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The endomembrane system (endo = “within”) is a group of membranes and organelles (Figure 1) in eukaryotic cells that works together to modify, package, and transport lipids and proteins. Although not technically within the cell, the plasma membrane is included in the endomembrane system because, as you will see, it interacts with the other endomembranous organelles. The endomembrane system does not include the membranes of either mitochondria or chloroplasts.
Figure 1 illustrates the connections of the endomembrane system as a (green) integral membrane protein in the ER is modified by attachment of a (purple) carbohydrate. Vesicles with the integral protein bud from the ER and fuse with the cis face of the Golgi apparatus. As the protein passes along the Golgi’s cisternae, it is further modified by the addition of more carbohydrates. After its synthesis is complete, it exits as integral membrane protein of the vesicle that bud from the Golgi’s trans face and when the vesicle fuses with the cell membrane the protein becomes integral portion of that cell membrane.
The endoplasmic reticulum (ER) (Figure 1) is a series of interconnected membranous sacs and tubules that collectively modifies proteins and synthesizes lipids. However, these two functions are performed in separate areas of the ER: the rough ER and the smooth ER, respectively.
The hollow portion of the ER tubules is called the lumen or cisternal space. The membrane of the ER, which is a phospholipid bilayer embedded with proteins, is continuous with the nuclear envelope.
The rough endoplasmic reticulum (RER) is so named because the ribosomes attached to its cytoplasmic surface give it a studded appearance when viewed through an electron microscope (Figure 2).
Ribosomes transfer their newly synthesized proteins into the lumen of the RER where they undergo structural modifications, such as folding or the acquisition of side chains. These modified proteins will be incorporated into cellular membranes—the membrane of the ER or those of other organelles—or secreted from the cell (such as protein hormones or enzymes). The RER also makes phospholipids for cellular membranes.
If the phospholipids or modified proteins are not destined to stay in the RER, they will reach their destinations via transport vesicles that bud from the RER’s membrane (Figure 1).
Since the RER is engaged in modifying proteins (such as enzymes, for example) that will be secreted from the cell, you would be correct in assuming that the RER is abundant in cells that secrete proteins. This is the case with cells of the liver, for example.
The smooth endoplasmic reticulum (SER) is continuous with the RER but has few or no ribosomes on its cytoplasmic surface. Functions of the SER include synthesis of carbohydrates, lipids, and steroid hormones; detoxification of medications and poisons; and storage of calcium ions.
In muscle cells, a specialized SER called the sarcoplasmic reticulum is responsible for storage of the calcium ions that are needed to trigger the coordinated contractions of the muscle cells.
Heart disease is the leading cause of death in the United States. This is primarily due to our sedentary lifestyle and our high trans-fat diets.
Heart failure is just one of many disabling heart conditions. Heart failure does not mean that the heart has stopped working. Rather, it means that the heart can’t pump with sufficient force to transport oxygenated blood to all the vital organs. Left untreated, heart failure can lead to kidney failure and failure of other organs.
The wall of the heart is composed of cardiac muscle tissue. Heart failure occurs when the endoplasmic reticula of cardiac muscle cells do not function properly. As a result, an insufficient number of calcium ions are available to trigger a sufficient contractile force.
Cardiologists (cardi = “heart”; ologist = “one who studies”) are doctors who specialize in treating heart diseases, including heart failure. Cardiologists can make a diagnosis of heart failure via physical examination, results from an electrocardiogram (ECG, a test that measures the electrical activity of the heart), a chest X-ray to see whether the heart is enlarged, and other tests. If heart failure is diagnosed, the cardiologist will typically prescribe appropriate medications and recommend a reduction in table salt intake and a supervised exercise program.
We have already mentioned that vesicles can bud from the ER and transport their contents elsewhere, but where do the vesicles go? Before reaching their final destination, the lipids or proteins within the transport vesicles still need to be sorted, packaged, and tagged so that they wind up in the right place. Sorting, tagging, packaging, and distribution of lipids and proteins takes place in the Golgi apparatus (also called the Golgi body), a series of flattened membranes (Figure 3).
The receiving side of the Golgi apparatus is called the cis face. The opposite side is called the trans face. The transport vesicles that formed from the ER travel to the cis face, fuse with it, and empty their contents into the lumen of the Golgi apparatus. As the proteins and lipids travel through the Golgi, they undergo further modifications that allow them to be sorted. The most frequent modification is the addition of short chains of sugar molecules. These newly modified proteins and lipids are then tagged with phosphate groups or other small molecules so that they can be routed to their proper destinations.
Finally, the modified and tagged proteins are packaged into secretory vesicles that bud from the trans face of the Golgi. While some of these vesicles deposit their contents into other parts of the cell where they will be used, other secretory vesicles fuse with the plasma membrane and release their contents outside the cell.
In another example of form following function, cells that engage in a great deal of secretory activity (such as cells of the salivary glands that secrete digestive enzymes or cells of the immune system that secrete antibodies) have an abundance of Golgi.
In plant cells, the Golgi apparatus has the additional role of synthesizing polysaccharides, some of which are incorporated into the cell wall and some of which are used in other parts of the cell.
Many diseases arise from genetic mutations that prevent the synthesis of critical proteins. One such disease is Lowe disease (also called oculocerebrorenal syndrome, because it affects the eyes, brain, and kidneys). In Lowe disease, there is a deficiency in an enzyme localized to the Golgi apparatus. Children with Lowe disease are born with cataracts, typically develop kidney disease after the first year of life, and may have impaired mental abilities.
Lowe disease is a genetic disease caused by a mutation on the X chromosome. The X chromosome is one of the two human sex chromosome, as these chromosomes determine a person’s sex. Females possess two X chromosomes while males possess one X and one Y chromosome. In females, the genes on only one of the two X chromosomes are expressed. Therefore, females who carry the Lowe disease gene on one of their X chromosomes have a 50/50 chance of having the disease. However, males only have one X chromosome and the genes on this chromosome are always expressed. Therefore, males will always have Lowe disease if their X chromosome carries the Lowe disease gene. The location of the mutated gene, as well as the locations of many other mutations that cause genetic diseases, has now been identified. Through prenatal testing, a woman can find out if the fetus she is carrying may be afflicted with one of several genetic diseases.
Geneticists analyze the results of prenatal genetic tests and may counsel pregnant women on available options. They may also conduct genetic research that leads to new drugs or foods, or perform DNA analyses that are used in forensic investigations.
Mycogone perniciosa causes wet bubble disease in Agaricus bisporus and various Agaricomycetes species. In a previous work, we identified 41 GH18 chitinase genes and other pathogenicity-related genes in the genome of M. perniciosa Hp10. Chitinases are enzymes that degrade chitin, and they have diverse functions in nutrition, morphogenesis, and pathogenesis. However, these important genes in M. perniciosa have not been fully characterized, and their functions remain unclear. Here, we performed a genome-wide analysis of M. perniciosa GH18 genes and analyzed the transcriptome profiles and GH18 expression patterns in M. perniciosa during the time course of infection in A. bisporus. Phylogenetic analysis of the 41 GH18 genes with those of 15 other species showed that the genes were clustered into three groups and eight subgroups based on their conserved domains. The GH18 genes clustered in the same group shared different gene structures but had the same protein motifs. All GH18 genes were localized in different organelles, were unevenly distributed on 11 contigs, and had orthologs in the other 13 species. Twelve duplication events were identified, and these had undergone both positive and purifying selection. The transcriptome analyses revealed that numerous genes, including transporters, cell wall degrading enzymes (CWDEs), cytochrome P450, pathogenicity-related genes, secondary metabolites, and transcription factors, were significantly upregulated at different stages of M. perniciosa Hp10 infection of A. bisporus. Twenty-three out of the 41 GH18 genes were differentially expressed. The expression patterns of the 23 GH18 genes were different and were significantly expressed from 3 days post-inoculation of M. perniciosa Hp10 in A. bisporus. Five differentially expressed GH18 genes were selected for RT-PCR and gene cloning to verify RNA-seq data accuracy. The results showed that those genes were successively expressed in different infection stages, consistent with the previous sequencing results. Our study provides a comprehensive analysis of pathogenicity-related and GH18 chitinase genes’ influence on M. perniciosa mycoparasitism of A. bisporus. Our findings may serve as a basis for further studies of M. perniciosa mycoparasitism, and the results have potential value for improving resistance in A. bisporus and developing efficient disease-management strategies to mitigate wet bubble disease.
The button mushroom (Agaricus bisporus) is one of the most widely cultivated and consumed edible mushrooms in the world. Production of button mushrooms in China has rapidly increased in recent years as a result of the expanded area of cultivation and the adoption of technology for improved commercial cultivation (He et al., 2014 Sonnenberg et al., 2017 Li et al., 2018 McGee, 2018). However, diseases caused by fungi, bacteria, and viruses are major constraints to A. bisporus production worldwide, often leading to serious economic losses (Fletcher et al., 1989 Largeteau and Savoie, 2010 Kouser et al., 2013). Wet bubble disease (WBD) is one of the most devastating diseases of A. bisporus, causing yield losses of 15% under favorable conditions and up to 75% or total crop loss in the most severe cases (Zhou et al., 2015). WBD is characterized by wet bubbles, malformation, white, fluffy mycelial growth, copious amber droplets (diseased carpophores exuding a brown malodorous liquid), and flocculent mycelia on most substrates (Fletcher et al., 1995 Sharma and Kumar, 2000 Umar et al., 2000). Mycogone perniciosa (teleomorph: Hypomyces perniciosus) is the causal agent of WBD, and it infects a variety of mushrooms (Zhang et al., 2017a,b Carrasco et al., 2019). M. perniciosa is a fungicolous fungus belonging to the order Hypocreales (Ascomycota) in the family Hypocreaceae.
M. perniciosa is mainly controlled by cultural practices and the application of fungicides (Brankica et al., 2009). Varying levels of WBD resistance have been identified in the A. bisporus germplasm collection in China however, no major resistance gene has been identified (Fu et al., 2016). The genome of M. perniciosa contains many genes implicated in pathogenicity (Li et al., 2019), but the regulation of these genes in the pathogenesis toward A. bisporus is still unclear. In addition, comparative genomics analysis of M. perniciosa has revealed gene expansion and positive selection of many genes, including GH18 chitinase, peptidase, and secondary metabolite genes (Li et al., 2019). Gene expansion and positive selection contribute to the evolution of virulence genes in microbial pathogens and to the adaptation to different environmental niches through the infection process and via escape from the host defense response (Yoshizaki et al., 2019).
Chitinase (EC 22.214.171.124) is a glycosyl hydrolase enzyme that degrades chitin (Hamid et al., 2013). Chitin is the main structural component of fungal cell walls and the exoskeleton of animals, including worms and arthropods (Mauch et al., 1988). Chitinases are found in various organisms, including those that do not contain chitin (Rathore and Gupta, 2015). Chitinases of pathogenic fungi not only play vital roles in spore germination, septum formation, cell division, and morphogenesis, but the enzymes are also important in the host interaction (Elad et al., 1982, 1983 Inbar and Chet, 1995 Chen, 2002 Adams, 2004). In addition to degradation of the host fungal cell wall, chitinases also inhibit hyphae growth and bud tube elongation (Gozia et al., 1993 Stressmann et al., 2004).
Chitinases are classified into two families, namely glycosyl hydrolases 18 and 19, based on amino acid sequence similarity (Henrissat and Bairoch, 1993). The GH18 chitinase gene family is widely distributed in bacteria, fungi, viruses, animals, and higher plants (Henrissat and Bairoch, 1993 Kawase et al., 2004 Seidl, 2008 Hartl et al., 2012 Adrangi and Faramarzi, 2013). Omics and bioinformatics analyses have demonstrated that most fungal chitinases have similar domains that generally contain a signal peptide sequence, a chitinase catalytic domain, a chitin-binding domain, and a short C-terminal domain (van Aalten et al., 2001). Among fungal pathogens, GH18 chitinase gene families are well characterized in Trichoderma species, Fusarium species, and Magnaporthe species (Häkkinen et al., 2012 Han et al., 2019). Currently, 30 chitinase genes have been reported within eight species of Trichoderma, including T. harzianum (30 genes), T. virens (29 genes), and T. atroviride (24 genes) (Kubicek et al., 2011, 2019).
The advent of next-generation sequencing technologies has increased the scalability, speed, and resolution of genomic sequencing and reduced genome sequencing cost (Faino et al., 2015). This has rapidly increased fungal genome availability for comparative genomics and genome-wide identification of gene families (Bartholomew et al., 2019). However, few studies have comprehensively examined the structure or the expression of GH18 chitinases of fungal pathogens infecting mushrooms.
In a previous work, we identified 41 GH18 chitinase genes in the genome of M. perniciosa (Li et al., 2019). However, the genome-wide identification, functional characterization, and expression of these GH18 chitinase genes were not considered. In this study, we present the first detailed and comprehensive analysis of the GH18 gene family in the genome of M. perniciosa. The analyses include chromosome location, phylogenetic analysis, protein structure, and motif composition. Furthermore, we performed a transcriptome analysis of the different infection stages of M. perniciosa on A. bisporus to identify pathogenicity-related differential gene expression and expression patterns of the GH18 genes. In addition, we selected five GH18 genes that represented different types and identified their expression levels by RT-PCR and gene cloning to verify the sequencing. The results can provide important information for identifying essential genes as potential antifungal targets in M. perniciosa. Further, the dataset generated in this study may provide a basis for identifying candidate resistant genes in A. bisporus against M. perniciosa and lay a foundation for further research to improve the resistance of A. bisporus to M. perniciosa.
In this section, you will explore the following questions:
- What is the role of enzymes in metabolic pathways?
- How do enzymes function as molecular catalysts?
Connection for AP ® Courses
Many chemical reactions in cells occur spontaneously, but happen too slowly to meet the needs of a cell. For example, a teaspoon of sucrose (table sugar), a disaccharide, in a glass of iced tea will take time to break down into two monosaccharides, glucose and fructose however, if you add a small amount of the enzyme sucrase to the tea, sucrose breaks down almost immediately. Sucrase is an example of an enzyme, a type of biological catalyst. Enzymes are macromolecules—most often proteins—that speed up chemical reactions by lowering activation energy barriers. Enzymes are very specific for the reactions they catalyze because they are polypeptides, enzymes can have a variety of shapes attributed to interactions among amino acid R-groups. One part of the enzyme, the active site, interacts with the substrate via the induced fit model of interaction. Substrate binding alters the shape of the enzyme to facilitate the chemical reaction in several different ways, including bringing substrates together in an optimal orientation. After the reaction finishes, the product(s) are released, and the active site returns to its original shape.
Enzyme activity, and thus the rate of an enzyme-catalyzed reaction, is regulated by environmental conditions, including the amount of substrate, temperature, pH, and the presence of coenzymes, cofactors, activators, and inhibitors. Inhibitors, coenzymes, and cofactors can act competitively by binding to the enzyme’s active site, or noncompetitively by binding to the enzyme’s allosteric site. An allosteric site is an alternate part of the enzyme that can bind to non–substrate molecules. Enzymes work most efficiently under optimal conditions that are specific to the enzyme. For example, trypsin, an enzyme in the human small intestine, works most efficiently at pH 8, whereas pepsin in the stomach works best under acidic conditions. Sometimes environmental factors, especially low pH and high temperatures, alter the shape of the active site if the shape cannot be restored, the enzyme denatures. The most common method of enzyme regulation in metabolic pathways is via feedback inhibition.
How can various factors, such as feedback inhibition, regulate enzyme activity?
Information presented and the examples highlighted in the section support concepts and Learning Objectives outlined in Big Idea 4 of the AP ® Biology Curriculum Framework. The learning objectives listed in the Curriculum Framework provide a transparent foundation for the AP ® Biology course, an inquiry-based laboratory experience, instructional activities, and AP ® Exam questions. A Learning Objective merges required content with one or more of the seven science practices.
|Big Idea 4||Biological systems interact, and these systems and their interactions possess complex properties.|
|Enduring Understanding 4.B||Competition and cooperation are important aspects of biological systems.|
|Essential Knowledge||4.B.1 Interactions between molecules affect their structure and function.|
|Science Practice||5.1 The student can analyze data to identify patterns or relationships.|
|Learning Objective||4.17 The student is able to analyze data to identify how molecular interactions affect structure and function.|
The idea that enzymes help chemical reactions to occur, but do not take part in the chemical reaction and are not changed by it can be confusing. Stress that an enzyme and substrate do not covalently bind to each other and the association is temporary. Figure 6.16 is useful in illustrating enzyme function. If two compounds are to be joined into one during the reaction, and they would anyway if left alone long enough, the enzyme molecule brings them close enough for the reaction to occur faster. If a large molecule is to be split into smaller units, the enzyme stresses the molecule and makes it easier for the covalent bonds holding the molecule to break. In both cases, the enzyme molecule subtlety changes its shape after attaching to the substrate (s). This creates an intermediate phase of the reaction and an enzyme-substrate complex. When the reaction is complete and the product(s) disassociate, the enzyme returns to its original shape.
The Science Practice Challenge Questions contain additional test questions for this section that will help you prepare for the AP exam. These questions address the following standards:
[APLO 2.15][APLO 4.8][APLO 2.16]
A substance that helps a chemical reaction to occur is a catalyst, and the special molecules that catalyze biochemical reactions are called enzymes. Almost all enzymes are proteins, made up of chains of amino acids, and they perform the critical task of lowering the activation energies of chemical reactions inside the cell. Enzymes do this by binding to the reactant molecules, and holding them in such a way as to make the chemical bond-breaking and bond-forming processes take place more readily. It is important to remember that enzymes don’t change the ∆G of a reaction. In other words, they don’t change whether a reaction is exergonic (spontaneous) or endergonic. This is because they don’t change the free energy of the reactants or products. They only reduce the activation energy required to reach the transition state (Figure 6.15).
Enzyme Active Site and Substrate Specificity
The chemical reactants to which an enzyme binds are the enzyme’s substrates . There may be one or more substrates, depending on the particular chemical reaction. In some reactions, a single-reactant substrate is broken down into multiple products. In others, two substrates may come together to create one larger molecule. Two reactants might also enter a reaction, both become modified, and leave the reaction as two products. The location within the enzyme where the substrate binds is called the enzyme’s active site . The active site is where the “action” happens, so to speak. Since enzymes are proteins, there is a unique combination of amino acid residues (also called side chains, or R groups) within the active site. Each residue is characterized by different properties. Residues can be large or small, weakly acidic or basic, hydrophilic or hydrophobic, positively or negatively charged, or neutral. The unique combination of amino acid residues, their positions, sequences, structures, and properties, creates a very specific chemical environment within the active site. This specific environment is suited to bind, albeit briefly, to a specific chemical substrate (or substrates). Due to this jigsaw puzzle-like match between an enzyme and its substrates (which adapts to find the best fit between the transition state and the active site), enzymes are known for their specificity. The “best fit” results from the shape and the amino acid functional group’s attraction to the substrate. There is a specifically matched enzyme for each substrate and, thus, for each chemical reaction however, there is flexibility as well.
The fact that active sites are so perfectly suited to provide specific environmental conditions also means that they are subject to influences by the local environment. It is true that increasing the environmental temperature generally increases reaction rates, enzyme-catalyzed or otherwise. However, increasing or decreasing the temperature outside of an optimal range can affect chemical bonds within the active site in such a way that they are less well suited to bind substrates. High temperatures will eventually cause enzymes, like other biological molecules, to denature , a process that changes the natural properties of a substance. Likewise, the pH of the local environment can also affect enzyme function. Active site amino acid residues have their own acidic or basic properties that are optimal for catalysis. These residues are sensitive to changes in pH that can impair the way substrate molecules bind. Enzymes are suited to function best within a certain pH range, and, as with temperature, extreme pH values (acidic or basic) of the environment can cause enzymes to denature.
Induced Fit and Enzyme Function
For many years, scientists thought that enzyme-substrate binding took place in a simple “lock-and-key” fashion. This model asserted that the enzyme and substrate fit together perfectly in one instantaneous step. However, current research supports a more refined view called induced fit (Figure 6.16). The induced-fit model expands upon the lock-and-key model by describing a more dynamic interaction between enzyme and substrate. As the enzyme and substrate come together, their interaction causes a mild shift in the enzyme’s structure that confirms an ideal binding arrangement between the enzyme and the transition state of the substrate. This ideal binding maximizes the enzyme’s ability to catalyze its reaction.
Link to Learning
View an animation of induced fit at this website.
- Production of energy by glycolysis will occur more slowly than normal skeletal muscles will function properly.
- Production of energy by glycolysis will not occur skeletal muscles will function properly.
- Production of energy by glycolysis will occur more erratically than normal skeletal muscles will not function properly.
- Production of energy by glycolysis will not occur skeletal muscles will not function properly.
When an enzyme binds its substrate, an enzyme-substrate complex is formed. This complex lowers the activation energy of the reaction and promotes its rapid progression in one of many ways. On a basic level, enzymes promote chemical reactions that involve more than one substrate by bringing the substrates together in an optimal orientation. The appropriate region (atoms and bonds) of one molecule is juxtaposed to the appropriate region of the other molecule with which it must react. Another way in which enzymes promote the reaction of their substrates is by creating an optimal environment within the active site for the reaction to occur. Certain chemical reactions might proceed best in a slightly acidic or non-polar environment. The chemical properties that emerge from the particular arrangement of amino acid residues within an active site create the perfect environment for an enzyme’s specific substrates to react.
You’ve learned that the activation energy required for many reactions includes the energy involved in manipulating or slightly contorting chemical bonds so that they can easily break and allow others to reform. Enzymatic action can aid this process. The enzyme-substrate complex can lower the activation energy by contorting substrate molecules in such a way as to facilitate bond-breaking, helping to reach the transition state. Finally, enzymes can also lower activation energies by taking part in the chemical reaction itself. The amino acid residues can provide certain ions or chemical groups that actually form covalent bonds with substrate molecules as a necessary step of the reaction process. In these cases, it is important to remember that the enzyme will always return to its original state at the completion of the reaction. One of the hallmark properties of enzymes is that they remain ultimately unchanged by the reactions they catalyze. After an enzyme is done catalyzing a reaction, it releases its product(s).
Science Practice Connection for AP® Courses
Think About It
AP Biology Investigation 13: Enzyme Activity. This investigation allows you to design and conduct experiments to explore the effects of environmental variables, such as temperature and pH, on the rates of enzymatic reactions.
This lab investigation is an application of LO 4.17 and Science Practice 5.1 because you will analyze experimental data to determine how various environment conditions affect enzyme structure and function and, thus, the rate of enzyme-catalyzed reactions.
An expanded lab investigation for enzymes, involving determining the effect of pH on the action of turnip peroxidase, is available from the College Board’s ® AP Biology Investigative Labs: An Inquiry-Based Approach, Investigation 13.
Control of Metabolism Through Enzyme Regulation
It would seem ideal to have a scenario in which all of the enzymes encoded in an organism’s genome existed in abundant supply and functioned optimally under all cellular conditions, in all cells, at all times. In reality, this is far from the case. A variety of mechanisms ensure that this does not happen. Cellular needs and conditions vary from cell to cell, and change within individual cells over time. The required enzymes and energetic demands of stomach cells are different from those of fat storage cells, skin cells, blood cells, and nerve cells. Furthermore, a digestive cell works much harder to process and break down nutrients during the time that closely follows a meal compared with many hours after a meal. As these cellular demands and conditions vary, so do the amounts and functionality of different enzymes.
Since the rates of biochemical reactions are controlled by activation energy, and enzymes lower and determine activation energies for chemical reactions, the relative amounts and functioning of the variety of enzymes within a cell ultimately determine which reactions will proceed and at which rates. This determination is tightly controlled. In certain cellular environments, enzyme activity is partly controlled by environmental factors, like pH and temperature. There are other mechanisms through which cells control the activity of enzymes and determine the rates at which various biochemical reactions will occur.
Regulation of Enzymes by Molecules
Enzymes can be regulated in ways that either promote or reduce their activity. There are many different kinds of molecules that inhibit or promote enzyme function, and various mechanisms exist for doing so. In some cases of enzyme inhibition, for example, an inhibitor molecule is similar enough to a substrate that it can bind to the active site and simply block the substrate from binding. When this happens, the enzyme is inhibited through competitive inhibition , because an inhibitor molecule competes with the substrate for active site binding (Figure 6.17). On the other hand, in noncompetitive inhibition , an inhibitor molecule binds to the enzyme in a location other than the active site, called an allosteric site, but still manages to prevent substrate binding to the active site. Some inhibitor molecules bind to enzymes in a location where their binding induces a conformational change that reduces the enzyme activity as it no longer effectively catalyzes the conversion of the substrate to product.
Some inhibitor molecules bind to enzymes in a location where their binding induces a conformational change that reduces the affinity of the enzyme for its substrate. This type of inhibition is called allosteric inhibition (Figure 6.18). Most allosterically regulated enzymes are made up of more than one polypeptide, meaning that they have more than one protein subunit. When an allosteric inhibitor binds to an enzyme, all active sites on the protein subunits are changed slightly such that they bind their substrates with less efficiency. There are allosteric activators as well as inhibitors. Allosteric activators bind to locations on an enzyme away from the active site, inducing a conformational change that increases the affinity of the enzyme’s active site(s) for its substrate(s).
Drug Discovery by Looking for Inhibitors of Key Enzymes in Specific Pathways
Enzymes are key components of metabolic pathways. Understanding how enzymes work and how they can be regulated is a key principle behind the development of many of the pharmaceutical drugs (Figure 6.19) on the market today. Biologists working in this field collaborate with other scientists, usually chemists, to design drugs.
Consider statins for example—which is the name given to the class of drugs that reduces cholesterol levels. These compounds are essentially inhibitors of the enzyme HMG-CoA reductase. HMG-CoA reductase is the enzyme that synthesizes cholesterol from lipids in the body. By inhibiting this enzyme, the levels of cholesterol synthesized in the body can be reduced. Similarly, acetaminophen is an inhibitor of the enzyme cyclooxygenase. While it is effective in providing relief from fever and inflammation (pain), its mechanism of action is still not completely understood.
How are drugs developed? One of the first challenges in drug development is identifying the specific molecule that the drug is intended to target. In the case of statins, HMG-CoA reductase is the drug target. Drug targets are identified through painstaking research in the laboratory. Identifying the target alone is not sufficient scientists also need to know how the target acts inside the cell and which reactions go awry in the case of disease. Once the target and the pathway are identified, then the actual process of drug design begins. During this stage, chemists and biologists work together to design and synthesize molecules that can either block or activate a particular reaction. However, this is only the beginning: both if and when a drug prototype is successful in performing its function, then it must undergo many tests from in vitro experiments to clinical trials before it can get FDA approval to be on the market.
- a drug that increases HMG-CoA reductase levels
- a drug that reduces cyclooxygenase levels
- a drug that reduces lipid levels in the body
- a drug that blocks the action of acetaminophen
Many enzymes don’t work optimally, or even at all, unless bound to other specific non-protein helper molecules, either temporarily through ionic or hydrogen bonds or permanently through stronger covalent bonds. Two types of helper molecules are cofactors and coenzymes . Binding to these molecules promotes optimal conformation and function for their respective enzymes. Cofactors are inorganic ions such as iron (Fe++) and magnesium (Mg++). One example of an enzyme that requires a metal ion as a cofactor is the enzyme that builds DNA molecules, DNA polymerase, which requires a bound zinc ion (Zn++) to function. Coenzymes are organic helper molecules, with a basic atomic structure made up of carbon and hydrogen, which are required for enzyme action. The most common sources of coenzymes are dietary vitamins (Figure 6.20). Some vitamins are precursors to coenzymes and others act directly as coenzymes. Vitamin C is a coenzyme for multiple enzymes that take part in building the important connective tissue component, collagen. An important step in the breakdown of glucose to yield energy is catalysis by a multi-enzyme complex called pyruvate dehydrogenase. Pyruvate dehydrogenase is a complex of several enzymes that actually requires one cofactor (a magnesium ion) and five different organic coenzymes to catalyze its specific chemical reaction. Therefore, enzyme function is, in part, regulated by an abundance of various cofactors and coenzymes, which are supplied primarily by the diets of most organisms.
In eukaryotic cells, molecules such as enzymes are usually compartmentalized into different organelles. This allows for yet another level of regulation of enzyme activity. Enzymes required only for certain cellular processes can be housed separately along with their substrates, allowing for more efficient chemical reactions. Examples of this sort of enzyme regulation based on location and proximity include the enzymes involved in the latter stages of cellular respiration, which take place exclusively in the mitochondria, and the enzymes involved in the digestion of cellular debris and foreign materials, located within lysosomes.
Feedback Inhibition in Metabolic Pathways
Molecules can regulate enzyme function in many ways. A major question remains, however: What are these molecules and where do they come from? Some are cofactors and coenzymes, ions, and organic molecules, as you’ve learned. What other molecules in the cell provide enzymatic regulation, such as allosteric modulation, and competitive and noncompetitive inhibition? The answer is that a wide variety of molecules can perform these roles. Some of these molecules include pharmaceutical and non-pharmaceutical drugs, toxins, and poisons from the environment. Perhaps the most relevant sources of enzyme regulatory molecules, with respect to cellular metabolism, are the products of the cellular metabolic reactions themselves. In a most efficient and elegant way, cells have evolved to use the products of their own reactions for feedback inhibition of enzyme activity. Feedback inhibition involves the use of a reaction product to regulate its own further production (Figure 6.21). The cell responds to the abundance of specific products by slowing down production during anabolic or catabolic reactions. Such reaction products may inhibit the enzymes that catalyzed their production through the mechanisms described above.
The production of both amino acids and nucleotides is controlled through feedback inhibition. Additionally, ATP is an allosteric regulator of some of the enzymes involved in the catabolic breakdown of sugar, the process that produces ATP. In this way, when ATP is abundant, the cell can prevent its further production. Remember that ATP is an unstable molecule that can spontaneously dissociate into ADP. If too much ATP were present in a cell, much of it would go to waste. On the other hand, ADP serves as a positive allosteric regulator (an allosteric activator) for some of the same enzymes that are inhibited by ATP. Thus, when relative levels of ADP are high compared to ATP, the cell is triggered to produce more ATP through the catabolism of sugar.
Ask students which inhibition is more effective at slowing or limiting the reaction? Relate this to the examples available and discuss why these would be used in specific instances.
Have the class research antimicrobial treatments that are based on enzyme inhibition, not on the administration of traditional antibiotics.
Enzymes are not changed by the chemicals they facilitate therefore, they can be used repeatedly. Yet, how do you keep them from catalyzing reactions when you do not need or want them to react anymore? If enzymes could not be controlled, the reactions would continue until the substrates were depleted, which is not a good situation for a living organism. Competitive and noncompetitive inhibition explains the control of enzyme activity. Research several examples of both in living organisms and explain why they are necessary. Amino acid production is one useful example. Amino acids are required for protein production, but too high a level of any amino acid is toxic, so the pathways must be controlled. Use the feedback inhibition of several pathways as examples.