Enzymology Methods


Enzymes are important catalysts throughout biology and are now being applied to chemical synthesis. Methods for analysing these catalysts through steady‐state kinetics are described, along with application of these methods to studies aimed at altering and/or inhibiting specific enzyme reactions.

Keywords: enzymes; steady‐state kinetics; enzyme assays; enzymology; biocatalysis

Figure 1.

Enzymes accelerate the rates of chemical reactions by stabilizing (lowering the potential energy of) the transition state of the reaction. The enzyme (E) and substrate (S) first combine to form the ES complex. The bound substrate is then converted to the bound transition state (ES*), which is energetically stabilized relative to the free transition‐state molecule (S*). The enzyme‐bound transition state is then converted to enzyme‐bound product (EP), and finally the product (P) is released. Adapted from Copeland RA (2000) Enzymes: A Practical Introduction to Structure, Mechanism and Data Analysis, 2nd edn. New York: Wiley, with permission of the author.

Figure 2.

Graphical determination of the kinetic constants Km and kcat. The production of product over time in an enzyme‐catalysed reaction proceeds with pseudo‐first‐order kinetics (a), slowing down as substrate supplies are diminished. The shaded area in (a) is shown on an expanded scale in (b). Here, the product increases linearly with time in the early phase of the reaction, and the initial velocity can therefore be estimated by the slope of the linear fit. The initial velocity thus measured increases with increasing initial concentration of substrate [S] over a finite range (c). A replot of the initial velocity as a function of substrate concentration (d) allows determination of the values of Km and Vmax by fitting to eqn . The value of kcat is then determined by dividing Vmax by the enzyme concentration [E].


Further Reading

Cleland WW (1967) Steady state kinetics. Advances in Enzymology 29: 1–65.

Copeland RA (2000) Methods for Protein Analysis: A Practical Guide to Laboratory Protocols, 2nd edn. New York: Chapman and Hall.

Copeland RA (1996) Enzymes: A Practical Introduction to Structure, Mechanism and Data Analysis. New York: Wiley‐VCH.

Cornish‐Bowden A (1995) Fundamentals of Enzyme Kinetics. London: Portland Press.

Davis JP and Copeland RA (1996) Protein engineering. In: Kirk–Othmer Encyclopedia of Chemical Technology, vol. 20, pp. 395–427. New York: Wiley Interscience.

Devlin JP (1997) High Throughput Screening: The Discovery of Bioactive Substances. New York: Marcel Dekker.

Fersht A (1999) Structure and Mechanism in Protein Sciences. New York: WH Freeman.

Jacobsen JR and Khosla C (1998) New directions in metabolic engineering. Current Opinion in Chemical Biology 2: 133–137.

MacArthur MW, Driscoll PC and Thornton JM (1994) NMR and crystallography – complementary approaches to structure determination. Trends in Biotechnology 12: 149–153.

Segal IH (1975) Enzyme Kinetics. New York: Wiley.

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Copeland, Robert A(Apr 2001) Enzymology Methods. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1038/npg.els.0002702]