Kinetic Assay Methods

R. Donald Allison1

1 University of Florida, Gainesville
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 3.5
DOI:  10.1002/0471140864.ps0305s05
Online Posting Date:  May, 2001
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The purpose of this unit is to provide a brief review of issues important in the design of initial‐rate assay methods. General aspects of kinetic assay design are discussed, including enzyme and substrate purity, concentration and stability. Also covered are issues such as continuous versus stop‐time assay formats, coupled enzyme assays, binding studies, and presentation of initial‐rate data.

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Table of Contents

  • General Aspects of Assay Design
  • Continuous Versus Stop‐Time Assays
  • Coupled Enzyme Assays
  • Binding Studies
  • Presentation of Initial‐Rate Data
  • Concluding Remarks
  • Literature Cited
  • Figures
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Literature Cited

   Allison, R.D. 1985. γ‐Glutamyl transpeptidase: Kinetics and mechanism. Methods Enzymol. 113:419‐437.
   Allison, R.D. and Meister, A. 1981. Evidence that transpeptidation is a significant function of γ‐glutamyl transpeptidase. J. Biol. Chem. 256:2988‐2992.
   Allison, R.D. and Purich, D.L. 1979. Practical considerations in the design of initial velocity enzyme rate assays. Methods Enzymol. 63:3‐22.
   Beechem, J.M. 1992. Global analysis of biochemical and biophysical data. Methods Enzymol. 210:37‐54.
   Bergmeyer, H.U. (ed.) 1983. Methods of Enzymatic Analysis, 3rd ed., Vols. 1‐4. Verlag Chemie, Deerfield Beach, Florida.
   Boehlein, S.K., Richards, N.G.J., and Schuster, S.M. 1994. Glutamine‐dependent nitrogen transfer in Escherichia coli asparagine synthetase B. J. Biol. Chem. 269:7450‐7457.
   Bowen, W.J. and Kerwin, T.P. 1955. The purification of myokinase with an ion exchange resin. Biochem. Biophys. 57:522‐524.
   Brand, L. and Johnson, M.L.(eds.). 1992. Numerical computer methods. Methods Enzymol. Vol. 210.
   Cleland, W.W. 1979a. Statistical analysis of enzyme kinetic data. Methods Enzymol. 63:103‐138.
   Cleland, W.W. 1979b. Substrate inhibition. Methods Enzymol. 63:500‐513.
   Cornish‐Bowden, A. and Endrenyi, L. 1981. Fitting of enzyme kinetic data without prior knowledge of weights. Biochem. J. 193:1005‐1008.
   Cornish‐Bowden, A. and Endrenyi, L. 1986. Robust regression of enzyme kinetic data. Biochem. J. 234:21‐29.
   Deans, N.L., Allison, R.D., and Purich, D.L. 1992. Steady‐state kinetic mechanism of bovine brain tubulin:tyrosine ligase. Biochem. J. 286:243‐251.
   Dybkaer, R. 1979. International Union of Pure and Applied Chemistry and International Federation of Clinical Chemistry. IUPAC Section of Clinical Chemistry; Commission on Quantities and Units in Clinical Chemistry and IFCC Committee on Standards, Expert Panel on Quantities and Units; Approved Recommendation (1978) Quantities and units in clinical chemistry. Clin. Chim. Acta 96:157F‐183F.
   Eisenthal, R. and Cornish‐Bowden, A. 1974. The direct linear plot. A new graphical procedure for estimating enzyme kinetic parameters. Biochem. J. 139:715‐720.
   Feldman, H.A. 1983. Statistical limits in Scatchard analysis. J. Biol. Chem. 258:12865‐12867.
   Fierke, C.A. and Hammes, G. 1995. Transient kinetic approaches to enzyme mechanisms. Methods Enzymol. 249:1‐32.
   Frieden, C. 1959. Glutamate dehydrogenase III. The order of substrate addition in the enzymatic reaction. J. Biol. Chem. 234:2391‐2396.
   Fromm, H.J. 1967. The use of competitive inhibitors in studying the mechanism of action of some enzyme systems utilizing three substrates. Biochim. Biophys. Acta 139:221‐230.
   Johnson, M.L. and Brand, L. (eds.) 1994. Numerical computer methods, part B. Methods Enzymol. 240:xi‐xiii, 1‐36, 51‐170, 181‐198, 311‐322, 781‐816.
   Klotz, I.M. 1982. Numbers of receptor sites from Scatchard graphs: Facts and fantasies. Science 217:1247‐1249.
   Klotz, I.M. 1983. Number of receptor sites from Scatchard and Klotz graphs: A constructive critique. Science 220:981.
   Klump, H., Di Ruggiero, J., Kessel, M., Park, J.‐B., Adams, M.W.W., and Robb, F.T. 1992. Glutamate dehydrogenase from the hyperthermophile Pyrococcus furiosus. J. Biol. Chem. 267:22681‐22685.
   Lienhard, G.E. and Secemski, I.I. 1973. P1,P5‐Di(adenosine‐5′)pentaphosphate, a potent multisubstrate inhibitor of adenylate kinase. Biol. Chem. 248:1121‐1123.
   Marmasse, C. 1963. Enzyme inhibition by excess substrate. Biochim. Biophys. Acta 77:530‐535.
   McClure, W.R. 1969. A kinetic analysis of coupled enzyme assays. Bio Chem. 8:2782‐2786.
   Nordlie, R.C. 1982. Kinetic examination of enzyme mechanisms involving branched reaction pathways: A detailed consideration of multifunctional glucose‐6‐phosphatase. Methods Enzymol. 87:319‐353.
   O'Sullivan, W.J. and Smithers, G.W. 1979. Stability constants for biologically important metal‐ligand complexes. Methods Enzymol. 63:294‐336.
   Purich, D.L. and Fromm, H.J. 1972. Inhibition of rabbit muscle adenylate kinase by the transition state analogue, P1,P4‐di(adenosine‐5′)tetraphosphate. Biochim. Biophys. Acta 276:563‐567.
   Rudolph, F.B. 1979. Product inhibition and abortive complex formation. Methods Enzymol. 63:411‐436.
   Scatchard, G. 1949. The attraction of proteins for small molecules and ions. Ann. N.Y. Acad. Sci. 51:660‐673.
   Seelig, G.F. and Meister, A. 1985. Glutathione biosynthesis: γ‐Glutamylcysteine synthetase from rat kidney. Methods Enzymol. 113:379‐390.
   Siano, D.B., Zyskind, J.W., and Fromm, H.J. 1975. A computer program for fitting and statistically analyzing initial rate data applied to bovine hexokinase type III isozyme. Arch. Biochem. Biophys. 170:587‐600.
   Silverstein, E. and Sulebele, G. 1969. Catalytic mechanism of pig heart mitochondrial malate dehydrogenase studied by kinetics at equilibrium. Biochemistry 8:2543‐2550.
   Storer, A.C. and Cornish‐Bowden, A. 1974. The kinetics of coupled enzyme reactions. Biochem. J. 141:205‐209.
   Tate, S.S. and Meister, A. 1978. Serine‐borate complex as a transition‐state inhibitor of γ‐glutamyl transpeptidase. Proc. Natl. Acad. Sci. U.S.A. 75:4806‐4809.
   Wilkinson, G.N. 1961. Statisticalestimations in enzyme kinetics. Biochem. J. 80:324‐332.
   Williams, J.W. and Morrison, J.F. 1979. The kinetics of reversible tight‐binding inhibition. Methods Enzymol. 63:437‐467.
Key References
   Bergmeyer, 1983. See above.
  Detailed collection of enzyme assay protocols and methods for a wide variety of enzymes, including descriptions of many special techniques: e.g., fluorometry, turbidimetry, luminometry, and microtechniques.
   Dixon, M. and Webb, E.C. 1979. Enzymes, 3rd ed. Academic Press, New York.
  Classic text on the basics of enzymology; Chapter 4 deals specifically with enzyme kinetics.
   Fromm, H.J. 1975. Initial Rate Enzyme Kinetics. Springer‐Verlag, New York.
  Valuable introductory text with considerable details on experimental design.
   Kuby, S.A. 1990. A Study of Enzymes,vol. I. CRC Press, Boca Raton, Florida
  Detailed text on steady‐state kinetics. Contains useful chapters on the effects of metal cofactors and transient phase kinetics.
   Purich, D.L. (ed.) 1979. Enzyme kinetics and mechanism, part A. Methods Enzymol. Vol. 63.
  Describes a variety of initial rate methods and inhibitor studies for characterizing enzyme‐catalyzed reactions.
   Purich, D.L. (ed.) 1980. Enzyme kinetics and mechanism, part B. Methods Enzymol. Vol. 64.
  Describes a number of isotopic probes for studying enzyme mechanisms and contains valuable chapters on allosterism, hysteresis, immobilized systems, and processivity.
   Purich, D. L. (ed.) 1982. Enzyme kinetics and mechanism, part C. Methods Enzymol. Vol. 87.
  Describes methods for characterizing intermediates in enzyme‐catalyzed reactions and using stereochemical probes for enzyme mechanisms; includes additional initial‐rate and inhibitor methods and discusses further uses of isotopic probes.
   Purich, D.L. (ed.) 1995. Enzyme kinetics and mechanism, part D. Methods in Enzymol. 249:3‐662.
  Covers in detail specialized topics in enzyme kinetics including transition‐state approaches, kinetic probes with site‐directed mutagenesis, partition analysis, positional isotope exchange, interfacial catalysis, and hydrogen tunneling.
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