Enzyme
Enzyme
Enzymes are biological catalysts. A catalyst is an agent which increases the rate of chemical reactions (the speed at which the reactions occur) without being used up or altered in the reaction. Enzymes are also proteins. They are able to function as catalysts because they have sites to which portions of other molecules can bind. The binding is typically relatively weak, so that molecules are not irreversibly bound. These weak binding interactions allow enzymes to accelerate specific reaction rates over and over again.
Louis Pasteur, a scientist who made a number of fundamentally important discoveries to science in general and microbiology in particular was among the first to study enzyme action. He incorrectly hypothesized that the conversion of sugar into alcohol by yeast was catalyzed by “ferments” that could not be separated from living cells. In 1897 the German biochemist Eduard Buchner (1860-1917) isolated the enzymes which catalyze alcoholic fermentation from living yeast cells, represented in the equation:
glucose → ethanol + carbon dioxide
The early twentieth century saw dramatic advancement in enzyme studies. Emil Fischer (1852-1919) recognized the importance of substrate shape for binding by enzymes. Leonor Michaelis (1875-1949) and Maud Menten introduced a mathematical approach for quantifying enzyme-catalyzed reactions. James Sumner (1887-1955) and John Northrop (1891-1987) were among the first to produce highly ordered enzyme crystals and firmly establish the protein nature of these biological catalysts. In 1937 Hans Krebs (1900-1981) postulated how a series of enzymatic reactions were coordinated in the citric acid cycle for the production of ATP from glucose metabolites. Today, enzymology is a central part of biochemical study.
Protein structural studies are a very active area of biochemical research, and it is known that the biological function (activity) of an enzyme is related to its structure. There are 20 common amino acids that make up the building blocks of all known enzymes. They have similar structures, differing mainly in their substituents. The organic substituent of an amino acid is called the R group.
The biologically common amino acids are designated as L-amino acids (see Figure 1). This is called Fischer projection. The amino acids are covalently joined via peptide bonds. Enzymes have many peptide linkages and range in molecular mass from 12,000 to greater than one million (see Figure 2).
Enzymes may also consist of more than a single polypeptide chain. Each polypeptide chain is called a subunit, and may have a separate catalytic function. Some enzymes have non-protein groups that are necessary for enzymatic activity. Metal ions and organic molecules called coenzymes are components of many enzymes. Coenzymes, which are tightly or covalently attached to enzymes, are termed prosthetic groups. Prosthetic groups contain critical chemical groups which allow the overall catalytic event to occur.
Enzymes bind their reactants (substrates) at special folds and clefts in their structures called active sites. Because active sites have chemical groups precisely located and orientated for binding the substrate, they generally display a high degree of substrate specificity. The active site of an enzyme consists of two key regions. The catalytic site, which interacts with the substrate during the reaction, and the binding site, the chemical groups of the enzyme which bind the substrate to allow chemical interaction at the catalytic site.
Although the details of enzyme active sites differ between different enzymes, there are common motifs. The active site represents only a small fraction of the total protein volume. The reason enzymes are so large is that many interactions are necessary for reaction. The crevice of the active site creates a microenvironment that is critical for catalysis. Environmental factors include polarity, hydrophobicity, and precise arrangement of atoms and chemical groups. In 1890 Emil Fischer compared the enzyme-substrate relationship to the fit between and lock and a key (see Figure 3). Fischer postulated that the active site and the enzyme have complimentary three-dimensional shapes. This model was extended by Daniel Koshland Jr. in 1958 by his so-called induced fit model, which reasoned that actives sites are complimentary to substrate shape only after the substrate is bound.
An example of enzyme action is a simple, uncatalyzed chemical reaction reactant → product B. As the concentration of reactant A is increased, the rate of product B formation increases. Rate of reaction is defined as the number of molecules of B formed per unit time. In the presence of a catalyst, the reaction rate is accelerated. For reactant to be converted to product, a thermodynamic energy barrier must be overcome. This energy barrier is known as the activation energy (Ea). A catalyst speeds up a chemical process by lowering the activation energy that the reactant must reach before being converted to product. It does this by allowing a different chemical mechanism or pathway which has a lower activation energy (see Figure 4).
Enzymes have high catalytic power, high substrate specificity, and are generally most active in aqueous solvents at mild temperature and physiological pH.
There are thousands of known enzymes, but nearly all can be categorized according to their biological activities into six major classes: oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases. Most enzymes catalyze the transfer of electrons, atoms, or groups of atoms, and are assigned names according to the type of reaction.
Thousands of enzymes have been discovered and purified to date. The structure, chemical function, and mechanism of hundreds of enzymes has given biochemists a solid understanding of how they work. In the 1930s, J.B.S. Haldane (1892-1964) described the principle that interactions between the enzyme and its substrate can be used to distort the shape of the substrate and induce a chemical reaction. The energy released and used to lower activation energies from the enzyme-substrate interaction is called the binding energy. The chemical form of the substrate at the top of the energy barrier is called a transition state. The maximum number of weak enzyme-substrate interactions is attained when the substrate reaches its transition state. Enzymes stabilize the transition state, allowing the reaction to proceed in the forward direction. The rate-determining process of a simple enzyme catalyzed reaction is the breakdown of the activated complex between the transition state and the enzyme. In 1889 the Swedish chemist Svante Arrhenius (1859-1927) showed the dependence of the rate of reaction on the magnitude of the energy barrier (activation energy). Reactions that consist of several chemical steps will have multiple activated complexes. The over-all rate of the reaction will be determined by the slowest activated complex decomposition. This is called the rate-limiting step.
In addition to enhancing the reaction rate, enzymes have the ability to discriminate among competing substrates. Enzyme specificity arises mainly from three sources. The first is the exclusion of other molecules from the active site because of the presence of incorrect functional groups, or the absence of necessary binding groups. Secondly, many weak interactions exist between the substrate and the enzyme. It is known that the requirement for many interactions is one reason enzymes are very large compared to the substrate. The noncovalent interactions that bind molecules to enzymes are similar to the intramolecular forces that control enzyme conformation. Induced conformational changes shift important chemical groups on the enzyme into close proximity for catalysis. Finally, stereospecificity arises from the fact that enzymes are built from only L-amino acids. They have active sites which are asymmetric and will bind only certain substrates.
Enzyme activity is controlled by many factors, including environment, enzyme inhibitors, and regulatory binding sites on the enzyme. Concerning the environment, enzymes generally have an optimum
KEY TERMS
Activation energy— The amount of energy required to convert the substrate to the transition state.
Active site— The fold or cleft of an enzyme which binds substrate and catalytically transforms it to product.
Hydrophobic— Nonpolar, insoluble in water.
Inducedfit— Enzyme conformational change caused by substrate binding and resulting in catalytic activity.
Lock-and-key— Geometrically complementary shapes of the enzyme (lock) and the substrate (key) resulting in specificity.
Peptide bond— Chemical covalent bond formed between two amino acids in a polypeptide.
Polypeptide— A long chain of amino acids linked by peptide bonds.
Rate of reaction— The quantity of substrate molecules converted to product per unit time.
Transition state— The activated form of the reactant occupying the highest point on the reaction coordinate.
Weak interactions— Noncovalent interactions between the substrate and the enzyme, allowing enzyme conformational change and catalysis.
pH range in which they are most active. The pH of the environment will affect the ionization state of catalytic groups at the active site and the ionization of the substrate. Electrostatic interactions are therefore controlled by pH. pH may also control the conformation of the enzyme through ionizable amino acids which are located distant from the active site, but are nevertheless critical for the three-dimensional shape of the macromolecule.
Enzyme inhibitors diminish the activity of an enzyme by altering the way substrates bind. Inhibitor structure may be similar to the substrate, but they react very slowly or not at all. Chemically, inhibitors may be large organic molecules, small molecules or ions. They are important because they can be useful for chemotherapeutic treatment of disease, and for providing experimental insights into the mechanism of enzyme action. Inhibitors work in either a reversible or an irreversible manner. Irreversible inhibition is characterized by extremely tight or covalent interaction with the enzyme, resulting in very slow dissociation and inactivation. Reversible inhibition is displayed by rapid dissociation of the inhibitor from the enzyme. There are three main classes of reversible inhibition, competitive inhibition, noncompetitive inhibition, and mixed inhibition. They are distinguishable by how they bind to enzymes and response to increasing substrate concentration. Enzyme inhibition is studied by examining reaction rates and how rates change in response to changes in experimental parameters.
Regulatory enzymes are characterized by increased or decreased activity in response to chemical signals. Metabolic pathways are regulated by controlling the activity of one or more enzymatic steps along that path. Regulatory control allows cells to meet changing demands for energy and biomolecules.
Enzymatic activity is regulated in four general ways. The first is termed allosteric control. Allosteric enzymes are those that have distinct binding sites for effector molecules, which control their rates of reaction. The second reglator of enzymatic activity involves proteins designated control proteins, which participate in cellular regulatory processes. For example, calmodulin is a Ca2+ binding protein which binds with high affinity and modulates the activity of many Ca2+-regulated enzymes. In this way, Ca2+ acts as to allow events like feedback control in metabolic pathways (see Figure 5). A third enzyme regulatory mechanism involves the reversible chemical modification that can be carried out with a variety of chemical groups. A common example is the transfer of phosphate groups, catalyzed by enzymes known as kinases (see Figure 6). Finally, proteolytic activation (activation of enzyme activity that occurs when part of the protein is removed) can regulate the activity of some enzymes. One example are zymogens, which are converted to an active form following the irreversible removal of a portion of the protein. Proteolytic enzymes are controlled by this latter mechanism.
See also Catalyst and catalysis; Krebs cycle.
Resources
BOOKS
Aehle, Wolfgang. Enzymes in Industry: Production and Applications. New York: Wiley, 2004.
Defelice, Karen. Enzymes for Digestive Health and Nutritional Wealth: The Practical Guide for Digestive Enzymes. Johnston IA: Thundersnow Interactive, 2003.
Silverman, Richard E. Organic Chemistry of Enzyme-Catalyzed Reactions, Revised 2nd. ed. New York: Academic Press, 2002.
Leonard D. Holmes