All living organisms continuously perform numerous biochemical reactions to sustain their presence. Most of these reactions require an input of energy to start, which is called the activation energy. Catalysts are chemicals that lower the activation energy. Even though catalysts facilitate a chemical reaction, they are not consumed by it. This means a catalyst can facilitate a specific chemical reaction over and over in succession, increasing the rate of that reaction.
Enzymes are biological catalysts that increase the speed and efficiency of biochemical reactions. Most enzymes are proteins but certain ribonucleic acid molecules also have catalytic properties. Each type of enzyme can only bind to one or a few specific substrates, therefore, there is a vast diversity of enzymes, all with distinct functions. The specific reaction of an enzyme depends on its active sites, which has shapes that closely matches the shape of its substrates. Upon substrate binding, the enzyme changes shape slightly in a way that allows the chemical reaction to happen more readily, thus reducing the activation energy of the reaction. Once the reaction takes place, the enzyme releases the products and returns to its original shape, which allows it to repeat the process again.
Catabolytic versus Anabolic Enzymes
Most enzymatic reactions can be categorized as either catabolic and anabolic, which have opposite functions. Enzymes that mediate catabolic reactions break down larger substances into multiple products. A well-known catabolic enzyme is lactase, which splits the disaccharide lactose in dairy products into monosaccharides galactose and glucose. Humans have large amounts of lactase in their small intestine after birth, however many individuals lose more than 90% of their lactase by early childhood1. This reduction diminishes the ability to digest lactose in an age-dependent manner, which is known as lactose intolerance2.
Anabolic enzymes combine multiple substances into a single product. For example, the enzyme DNA polymerase synthesizes DNA molecules by joining nucleotides together. Similarly, the enzyme glycogen synthase connects glucose molecules to generate glycogen. Insulin regulates the activity of glycogen synthase, therefore diabetic patients with low to no insulin levels cannot synthesize glycogen without insulin treatment3.
The shape and charge of the active site of an enzyme is tailored to the specific chemical reaction it catalyzes, therefore the enzyme activity is influenced by factors that affect its active site. Hence, enzymes require specific conditions to work at optimal levels and depending on their function and location, these conditions can vary greatly. These factors include pH and temperature. For example, active sites of enzymes often contain acidic or basic amino acids. Deviations from the optimum pH alter the charges in the active site, making it difficult for the substrates to interact with the enzyme. This property has been used for centuries to preserve food by pickling, which reduces the pH of the pickled food item to a level that effectively inhibits microbial enzymatic activity. Similarly, each enzyme works at an optimal temperature range. If the temperature shifts below or above this range, it alters the shape of the active site, preventing it from effectively interacting with its substrates. In most cases, returning to the optimal temperature restores the enzyme’s original shape and function, however, temperatures that are too high can denature or irreversibly damage the enzyme structure. Again, these properties are useful for food safety; we cook meat thoroughly to kill harmful bacteria by denaturing their enzymes and refrigerate most food items to slow down enzymatic activity of microorganisms.
Extremophiles, or organisms found in extreme conditions that are lethal to most forms of life, have enzymes capable of functioning at those extremes. For instance, Thermus aquaticus is a bacterium found in hot springs and hydrothermal vents reaching temperatures at which most proteins would denature. Taq polymerase is a thermostable DNA polymerase found in T. aquaticus, which has allowed the development of polymerase chain reaction (PCR), a widely-used laboratory method for rapid DNA replication. PCR requires high temperatures to separate the strands of the DNA double helix to be replicated. Taq polymerase remains stable at these high temperatures while most enzymes would be degraded. PCR cycles between high temperatures that break the DNA into single strands and lower temperatures that allow the polymerase to build new double stranded DNA.
Molecular Factors that Influence Enzyme Activity
In addition to specific pH and temperature ranges, some enzymes function better in the presence of other molecules or ions. Coenzymes are biomolecules that bind to the active site of the enzyme and participate in catalysis without getting consumed by the reaction. Coenzymes often transport electrons, specific atoms and functional groups, and thus act as intermediary carriers. B vitamins act as coenzymes for numerous biochemical reactions. For instance, coenzyme A (coA), which is the active form of pantothenic acid, is required by 4% of all mammalian enzymes4. Cofactors are often classified as inorganic substances, such as ions, that are required for the enzymatic function. For example, all DNA polymerase enzymes require Mg2+ or Mn2+ for DNA replication as well as excision of incorrectly incorporated nucleotides5.
There are also other molecules that are not necessary for an enzyme to function but are capable of altering the enzymatic activity. Substances that increase the enzyme activity are called activators, whereas others that decrease the enzyme activity are called inhibitors. These molecules bind to enzymes either at the active site or other locations on the enzyme known as allosteric sites. Sometimes these molecules can bind to the exact location in the active site as the substrate, and thus compete with the substrate for the enzyme binding. Others, especially the substances that bind to allosteric sites, can alter enzyme function without blocking the substrate binding to the active site, and are thus non-competitive. Moreover, binding of inhibitors can be reversible or irreversible. When the interaction is reversable, the enzyme can resume its function once the inhibitor detaches. However, when the binding is irreversible, the inhibitor never detaches from the enzyme and the enzyme becomes irreparably damaged and needs to be replaced. Nevertheless, enzyme inhibitors are useful as well. For instance, inhibitors have been utilized as antibiotics to inhibit bacterial growth, and as chemotherapeutics to inhibit the uncontrolled division of cancer cells6.
Measuring Enzyme Function
One method used by scientists to study the effects of different conditions on enzyme function is to quantify the reaction rate, which is how fast the enzyme is catalyzing the reaction. Therefore, a higher reaction rate indicates that the enzyme is catalyzing reactions more efficiently, whereas a lower rate means that it is working less efficiently. To determine the reaction rate, the reaction velocity is calculated by measuring the amount of product made per unit time. This is done by measuring the product concentration starting right at the beginning of the reaction when the product concentration is increasing linearly, until the enzyme is completely saturated with the substrate and the catalysis reaches a steady level. Once the baseline reaction rate is determined, it can be used as a reference point, or control, when comparing between a variety of treatments. We can assess reaction rates as relative or absolute measures. A relative reaction rate is a comparative measure that shows differences between two or more conditions, such as treatments in a single experiment, but does not give a value for what is being measured. An absolute reaction rate relies on a quantitative measure of the progress of the reaction. An easy way to understand the difference between relative and absolute reaction rates is to consider measuring the speed of a car in a race. A relative method would compare by assessing the ranking of cars at the finish line, whereas the absolute rate would assess and compare the speed of individual cars in miles per hour.
Since enzymes are so widely used, researchers have begun looking for ways to create and modify enzymes to suit specific needs, known as protein engineering. Recombinant DNA technology allows amino acid sequences to be changed in order to alter an enzyme’s shape or active site affinity. This has been used to create highly sensitive diagnostic tests for various conditions, as well as for bioremediation efforts and waste management tools7.
- Swagerty DL Jr, Walling AD, Klein RM. Lactose intolerance. Am Fam Physician. 2002, Vol. 65, 9: 1845-50.
- Lomer MC, Parkes GC, Sanderson JD. Review article: lactose intolerance in clinical practice--myths and realities. Aliment Pharmacol Ther. 2008, Vol. 27, 2: 93-103.
- AR, Saltiel. New perspectives into the molecular pathogenesis and treatment of type 2 diabetes. Cell. 2001, Vol. 104, 4: 517-29.
- DO, Kennedy. B Vitamins and the Brain: Mechanisms, Dose and Efficacy--A Review. Nutrients. 2016, Vol. 8, 2: 68 doi:10.3390/nu8020068.
- Vashishtha AK, Wang J, Konigsberg WH. Different Divalent Cations Alter the Kinetics and Fidelity of DNA Polymerases. J Biol Chem. 2016, Vol. 291, 40: 20869-20875.
- Watson ID, Stewart MJ, Platt DJ. Clinical pharmacokinetics of enzyme inhibitors in antimicrobial chemotherapy. Clin Pharmacokinet. 1988, Vol. 15, 3: 133-64.
- Ahuja SK, Ferreira, GM, Moreira AR. Utilization of Enzymes for Environmental Applications. Crit Rev Biotech. 2004, Vols. 2-3, 125-54.