A microbial cell could be viewed as a microscopic factory, complete with basic building materials, a source of energy, and a genetic “blueprint” for running its extensive network of metabolic reactions. But the chemical reactions of life would never proceed without a special class of proteins called enzymes. Enzymes are a remarkable example of catalysts, chemicals that increase the rate of a chemical reaction without becoming part of the products or being consumed in the reaction. Do not make the mistake of thinking that an enzyme creates a reaction. Because of the free energy inherent in molecules, a reaction could occur spontaneously at some point even without an enzyme but at a very slow rate. A study of the enzyme urease shows that it increases the rate of the breakdown of urea by a factor of 100 trillion as compared to an uncatalyzed reaction. Because most uncatalyzed metabolic reactions do not occur fast enough to sustain cell processes, enzymes are indispensable to life. Other major qualities of enzymes are summarized in table 1.
Table1. Checklist of Enzyme Characteristics
How Do Enzymes Work?
We have said that an enzyme speeds up the rate of a metabolic reaction, but just how does it do this? During a chemical reaction, reactants are converted to products by bond formation or breakage. A certain amount of energy is required to initiate every such reaction, which limits its rate. This resistance to a reaction, which must be overcome for a reaction to proceed, is measurable and is called the energy of activation or activation energy (8.1 Making Connections). In the laboratory, overcoming this initial resistance can be achieved by
● increasing thermal energy (heating) to increase molecular velocity,
● increasing the concentration of reactants to increase the rate of molecular collisions, or
● adding a catalyst.
In most living systems, the first two alternatives are not feasible, because elevating the temperature is potentially harmful, and higher concentrations of reactants are often not practical. This leaves only the action of catalysts, and enzymes fill this need efficiently and potently.
At the molecular level, an enzyme promotes a reaction by serving as a physical site upon which the reactant molecules, called substrates, can be positioned for various interactions. The enzyme is much larger in size than its substrate, and it presents a uniquely shaped pocket that fits only that particular substrate. Although an enzyme binds to the substrate and participates directly in changes to the substrate, it does not become a part of the products, it is not used up by the reaction, and it can function over and over again. Enzyme speed, defined as the number of substrate molecules con verted per enzyme per second, is well documented. Speeds range from several million for catalase to a thousand for lactate dehydrogenase. A key to understanding the roles of enzymes in metabolism is their structure.
Enzyme Structure
Enzymes are composed largely of protein molecules, although certain types of RNA can function as nonenzyme catalysts (8.2 MakingConnections). Enzymes can be classified as simple or conjugated. Simple enzymes consist of protein alone, whereas conjugated enzymes (figure 1) contain protein and nonprotein molecules. A conjugated enzyme, sometimes referred to as a holoenzyme, is a combination of a protein, now called the apoenzyme, and one or more cofactors (table 2). Cofactors are either organic molecules, called coenzymes, or inorganic elements (metal ions) that many enzymes require to become functional. In some enzymes, the cofactor is loosely associated with the apoenzyme by noncovalent bonds; in others, it is linked by covalent bonds.
Fig1. Conjugated enzyme structure. All examples have an apoenzyme (polypeptide or protein) component and one or more cofactors.
Table2. Selected Enzymes, Catalytic Actions, and Cofactors*
Apoenzymes: Specificity and the Active Site
Apoenzymes range in size from small polypeptides with about 100 amino acids and a molecular weight of 12,000 to large poly peptide conglomerates with thousands of amino acids and a molecular weight of over 1 million. Like all proteins, an apoenzyme exhibits levels of molecular complexity called the primary, sec ondary, tertiary, and, in larger enzymes, quaternary organization (figure 8.3). As we saw in chapter 2, the first three levels of structure arise when a single polypeptide chain undergoes an automatic folding process and achieves stability by forming disulfide and other types of bonds. Folding causes the surface of the apoenzyme to acquire three-dimensional features that create the enzyme’s specificity for substrates. The actual pocket where the substrate binds is called the active site, or catalytic site, and there can be from one to several such sites (figure2). Because each type of enzyme has a different primary structure (type and sequence of amino acids), this gives rise to variations in folding and unique active sites.
Fig2. How the active site and specificity of the apoenzyme arise.
Enzyme–Substrate Interactions
For a reaction to take place, a temporary enzyme–substrate union must occur at the active site. There are several explanations for the way this happens (figure 3). The specificity is often described as a “lock-and-key” fit in which the substrate is inserted into the active site’s pocket. This is a useful analogy, but an enzyme is not a rigid lock mechanism. It is likely that the enzyme actually helps the substrate move into the active site through slight changes in its shape. This is called an induced fit (figure3d). The bonds formed between the substrate and enzyme are weak and, of necessity, easily reversible. Once the enzyme–substrate complex has formed, appropriate reactions occur on the substrate, often with the aid of a cofactor, and a product is formed and released. The enzyme can then attach to another substrate molecule and repeat this action.
Fig. Enzyme–substrate reactions. (a) When the enzyme and substrate come together, the substrate (S) will have a correct fit and position with respect to the enzyme (E). (b) When the ES complex is formed, it enters a transition state. During this temporary but tight interlocking union, the enzyme participates directly in breaking or making bonds. (c) Once the reaction is complete, the enzyme releases the products. (d) The induced-fit model proposes that the enzyme recognizes its substrate and adapts slightly to it so that the final binding is even more precise.
Cofactors: Supporting the Work of Enzymes
In chapter 7, you learned that microorganisms require specific metal ions called trace elements and certain organic growth factors. In many cases, the need for these substances arises from their roles as cofactors (table 2). The metallic cofactors, including iron, copper, magnesium, manganese, zinc, cobalt, selenium, and many others, participate in precise functions between the enzyme and its substrate. In general, metals activate enzymes, help bring the active site and substrate close together, and participate directly in chemical reactions with the enzyme–substrate complex.
Coenzymes are organic cofactors that work in conjunction with the apoenzyme to perform a necessary alteration of a substrate. The general function of a coenzyme is to remove a chemical group from one substrate molecule and add it to another substrate, thereby serving as a temporary carrier of this group (figure 4). The specific activities of coenzymes are many and varied. we shall see that coenzymes carry and transfer hydrogen atoms, electrons, carbon dioxide, and amino groups. Some of the most common components of coenzymes are vitamins, which ex plains why vitamins are important to nutrition and may be required as growth factors for living things. Vitamin deficiencies prevent the complete holoenzyme from forming, which compromises both the chemical reaction and the structure or function that is dependent upon that reaction.
Fig4. The carrier functions of coenzymes. Many enzymes use coenzymes to transfer chemical groups from one substrate to another.
Classification of Enzyme Functions
Enzymes are classified and named according to characteristics such as site of action, type of action, and substrate (8.3 Making Connections).
Location and Regularity of Enzyme Action
Enzymes perform their tasks either inside or outside of the cell in which they were produced. After initial synthesis in the cell, exoenzymes are transported extracellularly, where they break down large food molecules or harmful chemicals (see discussion of hydrolysis reactions under the next heading). Examples of exoenzymes are cellulase, amylase, and penicillinase. By contrast, endoenzymes are retained intracellularly and function there (figure 5). Most enzymes of the metabolic pathways are of this variety.
Fig5. Types of enzymes, as described by their location of action. (a) Exoenzymes are transported outside the cell to become active. (b) Endoenzymes remain in the cell and function there.
In terms of their presence in the cell, enzymes are not all produced in equal amounts or at equal rates. Some, called constitutive enzymes (figure 6a), are continually present in relatively constant amounts, regardless of the amount of substrate. The enzymes involved in utilizing glucose, for example, are very important in metabolism and thus are constitutive. Other enzymes are regulated enzymes (figure 6b), the production of which is either turned on (induced) or turned off (repressed) in response to changes in concentration of the substrate or product. The level of inducible and repressible enzymes is controlled by variations in expression of the genes for these proteins.
Fig6. Constitutive and regulated enzymes (a) Constitutive enzymes are present in constant amounts in a cell. The addition of more substrate does not increase the numbers of these enzymes. (b) The concentration of some regulated enzymes in a cell increases or decreases in response to substrate levels.
Synthesis and Hydrolysis Reactions A growing cell is in a constant state of synthesis. All such anabolic reactions require enzymes (synthases) to form covalent bonds between smaller substrate molecules. Also known as condensation (dehydration) reactions, synthesis reactions release one water molecule for each bond made (figure 7a). Catabolic reactions are also highly active in a growing cell and require their own, different set of enzymes. For example, large substrates must be broken down or digested by enzymes into smaller molecules so they can be used. Because the breaking of bonds usually requires the input of water, digestion is also termed a hydrolysis* reaction (figure 7b).
Fig7. Examples of enzyme-catalyzed synthesis and hydrolysis reactions.
The Sensitivity of Enzymes to Environmental Conditions
The activity of an enzyme is highly influenced by the cell’s environment. In general, enzymes operate only under the natural temperature, pH, and osmotic pressure of an organism’s habitat. When enzymes are subjected to departures from these normal conditions, they tend to be chemically unstable, or labile. Low temperatures inhibit catalysis, and high temperatures can denature the apoenzyme. Denaturation is a process by which the bonds that collectively maintain the native shape of the apoenzyme are disrupted. This loss causes extreme distortion of the enzyme’s shape and prevents the substrate from attaching to the active site. Such nonfunctional enzymes block metabolic reactions and can lead to cell death. Low or high pH and certain chemicals (heavy metals, alcohol) are also denaturing agents.
