Enzyme inhibition

Enzyme inhibitors are molecules that decrease the activity of enzymes, and knowledge about inhibitors can, for example, be used in developing drugs or in the study of biochemical pathways, because inhibitors provide a way to interfere with these pathways. Enzyme inhibitors can be either irreversible or reversible; irreversible inhibitors decrease enzymatic activity by destroying the enzyme through various mechanisms, while reversible inhibitors keep the enzyme functional. The inhibitors we will study here are reversible inhibitors [1].

Types of inhibition

The mechanisms of enzyme inhibitors can be classified into 3 major groups: Competitive inhibitors, uncompetitive inhibitors, and mixed inhibitors. Competitive inhibitors work by binding to the active site of the enzyme in competition with the substrate; uncompetitive inhibitors bind to the enzyme-substrate complex at a site distinct from the active site, but they cannot bind to the enzyme alone, and mixed inhibitors can bind to both the enzyme and the enzyme-substrate complex at a site distinct from the active site [1].

The mechanisms of enzyme inhibition can be thought of as an extension to the Michaelis-Menten mechanism and competitive and un-competitive inhibition can be regarded as a special case of mixed inhibition (see Figure 1a), where KI and K’I are the dissociation constants of the EI and ESI K I and K prime I are the dissociation constants of the E I and E S I complex, respectively. Using the same approach as that used for deriving the Michaelis-Menten equation (for a detailed derivation, see [2]), the following equation for mixed inhibition can be obtained:

V0 = Vmax•[S] / Km•α+[S]•α’ V 0 equals V max times the substrate concentration divided by K m times alpha plus the substrate's concentration times alpha prime

where α = 1+[I]/KI and α’ = 1+[I]/K’I alpha equals one plus the inhibitor concentration and alpha prime equals one plus the inhibitor concentration divided by K prime I

Just like the Michealis-Menten equation, this equation can be rearranged to fit a double-reciprocal plot:

1/V0 = α’/Vmax + Km•α/Vmax • 1/[S] one divided by V 0 equals alpha prime divided by V max plus K m times alpha divided by V max times the reciprocal of the substrate's concentration

If α > 1 and α’ > 1, the inhibition is mixed; for competitive inhibition, α’ = 1; for uncompetitive inhibition, α = 1 if alpha and alpha prime are bigger than one, the inhibition is mixed; for competitive inhibition, alpha prime equals one; for uncompetitive inhibition, alpha equals one. Thus, 3 different equations are obtained for the 3 different types of inhibition, and a Lineweaver-Burk plot of the kinetic data can reveal the type of inhibition that the inhibitor performs (see Figure 1.b, 1.c, and 1.d).

At the top, Figure 1a presents the overall enzymatic reaction and enzyme inhibition mechanisms. The reaction unfolds in multiple steps, beginning with enzyme-substrate formation, reversible in both directions. Inhibitor addition to the enzyme and enzyme-substrate complex is illustrated, with K I and K prime I representing dissociation constants for enzyme-inhibitor and enzyme-substrate-inhibitor complexes, respectively. The enzyme inhibition mechanism, depicted below the overall reaction, showcases the reversible formation of the enzyme-substrate-inhibitor complex from the enzyme-inhibitor complex and substrate. Figures 1b, 1c, and 1d beneath illustrate Lineweaver-Burk plots representing three major types of inhibition: competitive, mixed, and uncompetitive. Each plot features 1 divided by V on the y-axis and 1 divided by the substrate concentration on the x-axis.

Figure 1: The overall enzymatic reaction and the extension of the enzyme inhibition mechanism.

Methanol poisoning

The enzyme alcohol dehydrogenase is not completely specific for ethanol; it also catalyzes the formation of aldehydes from other alcohols. One of these alcohols is methanol, which is metabolized into formaldehyde and other toxic compounds that can cause blindness or death. Methanol poisoning is quite common, and can be caused by the ingestion of homemade alcohol. Methanol and ethanol are thus competitive substrates, and ethanol is actually used to prevent poising after the ingestion of methanol, because it inhibits ADH in catalyzing the oxidation of this compound.

Calculation of kinetic parameters

See the following pages for details of how to calculate the kinetic parameters for different inhibitors:

Competitive inhibition

Un-competitive inhibition

Mixed/non-competitive inhibition


  1. Lehninger, Albert L.; Nelson, David L.; Cox, Michael M. (2008). Principles of Biochemistry (5th ed.). New York, NY: W.H. Freeman and Company. ISBN 978-0-7167-7108-1.

  2. Atkins, Peter W.; de Paula, Julio; Friedman, Ronald (2009). Quanta, Matter, and Change: A molecular approach to physical chemistry. Oxford University Press. ISBN 978-0-19-920606-3.

  3. Beatty, L., Green, R., Magee, K. and Zed, P. (2013) A Systematic Review of Ethanol and Fomepizole Use in Toxic Alcohol Ingestions. Emerg. Med. Int. 2013, 638057.