Role of Enzyme Inhibitors

Studying enzyme inhibitors is not only critical for understanding normal body processes, but also for pharmaceutical and drug development research. Uncover the role of enzyme inhibitors including the importance of enzyme inhibitors, the advantages of using small molecule inhibitors over RNA interference (RNAi), and learn how enzymes work.

• Why Are Enzyme Inhibitors Important?
• Advantages of Small Molecule Inhibitors vs RNAi
• How Do Enzymes Work?

Why Are Enzyme Inhibitors Important?

Enzyme inhibitors are important to help regulate enzyme activity. Although enzymes are absolutely essential for life, abnormally high enzyme activity can lead to certain health issues and diseases. Hence, overactive enzymes are attractive targets for the development of inhibitor molecules to alleviate these diseases.  Manipulation of enzyme catalysis with inhibitors is critical for prevention of infectious diseases, intervention in cell cycle and cell growth, treatment of hypertension, control of inflammatory responses, and more. Besides acting as therapeutic agents, enzyme inhibitors also play important roles in biological and clinical research.

Various roles of enzyme inhibitors include:

• Serving as major control mechanisms in biological systems
• Regulating metabolic activities
• Blocking or slowing down the rate of biochemical reactions
• Inhibiting specific enzymes which is a mechanism of action for many therapeutically important drugs
• Reversibly inhibiting enzyme purification (reversible inhibitors)
• Identifying active site amino acids (covalent inhibitors)
• Localizing and identifying intracellular sites of enzymes (immobilized and fluorochrome-tagged inhibitors)
• Acting as potent poisons, pesticides, and herbicides

Advantages of Small Molecule Inhibitors vs RNAi

Chemical biology has been successfully used in both gain-of-function and loss-of-function approaches to study a variety of biological processes. For example, in chemical genetics, either small organic molecules or peptides are used to activate or inhibit specific proteins/enzymes involved in specific signaling pathways. This allows researchers to analyze the phenotype when a specific cellular protein is induced or suppressed.

Small molecules offer a powerful approach to temporally and spatially modulate individual proteins and processes that can control biological phenotypes. These molecules have also helped to advance our understanding of the biological pathways that influence stem cell fate. They are used to modulate self-renewal, survival, direct reprogramming, and differentiation of pluripotent stem cells. Further, small molecules have helped to identify signaling molecules that define and maintain the extensive intracellular communication networks that control cell growth, differentiation, and metabolism.

Compared to other approaches, such as overexpressing genes from plasmid DNA or genetic knockdowns, chemical genetics is technically simple to perform in cell culture and requires fewer resources and less time. Compared to the RNAi approach, small molecules provide the advantage of acting quickly and directly on the protein target, and the effects can often be reversed rapidly by simply washing (Figure 1). The fast action of most small molecules also makes them ideal for live imaging. In addition, the use of small molecules can provide dose-response information.

Figure 1. Comparison of the sites of action of (1) RNAi and (2) small molecule inhibitors.

Some advantages of small molecule inhibitors over RNAi are described in Table 1.

Table 1. Advantages of Small Molecule Inhibitors.

RNAi	Small Molecule Inhibitors
Challenge with primary cells and difficult-to-transfect cells Challenge with messenger RNAs (mRNAs) encoding proteins with a long half-life Challenge with mRNAs with strong secondary structure and difficult to knock down Gradual protein depletion; slowly reversible Difficult to determine dose response during gradual depletion	Frequently cell-permeable No need for transfection agents Small molecules act by inhibiting or inactivating specific proteins Fast, often inhibitor-based drugs are reversible inhibitors Dose response is informative Dose can be easily manipulated

How Do Enzymes Work?

Enzymes work through interactions of their active site and its substrate.

Active Sites and Substrates

Enzymes are specific in the reactions that they catalyze: each enzyme usually acts on a single substrate or a pair of substrates (in the case of bimolecular reactions). However, some enzymes may act on closely related substrate molecules, often with different efficiencies. Enzymes possess a small region (typically only about 20 amino acids), known as the active site, where the reaction occurs. Enzymes catalyze biological reactions by binding the substrate(s) to the active site. This binding changes the distribution of electrons in the chemical bonds of the substrate(s), lowering the activation energy of the reaction and enabling the generation of the final product. This product is released from the active site, allowing the enzyme to regenerate itself for another reaction cycle. The substrate can interact with the active site through ionic interactions, hydrogen bonding, hydrophobic nonpolar interactions, and coordinate covalent bonding to a metal ion activator.

Enzyme-Substrate Binding

The active site organization is complementary to that of the substrate molecule, which allows only one substrate to bind to the site. However, in a few cases, similar molecules can also act as substrates, but with less than optimal reaction kinetics. This is popularly known as the lock-and-key model. However, not all reactions can be explained based on the lock-and-key model. Hence, an induced fit theory was proposed, which takes into account that substrates also play a role in determining the final shape of the enzyme. This means the enzyme shape can be flexible (Figure 2). In the induced fit model of enzyme-substrate binding, the shape of the active site of the unbound enzyme is not the exact complement of the shape of the substrate.

Figure 2. (1) Lock–and-key and (2) induced fit models of enzyme-substrate binding.

Based on the induced fit theory, one can explain the phenomenon where certain compounds can bind to the enzyme but fail to react rapidly because the enzyme structure has been distorted or the molecule is too small to induce a proper alignment. However, the enzyme does bind to the substrate, and, after binding, a conformational change occurs in the active site that results in a new shape, which is complementary to the shape of the substrate. Since almost all enzymes are proteins, the nature of the amino acid side chains in the vicinity of the active site is also very important.

Enzyme Specificity

Enzymes may have varying degrees of specificity for multiple substrates. Some enzymes have absolute specificity for only one substrate. Others may react with substrates with similar functional groups, side chains, or positions on a chain. The least specific enzymes catalyze a reaction at a particular chemical bond regardless of any other structural features.

Enzyme inhibitors such as small molecule inhibitors can be used to inhibit these enzyme-substrate binding interactions. Learn more about terms and calculations used in enzyme inhibitor research to plan your next experiment.