Enzyme Kinetics: Transient Phase


Transient kinetic methods allow events occurring at the active site of an enzyme to be observed directly by monitoring reactions on a short time scale and with a sufficiently high concentration of enzyme to witness product formed in a single enzyme turnover. Rapid mixing methods provide unique kinetic information to define the reaction mechanism in terms that can be directly related to structure.

Keywords: enzyme intermediates; enzyme kinetics; pre‐steady state; transient kinetics; stopped flow; chemical‐quench‐flow

Figure 1.

Kinetics of a pre‐steady‐state burst. The kinetics of product formation were simulated according to eqn with a concentration of 300 μmol L−1 substrate, 1 μmol L−1 enzyme and the following constants: k1=5 μmol−1s−1, k−1=0.1s−1, k2=100s−1 and variable values of k−2 and k3 summarized in the table. These simulations illustrate the reduction of burst amplitude due to reversal of the chemistry step (k−2) and fast product release (k3).

Figure 2.

Kinetics of a single‐turnover of EPSP synthase. This figure shows a single turnover performed with 10 μmol L−1 enzyme and 3.5 μmol L−1 PEP and 100 μmol L−1S3P. Note the transient formation of an enzyme‐bound intermediate and its conversion to the product, EPSP. The curves were simulated by numerical integration of the full kinetic model involving 12 rate constants governing the six‐step mechanism. EPSP: 5‐enolpyruvoyl‐shikimate‐3‐3‐phosphate; PEP: phosphoenol pyruvate; S3P: shikimate‐3‐phosphate. Data are redrawn from Anderson et al., (1988).

Figure 3.

Kinetics of dTTP incorporation by the human mitochondrial DNA polymerase. (a) The time dependence of incorporation is shown at several concentrations of dTTP: 0.1, 0.5, 1, 5 and 10 μmol L−1 (lower to upper curves). The smooth lines represent the best global fit by computer simulation using KinTekSim. (b) The concentration dependence of the rate of the burst is shown with a fit to a hyperbola (eqn ) to obtain the maximum rate of 25s−1 and the Kd=0.6 μmol L−1. Data are redrawn from Johnson and Johnson .



Anderson KS and Johnson KA (1990a) ‘Kinetic competence’ of the 5‐enolpyruvoylshikimate‐3‐phosphate synthase tetrahedral intermediate. Journal of Biological Chemistry 265: 5567–5572.

Anderson KS and Johnson KA (1990b) Kinetic and structural analysis of enzyme intermediates: lessons from EPSP synthase. Chemical Reviews 90: 1131–1149.

Anderson KS, Sikorski JA and Johnson KA (1988) A tetrahedral intermediate in the EPSP synthase reaction observed by rapid quench kinetics. Biochemistry 27: 7395–7406.

Anderson KS, Miles EW and Johnson KA (1991) Serine modulates substrate channeling in tryptophan synthase. A novel intersubunit triggering mechanism. Journal of Biological Chemistry 266: 8020–8033.

Brandis JW, Edwards SG and Johnson KA (1996) Slow rate of phosphodiester bond formation accounts for the strong bias that Taq DNA polymerase shows against 2′,3′‐dideoxynucleotide terminators. Biochemistry 35: 2189–2200.

Gilbert SP, Moyer ML and Johnson KA (1998) Alternating site mechanism of the kinesin ATPase. Biochemistry 37: 792–799.

Johnson KA (1983) The pathway of ATP hydrolysis by dynein. Kinetics of a presteady state phosphate burst. Journal of Biological Chemistry 258: 13825–13832.

Johnson KA (1992) Transient‐state kinetic analysis of enzyme reaction pathways. The Enzymes XX: 1–61.

Johnson KA (1993) Conformational coupling in DNA polymerase fidelity. Annual Review of Biochemistry 62: 685–713.

Johnson KA (1995) Rapid quench kinetic analysis of polymerases, adenosinetriphosphatases, and enzyme intermediates. Methods in Enzymology 249: 38–61.

Johnson AA and Johnson KA (2001) Fidelity of nucleotide incorporation by human mitochondrial DNA polymerase. Journal of Biological Chemistry 276: 38090–38096.

Johnson AA, Ray AS, Hanes J et al. (2001) Toxicity of antiviral nucleoside analogs and the human mitochondrial DNA polymerase. Journal of Biological Chemistry 276: 40847–40857.

Johnson KA and Taylor EW (1978) Intermediate states of subfragment 1 and actosubfragment 1 ATPase: reevaluation of the mechanism. Biochemistry 17: 3432–3442.

Further Reading

Johnson KA (ed.) (2003) Kinetic Analysis of Macromolecules: A Practical Approach. Oxford: Oxford University Press.

Kati WM, Johnson KA, Jerva LF and Anderson KS (1992) Mechanism and fidelity of HIV reverse transcriptase. Journal of Biological Chemistry 267: 25988–25997.

Bevilacqua PC, Kierzek R, Johnson KA and Turner DH (1992) Dynamics of ribozyme binding of substrate revealed by fluorescence detected stopped‐flow. Science 258: 1355–1358.

Spence RA, Kati WM, Anderson KS and Johnson KA (1995) Mechanism of inhibition of HIV‐1 reverse transcriptase by nonnucleoside inhibitors. Science 267: 988–993.

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Johnson, Kenneth A(May 2005) Enzyme Kinetics: Transient Phase. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1038/npg.els.0000721]