the late 1970s with the ELISA application, that’s probably the reason why so many researchers call microplates “elisa plates.”

References

  1. Ballicora MA, Frueauf JB, Fu YB, Schurmann P, Preiss J (2000) Activation of the potato tuber ADP-glucose pyrophosphorylase by thioredoxin. J Biol Chem 275:1315–1320.

    Article  PubMed  CAS  Google Scholar 

  2. Bergmeyer H (1987) Methods of Enzymatic Analysis. VCH Weinheim, Germany.

    Google Scholar 

  3. Bieniawska Z, Barratt DHP, Garlick AP, Thole V, Kruger NJ, Martin C, Zrenner R, Smith AM (2007) Analysis of the sucrose synthase gene family in Arabidopsis. Plant J 49:810–828.

    Article  PubMed  CAS  Google Scholar 

  4. Bisswanger H (2002) Enzyme Kinetics, Principals and Methods. Wiley-VCH Weinheim, Germany.

    Book  Google Scholar 

  5. Blasing OE, Ernst K, Streubel M, Westhoff P, Svensson P (2002) The non-photosynthetic phosphoenolpyruvate carboxylases of the C4 dicot Flaveria trinervia – implications for the evolution of C4 photosynthesis. Planta 215:448–456.

    Article  PubMed  Google Scholar 

  6. Briggs G, Haldane J (1925) A note on the kinetics of enzyme action. Biochem J 19:338–339.

    PubMed  CAS  Google Scholar 

  7. Burrin D (1993) Spectroscopic techniques. In: Wilson K, Goulding K (eds). A Biologist’s Guide to Principals and Techniques of Practical Biochemistry. University Press, Cambridge, UK.

    Google Scholar 

  8. Chen SX, Harmon AC (2006) Advances in plant proteomics. Proteomics 6:5504–5516.

    Article  PubMed  CAS  Google Scholar 

  9. Chiadmi M, Navaza A, Miginiac-Maslow M, Jacquot JP, Cherfils J (1999) Redox signalling in the chloroplast: structure of oxidized pea fructose-1,6-bisphosphate phosphatase. EMBO J 18:6809–6815.

    Article  PubMed  CAS  Google Scholar 

  10. Ching TM (1982) A sensitive and simple assay of starch synthase activity with pyruvate-kinase and luciferase. Anal Biochem 122:139–143.

    Article  PubMed  CAS  Google Scholar 

  11. Cole HA, Wimpenny JW, Hughes DE (1967) ATP pool in Escherichia coli .I. Measurement of pool using a modified luciferase assay. Biochim Biophys Acta 143:445–453.

    Article  PubMed  CAS  Google Scholar 

  12. Cornish-Bowden A (2004) Fundamentals of Enzyme Kinetics. Portland Press, London, UK.

    Google Scholar 

  13. Estevez M, Skarda J, Spencer J, Banaszak L, Weaver TM (2002) X-ray crystallographic and kinetic correlation of a clinically observed human fumarase mutation. Prot Sci 11: 1552–1557.

    Article  CAS  Google Scholar 

  14. Fan F, Wood KV (2007) Bioluminescent assays for high-throughput screening. Assay Drug Dev Technol 5:127–136.

    Article  PubMed  CAS  Google Scholar 

  15. Fridman E, Carrari F, Liu YS, Fernie AR, Zamir D (2004) Zooming in on a quantitative trait for tomato yield using interspecific introgressions. Science 305:1786–1789.

    Article  PubMed  CAS  Google Scholar 

  16. Gibon Y, Blaesing OE, Hannemann J, Carillo P, Hohne M, Hendriks JHM, Palacios N, Cross J, Selbig J, Stitt M (2004) A robot-based platform to measure multiple enzyme activities in Arabidopsis using a set of cycling assays: Comparison of changes of enzyme activities and transcript levels during diurnal cycles and in prolonged darkness. Plant Cell 16: 3304–3325.

    Article  PubMed  CAS  Google Scholar 

  17. Gibon Y, Usadel B, Blaesing OE, Kamlage B, Hoehne M, Trethewey R, Stitt M (2006) Integration of metabolite with transcript and enzyme activity profiling during diurnal cycles in Arabidopsis rosettes. Genome Biology 7:R76.

    Article  PubMed  Google Scholar 

  18. Gibon Y, Vigeolas H, Tiessen A, Geigenberger P, Stitt M (2002) Sensitive and high throughput metabolite assays for inorganic pyrophosphate, ADPGlc, nucleotide phosphates, and glycolytic intermediates based on a novel enzymic cycling system. Plant J 30:221–235.

    Article  PubMed  CAS  Google Scholar 

  19. Gomes A, Fernandes E, Lima J (2006) Use of fluorescence probes for detection of reactive nitrogen species: A review. J Fluorescence 16:119–139.

    Article  CAS  Google Scholar 

  20. Greenberg LJ (1962) Fluorometric measurement of alkaline phosphatase and aminopeptidase activities in order of 10–14 mole. Biochem Biophys Res Comm 9:430–435.

    Article  PubMed  CAS  Google Scholar 

  21. Greis KD (2007) Mass spectrometry for enzyme assays and inhibitor screening: an emerging application in pharmaceutical research. Mass Spec Rev 26:324–339.

    Article  CAS  Google Scholar 

  22. Gronwald JW, Plaisance KL (1998) Isolation and characterization of glutathione S-transferase isozymes from sorghum. Plant Physiol 117:877–892.

    Article  PubMed  CAS  Google Scholar 

  23. Gutteridge A, Thornton JM (2005) Understanding nature’s catalytic toolkit. Trends Biochem Sci 30:622–629.

    Article  PubMed  CAS  Google Scholar 

  24. Hausler RE, Fischer KL, Flugge UI (2000) Determination of low-abundant metabolites in plant extracts by NAD(P)H fluorescence with a microtiter plate reader. Anal Biochem 281: 1–8.

    Article  PubMed  CAS  Google Scholar 

  25. Hendriks JHM, Kolbe A, Gibon Y, Stitt M, Geigenberger P (2003) ADP-glucose pyrophosphorylase is activated by posttranslational redox-modification in response to light and to sugars in leaves of Arabidopsis and other plant species. Plant Physiol 133:838–849.

    Article  PubMed  CAS  Google Scholar 

  26. Kaiser WM, Huber SC (2001) Post-translational regulation of nitrate reductase: mechanism, physiological relevance and environmental triggers. J Exp Bot 52:1981–1989.

    Article  PubMed  CAS  Google Scholar 

  27. Kappes T, Hauser PC (2000) Recent developments in electrochemical detection methods for capillary electrophoresis. Electroanalysis 12:165–170.

    Article  CAS  Google Scholar 

  28. Koshland DE, Nemethy G, Filmer D (1966) Comparison of experimental binding data and theoretical models in proteins containing subunits. Biochemistry 5:365–385.

    Article  PubMed  CAS  Google Scholar 

  29. Lienhard GE, Secemski, II (1973) P1,P5-Di(Adenosine-5′)Pentaphosphate, a Potent Multisubstrate Inhibitor of Adenylate Kinase. J Biol Chem 248:1121–1123.

    PubMed  CAS  Google Scholar 

  30. Lorimer GH, Badger MR, Andrews TJ (1977) D-Ribulose-1,5-Bisphosphate Carboxylase-Oxygenase – Improved Methods for Activation and Assay of Catalytic Activities. Anal Biochem 78:66–75.

    Article  PubMed  CAS  Google Scholar 

  31. Lowry CV, Kimmey JS, Felder S, Chi MMY, Kaiser KK, Passonneau PN, Kirk KA, Lowry OH (1978) Enzyme patterns in single human muscle-fibers. J Biol Chem 253:8269–8277.

    PubMed  CAS  Google Scholar 

  32. Lowry OH, Rock MK, Schulz DW, Passonneau JV (1961) Measurement of pyridine nucleotides by enzymatic cycling. J Biol Chem 236: 2746–2755.

    PubMed  CAS  Google Scholar 

  33. McIntosh CA, Oliver DJ (1992) NAD+-linked isocitrate dehydrogenase – isolation, purification, and characterization of the protein from pea mitochondria. Plant Physiol 100:69–75.

    Article  PubMed  CAS  Google Scholar 

  34. Michaelis L, Menton M (1913) Die kinetik der invertinwirkung. Biochemische Zeitschrift 49:333–369.

    CAS  Google Scholar 

  35. Monod J, Wyman J, Changeux JP (1965) On nature of allosteric transitions – a plausible model. J Mol Biol 12:88–118.

    Article  PubMed  CAS  Google Scholar 

  36. Morcuende R, Bari R, Gibon Y, Zheng WM, Pant BD, Blasing O, Usadel B, Czechowski T, Udvardi MK, Stitt M, Scheible WR (2007) Genome-wide reprogramming of metabolism and regulatory networks of Arabidopsis in response to phosphorus. Plant Cell Environ 30:85–112.

    Article  PubMed  CAS  Google Scholar 

  37. Outlaw WH, Manchester J (1980) Conceptual error in determination of NAD+-malic enzyme in extracts containing NAD+-malic dehydrogenase. Plant Physiol 65:1136–1138.

    Article  PubMed  CAS  Google Scholar 

  38. Schaffer AA, Levin I, Oguz I, Petreikov M, Cincarevsky F, Yeselson Y, Shen S, Gilboa N, Bar M (2000) ADPglucose pyrophosphorylase activity and starch accumulation in immature tomato fruit: the effect of a Lycopersicon hirsutum-derived introgression encoding for the large subunit. Plant Sci 152:135–144.

    Article  CAS  Google Scholar 

  39. Scheible WR, GonzalezFontes A, Lauerer M, MullerRober B, Caboche M, Stitt M (1997) Nitrate acts as a signal to induce organic acid metabolism and repress starch metabolism in tobacco. Plant Cell 9:783–798.

    Article  PubMed  CAS  Google Scholar 

  40. Schmidt O, Bassler M, Kiesel P, Knollenberg C, Johnson N (2007) Fluorescence spectrometer-on-a-fluidic-chip. Lab on a Chip 7:626–629.

    Article  PubMed  CAS  Google Scholar 

  41. Schwarz MA, Hauser PC (2001) Recent developments in detection methods for microfabricated analytical devices. Lab on a Chip 1: 1–6.

    Article  PubMed  CAS  Google Scholar 

  42. Selwyn MJ (1965) A simple test for inactivation of an enzyme during assay. Biochim Biophys Acta 105:193–195.

    PubMed  CAS  Google Scholar 

  43. Shen JB, Ogren WL (1992) Alteration of Spinach Ribulose-1,5-Bisphosphate Carboxylase Oxygenase Activase Activities by Site-Directed Mutagenesis. Plant Physiol 99:1201–1207.

    Article  PubMed  CAS  Google Scholar 

  44. Slade AJ, Fuerstenberg SI, Loeffler D, Steine MN, Facciotti D (2005) A reverse genetic, nontransgenic approach to wheat crop improvement by TILLING. Nature Biotechnol 23: 75–81.

    Article  CAS  Google Scholar 

  45. Stitt M, Gibon Y, Lunn JE, Piques M (2007) Multilevel genomics analysis of carbon signalling during low carbon availability: coordinating the supply and utilisation of carbon in a fluctuating environment. Funct Plant Biol 34:526–549.

    Article  CAS  Google Scholar 

  46. Storer AC, Cornishb.A (1974) Kinetics of coupled enzyme reactions – applications to assay of glucokinase, with glucose-6-phosphate dehydrogenase as coupling enzyme. Biochem J 141:205–209.

    PubMed  CAS  Google Scholar 

  47. Studart-Guimaraes C, Gibon Y, Frankel N, Wood CC, Zanor MI, Fernie AR, Carrari F (2005) Identification and characterisation of the alpha and beta subunits of succinyl CoA ligase of tomato. Plant Mol Biol 59:781–791.

    Article  PubMed  CAS  Google Scholar 

  48. Sulpice R, Tschoep H, Von Korff M, Bussis D, Usadel B, Hohne M, Witucka-Wall H, Altmann T, Stitt M, Gibon Y (2007) Description and applications of a rapid and sensitive non-radioactive microplate-based assay for maximum and initial activity of D-ribulose-1,5-bisphosphate carboxylase/oxygenase. Plant Cell Environ 30:1163–1175.

    Article  PubMed  CAS  Google Scholar 

  49. Tcherkez GGB, Farquhar GD, Andrews TJ (2006) Despite slow catalysis and confused substrate specificity, all ribulose bisphosphate carboxylases may be nearly perfectly optimized. Proc Nat Acad Sci U S A 103:7246–7251.

    Article  CAS  Google Scholar 

  50. Teipel JW, Hill RL (1971) Subunit interactions of fumarase. J Biol Chem 246: 4859–4865.

    PubMed  CAS  Google Scholar 

  51. Tiessen A, Hendriks JHM, Stitt M, Branscheid A, Gibon Y, Farre EM, Geigenberger P (2002) Starch synthesis in potato tubers is regulated by post-translational redox modification of ADP-glucose pyrophosphorylase: a novel regulatory mechanism linking starch synthesis to the sucrose supply. Plant Cell 14:2191–2213.

    Article  PubMed  CAS  Google Scholar 

  52. Usadel B, Blasing OE, Gibon Y, Poree F, Hohne M, Gunter M, Trethewey R, Kamlage B, Poorter H, Stitt M (2008) Multilevel genomic analysis of the response of transcripts, enzyme activities and metabolites in Arabidopsis rosettes to a progressive decrease of temperature in the non-freezing range. Plant Cell Environ 31:518–547.

    Article  PubMed  CAS  Google Scholar 

  53. Warburg O, Christian W, Griese A (1935) Wasserstoffubertragendes co-ferment seine zusammensetzung und wirkungsweise. Biochemische Zeitschrift 282:157–165.

    CAS  Google Scholar 

  54. Zhang Q, Xu JJ, Chen HY (2006) Glucose microfluidic biosensors based on immobilizing glucose oxidase in poly(dimethylsiloxane) electrophoretic microchips. J Chromatography A 1135:122–126.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

A.R. acknowledges support from the U.S. Department of Energy (DOE), Office of Science, Biological and Environmental Research (BER) program as part of its Program for Ecosystem Research (PER) and contract No. DE-AC02-98CH10886 to Brookhaven National Laboratory. Y.G. acknowledges Mark Stitt, Melanie Höhne, Jan Hannemann, John Lunn, Hendrik Tschoep, Ronan Sulpice, Marie-Caroline Steinhauser, and support from the Max Planck Society and the German Ministry for Research and Technology (GABI 0313110).

Author information

Authors and Affiliations

  1. Department of Environmental Sciences, Brookhaven National Laboratory, Upton, NY 11973-5000, USA

    Alistair Rogers

  2. Max Planck Institute for Molecular Plant Physiology, University of Illinois at Urbana Champaign, Department 1, 14059 Golm-Potsdam, Germany

    Yves Gibon

Authors
  1. Alistair Rogers
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yves Gibon
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

    Rights and permissions

    Reprints and permissions

    Copyright information

    © 2009 Springer Science+Business Media, LLC

    About this chapter

    Cite this chapter

    Rogers, A., Gibon, Y. (2009). Enzyme Kinetics: Theory and Practice. In: Schwender, J. (eds) Plant Metabolic Networks. Springer, New York, NY. https://doi.org/10.1007/978-0-387-78745-9_4

    Download citation

    Publish with us

    Policies and ethics

    Search

    Navigation