Enzyme inhibitors are molecules that bind to the enzyme and reduce the catalytic activity of enzymes. There are many types of inhibitors, including nonspecific, irreversible or reversible (competitive, uncompetitive and non-competitive inhibitors).
Non-specific inhibitors can inhibit multiple enzyme targets by forming the aggregate. One mechanism for this nonspecific interaction is the formation of colloidal aggregates by self-association of low molecular weight compounds in aqueous solutions. These aggregates tend to sequester protein, presumably by partially unfolding it and thus inhibiting its function.
Irreversible inhibitors usually react with the enzyme by forming covalent bonds; thus dissociating very slowly from its target enzyme.[2, 3] There are some important drugs act as irreversible inhibitors to the enzyme. One example is penicillin, as a first discovered antibiotic, can covalently bind to the enzyme transpeptidase by mimic the normal substrate the D-Ala-D-Ala peptide. It is therefore preventing the synthesis of bacterial cell walls and killing the bacteria. (Fig.1-2) It is very useful to identify if inhibitors are irreversible or not. One method is through dilution, namely measuring the recovery of enzymatic activity after a rapid and large dilution of the enzyme-inhibitor complex. [3, 4] (Fig.3)
Figure 1. Transpeptidation Reaction. An acyl-enzyme intermediate is formed in the transpeptidation reaction leading to cross-link formation. Figure is from the following published study: Reference 2.
Figure 2. Conformations of Penicillin and a Normal Substrate. The conformation of penicillin in the vicinity of its reactive peptide bond (A) resembles the postulated conformation of the transition state of R-d-Ala-d-Ala (B) in the transpeptidation reaction. Figure is from the following published study: Reference 2.
Figure 3. Hypothetical time-courses for activity recovery after rapid dilution of enzyme-bound inhibitor into a solution containing saturating substrate concentration. C, Control sample having no inhibitor present; R, Reversible inhibitor bound as E$I complex at zero-time; SR, Slowly-reversible inhibitor bound as E$In complex at zero-time, but must isomerize through one or more intermediate states to form E$I complex, which then releases inhibitor; I, Irreversible inhibitor bound as E–I complex at zero-time. The actual time-scale for inhibitor release will depend to the transit time for isomerization of enzyme-inhibitor complexes to that enzyme species from which inhibitor is released. Figure is from the following published study: Reference 3.
In contrast with irreversible inhibitors, reversible inhibitors have a rapid dissociation to the enzyme and bind non-covalently. [2, 3] Reversible inhibitors contain three types of inhibitors, competitive, uncompetitive and non-competitive, depend on whether the inhibitors compete with substrate binding to the enzyme or binding the enzyme-substrate complex. (Fig.4) Competitive inhibitors can compete with substrate to bind the active site of the enzyme and this binding state can be relieved by increasing the substrate concentration. On the contrary, noncompetitive inhibitors can bind to the enzyme simultaneously with the substrate and this inhibition cannot be overcome by increasing the substrate concentration. Uncompetitive inhibitors can only bind to the enzyme-substrate complex, in which a binding site is formed.
Figure 4. Three types of reversible inhibitors: A. competitive; B. noncompetitive; C. uncompetitive inhibitors. Figure is from the following published study: Reference 3.
These three types of inhibitions can be determined by Michaelis-Menten kinetics. Since the competitive inhibition can be overcome by a sufficiently high concentration of substrate, Vmax can be maintained in the same value and Km value (Km = [S] at Vmax/2) is increased in the presence of a competitive inhibitor. Noncompetitive inhibitors inactive the enzyme, lower the concentration of functional enzyme, so Vmax is declined and Km is unchanged. Uncompetitive inhibitors work best when substrate concentration is high, both Vmax and Km values are therefore decreased.[2, 5](Fig.5)
Figure 5. A. Competitive inhibition is characterized by an increase in Km for the substrate and no change of Vmax. B. Non-competitive inhibition is characterized by no change in the Km value and a decrease in Vmax. C. Uncompetitive inhibition is characterized by a decrease in the apparent Km value and a decreased Vmax. Figure is from the following published study: Reference 5.
Among three types of reversible inhibitors, uncompetitive inhibitors are rare, but some potent drugs have been regarded as uncompetitive inhibitors to the enzyme.(Table.1) Many enzyme-substrate complexes are extremely short-lived in the enzyme-catalyzed reactions, so uncompetitive inhibitors have much less opportunities to bind the target than other two types of reversible inhibitors.  One example is about the first uncompetitive inhibitor-blebbistatin of myosin, binding at a site other than the nucleotide- or actin filament-binding sites, which opens and closes during the contractile cycle. Blebbistatin can stabilize the metastable or ‘transition’ state of myosin, representing a long-live complex of myosin with ADP and inorganic phosphate. [3, 6]
Table 1. Several clinically useful uncompetitive inhibitors. Table is from the following published study: Reference
Blog by Tina(Xiangrong) CHEN
- Habig, M., et al., Efficient elimination of nonstoichiometric enzyme inhibitors from HTS hit lists. Journal of biomolecular screening, 2009. 14(6): p. 679-689.
- Berg, J.M., J.L. Tymoczko, and L. Stryer, Biochemistry New York. NY: WH Freeman, 2002.
- Purich, D.L., Enzyme kinetics: catalysis and control: a reference of theory and best-practice methods. 2010: Elsevier.
- Copeland, R.A., Evaluation of enzyme inhibitors in drug discovery: a guide for medicinal chemists and pharmacologists. 2013: John Wiley & Sons.
- Kenakin, T.P., Chapter 6 – Enzymes as Drug Targets, in Pharmacology in Drug Discovery. 2012, Academic Press: Boston. p. 105-124.
- Allingham, J.S., R. Smith, and I. Rayment, The structural basis of blebbistatin inhibition and specificity for myosin II. Nature structural & molecular biology, 2005. 12(4): p. 378-379.