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 1.a), where KI and K’I are the dissociation constants of the EI and ESI 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’

where α = 1+[I]/KI and α’ = 1+[I]/K’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]

If α > 1 and α’ > 1, the inhibition is mixed; for competitive inhibition, α’ = 1; for uncompetitive inhibition, α = 1. 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)[1,2].

At the top is figure 1.a that illustrates the overall enzymatic reaction and the mechanisms of enzyme inhibition as an extension to this. The enzymatic reaction is made up of different steps illustrated in the reaction from left to right, with the formation of enzyme-substrate as the first step in the overall reaction. This step goes both ways, illustrated with a two-directional arrow, with the reversed reaction being the dissociation of the enzyme-substrate into enzyme and substrate again. The next step is the dissociation of the enzyme-substrate into enzyme and product. The addition of inhibitors to the enzyme and the enzyme-substrate complex in the overall enzymatic reaction is also illustrated, where K I and K’ I  are the dissociation constants of the enzyme-inhibitor and enzyme-substrate-inhibitor complex, respectively. In addition to this reaction is the enzyme inhibition mechanism which is illustrated below the overall reaction. This reaction is the formation of the enzyme-substrate-inhibitor complex, created from the enzyme-inhibitor complex and substrate. This reaction also goes both ways, illustrated with a two-directional arrow, with the reversed reaction being the dissociation of the enzyme-inhibitor-substrate complex into enzyme-inhibitor complex and substrate. Beneath this illustration are figure 1.b, 1.c, and 1.d. These figures show three different Lineweaver-Burk plots showing the 3 major types of inhibition. Figure 1.b illustrates the competitive inhibition, figure 1.c illustrates the mixed inhibition, and figure 1.d illustrates the un-competitive inhibition. All plots have 1 divided by V on the y-axis and 1 divided by the concentration of substrate on the x-axis. Figure 1.b is a graph with three different lines which represent different datasets with varying inhibitor concentrations and they, therefore, have different slopes. All three lines intersect at the same spot on the y-axis. Next to this plot is figure 1.c which is another graph showing the mixed inhibition as three different lines. These lines represent different datasets with varying inhibitor concentrations and the lines all have different slopes and different y-intercepts. Next to this plot is figure 1.d which is a graph showing the uncompetitive inhibition as three different lines. These lines represent different datasets with varying uncompetitive inhibitor concentrations and the lines have the same slopes. However, the three lines intersect at varying spots on the y-axis

Figure 1: Figure 1.a; The overall enzymatic reaction and the extension of the enzyme inhibition mechanism. Figure 1b, c, and d; Lineweaver-Burk plots showing the 3 major types of inhibition. If the y-intersect is the same, but the slopes differ, the inhibitor is competitive. If both the slopes and the y-intersects differ, the inhibitor is mixed. If the slopes are the same, but the y-intersects differ, the inhibitor is uncompetitive.

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 [3]. 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 [4].

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

References

  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.