Cell Biology PDF 2024-2025

Cell Biology Cell Biology 2024-2025 1  Cell Biology No. Content Page no. 1 Cell Origin & Diversity 3 2 Cell Architecture 7 3 Membrane Transport 25 4 Genetic Material 32...

Cell Biology Cell Biology 2024-2025 1  Cell Biology No. Content Page no. 1 Cell Origin & Diversity 3 2 Cell Architecture 7 3 Membrane Transport 25 4 Genetic Material 32 5 Cell Cycle 38 6 Enzymes 52 7 Cell Death 57 8 References 58 2  Cell Biology Cell Origin and Diversity The discovery of cells The old theory of spontaneous generation states that cells could arise from non-cellular materials. Later, in 1959, Pasteur disproved that Spontaneous Generation and proved that some microorganisms are airborne and are responsible for the life we see. The origin of the idea that living organisms are made of cells is often traced back to observations of thin slices of plant cork cells made by Robert Hooke in 1665. According to one of the beliefs of modern biology, all living organisms have evolved from a single, common ancestral cell that lived more than three billion years ago. Because it gave rise to all the living organisms that we know of, this ancient cell is often referred to as the last universal common ancestor (or LUCA). The Cell Theory 1) All organisms are composed of one or more cells. 2) The cell is the structural unit of life. 3) Cells can arise only by division from a preexisting cell. Living cells basic properties 1) Cells Complexity and Organization: The more complex a structure, the greater the number of parts that must be in their proper place, the less tolerance of errors in the nature and interactions of the parts, and the more regulation or control that must be exerted to maintain the system. 2) Energy use: Cells expend an enormous amount of energy simply breaking down and rebuilding the macromolecules and organelles of which they are made (i.e. metabolism). This continual “turnover,” as it is called, maintains the integrity of cell components in the face of inevitable wear and tear and enables the cell to respond rapidly to changing conditions. Cell Origin & Diversity 3  Cell Biology 3) Self- regulation or robustness: In addition to requiring energy, maintaining a complex, ordered state requires constant regulation. For example, failure of a cell to regulate and correct a mistake when it duplicates its DNA may result in a mutation, or a breakdown in a cell’s growth-control safeguards can transform the cell into a cancer cell with the capability of destroying the entire organism. 4) Sensitivity to different stimuli: All living cells in different organisms respond to various stimuli but in different ways. 5) Growth and Development: All organisms grow in size, change and develop into more complex organism. 6) Reproduction: Just as individual organisms are generated by reproduction, so too are individual cells. Cells reproduce by division, a process in which the contents of a “mother” cell are distributed into two “daughter” cells. 7) Homeostasis: All organisms have stable internal conditions which must be maintained in order to remain alive. These include temperature, water content, heartbeat, and other such things. In a way, this has to do with energy use, because a certain level of energy must be kept within the body at all times. For this, obviously, humans must then ingest food on a regular basis. 8) Heredity and Genetic Program: All organisms on earth possess a genetic system that is based on the replication of a long, complex molecule called DNA. Genes are more than storage lockers for information: they constitute the blueprints for constructing cellular structures, the directions for running cellular activities, and the program for making more of themselves. The molecular structure of genes allows for changes in genetic information (mutations) that lead to variation among individuals. This mechanism allows for adaptation and evolution over time and for the distinguishing characteristics of living things. Cell Origin & Diversity 4  Cell Biology 9) Cells Carry Out a Variety of Chemical Reactions: All chemical changes that take place in cells require enzymes—molecules that greatly increase the rate at which a chemical reaction occurs. The sum total of the chemical reactions in a cell represents that cell’s metabolism. The Cell Diversity According to one of the beliefs of modern biology, all living organisms have evolved from a single, common ancestral cell that lived more than three billion years ago. Because it gave rise to all the living organisms that we know of, this ancient cell is often referred to as the last universal common ancestor (or LUCA). There were two basic classes of cells—prokaryotic and eukaryotic— distinguished by their size and the types of internal structures, or organelles, they contain (Table 1). The main difference is that the genetic material of a prokaryotic cell is present in a nucleoid: a poorly defined region of the cell that lacks a boundary membrane to separate it from the surrounding cytoplasm. In contrast, eukaryotic cells possess a nucleus: a region bounded by a complex membranous structure called the nuclear envelope (Figure 1). FIGURE 1: Cell diversity showing prokaryotic and eukaryotic cell Cell Origin & Diversity 5  Cell Biology Table 1: A Comparison between Prokaryotic and Eukaryotic Cells Nucleus Prokaryotes Eukryotes Nucleus No nucleus True Nuclear Absent Present membrane DNA Circular Linear Histones Absent Present Chromosomes No chromosomes DNA packed in chromosomes Organelles Absent Present Eukaryotes comprise all members of the plant and animal kingdoms (Figure 2 and 3, Table 2), as well as protozoans; which are exclusively unicellular and include fungi and amoebae. Table 2: Different types of Eukaryotes; Plant and Animal Cells Nucleus Plant Cells Animal Cells Size 10 to 100 micrometers 10 to 30 micrometers Energy from chloroplasts from mitochondrion Cell Wall Present (rigid, provides stability) Absent Large Central Present Absent Vacuole FIGURE 2: Plant Cell. Cell Origin & Diversity 6  Cell Biology Cell Architecture The components of an eukaryotic cell are: cell membrane, intracellular components (including cytoplasm, nucleus, endomembrane system, and cytoskeleton), as well as, surface appendages in some eukaryotic cells (Figure 3). Figure 3: Eukaryotic Animal Cell Cell Architecture 7  Cell Biology 1) Cell Membrane All cell membranes have the same basic architecture: a phospholipid bilayer in which proteins are embedded. Each cell membrane has its own set of proteins that allow it to carry out many different functions, including membrane transport, cell signaling, and connecting cells into tissues. An Overview of Cell Membrane Functions a) Compartmentalization: Cell membranes are continuous, unbroken sheets and, as such, certainly enclose compartments by separating the inside from the outside. The plasma membrane defines the cell and separates the inside from the outside. In eukaryotes, cell membranes also define intracellular organelles such as the nucleus, mitochondrion, and lysosome. b) Scaffold for biochemical activities: Because of their construction, biomembranes provide the cell with an extensive framework or scaffolding within which components can be ordered for effective interaction. c) Providing a selectively permeable barrier: Biomembranes prevent the unrestricted exchange of molecules from one side to the other. At the same time, promote the movement of select elements into and out of the enclosed living space. d) Transporting solutes: The plasma membrane contains the machinery (transport protein) for physically transporting substances from one side of the membrane to another, often from a region where the solute is present at low concentration into a region where that solute is present at much higher concentration. e) Signal transduction: The plasma membrane plays a critical role in the response of a cell to external stimuli via protein receptors that combine with specific molecules (ligands) or respond to other types of stimuli such as light or mechanical tension. Cell Architecture 8  Cell Biology f) Intercellular interaction: The plasma membrane allows cells to recognize and signal one another, to adhere when appropriate, and to exchange materials and information, via proteins within the plasma membrane that may also facilitate the interaction between extracellular materials and the intracellular cytoskeleton. g) Energy transduction (a process by which one type of energy is converted to another type): In eukaryotes, the machinery for energy conversions is contained within membranes of chloroplasts and mitochondria to transfer chemical energy from carbohydrates to ATP (not plasma membrane). A Brief History of Studies on Plasma Membrane Structure In 1935, Davson and Danielli proposed that the plasma membrane was composed of a lipid bilayer lined on both its inner and outer surface by a layer of globular proteins. The phospholipid bilayer is a two-layer, sheet-like structure in which the polar head groups of phospholipids are exposed to the aqueous media on either side and the nonpolar fatty acyl chains are in the center; the foundation for all biomembranes. This would be the thermodynamically favored arrangement, because the polar head groups of the lipids could interact with surrounding water molecules, just as the hydrophobic fatty acyl chains would be protected from contact with the aqueous environment (Figure 4a). However, Davson and Danielli model showed many drawbacks like it could not account for the semi-permeability of the cell membrane, i.e. the hydrophobic core of the lipid bilayer prevents most water-soluble substances from crossing from one side of the membrane to the other. Also a membrane with an outside layer of proteins would be an unstable structure, because proteins, like phospholipids, have both hydrophilic and hydrophobic regions (amphipathic properties). In 1972, fluid mosaic model of biomembranes was proposed in which the phospholipid bilayer remained the core of the membrane, but attention was focused on its physical state, affirming that the bilayer in some respects behaves like a two- Cell Architecture 9  Cell Biology dimensional fluid, with individual lipid molecules able to move laterally within the plane of the membrane (Figure 4b). Later, the external surface of most membrane proteins, as well as a small percentage of the phospholipids, was suggested to contain short chains of sugars (carbohydrates), making them glycoproteins and glycolipids (Figure 4c). (C) FIGURE 4: Structure of the Cell Membrane. (a) Davson and Danielli model, (b) fluid mosaic model in 1972, (c) current accepted model. Cell Architecture 10  Cell Biology The Chemical Composition of Membranes a) Membrane Proteins: Membrane proteins can be classified on the basis of their position with respect to the membrane into three categories (Figure 5): i. Integral proteins: that penetrate the lipid bilayer. Integral proteins are transmembrane proteins; that is, they pass entirely through the lipid bilayer and thus have domains that protrude from both the extracellular and cytoplasmic sides of the membrane, or unilateral proteins; that reach only part way across the membrane. ii. Peripheral proteins: that are located entirely outside of the lipid bilayer, on either the cytoplasmic or extracellular side, yet are associated with the surface of the membrane by non-covalent bonds. iii. Lipid-anchored proteins: that are located outside the lipid bilayer, on either the extracellular or cytoplasmic surface, but are covalently linked to a lipid molecule that is situated within the bilayer. (a) (b) (c) (d) FIGURE 5: Three classes of membrane proteins. (a) Unilateral integral protein, (b) Peripheral protein, (c) Bilateral integral protein, (d) Lipid-anchored protein. Cell Architecture 11  Cell Biology b) Membrane Lipids: (Figure 6) Membranes contain a wide diversity of lipids, all of which are amphipathic; that is, they contain both hydrophilic and hydrophobic regions. There are three main types of membrane lipids: phosphoglycerides, sphingolipids, and cholesterol. i. Phosphoglycerides: Most membrane lipids contain a phosphate group, which makes them phospholipids. Because most membrane phospholipids are built on a glycerol backbone, they are called phosphoglycerides. Only two of the hydroxyl groups of the glycerol are esterified to fatty acids; the third is esterified to a hydrophilic phosphate group. Without any additional substitutions beyond the phosphate and the two fatty acyl chains, the molecule is called phosphatidic acid, which is virtually absent in most membranes. Instead, membrane phosphoglycerides have an additional group linked to the phosphate, most commonly either choline (forming phosphatidylcholine, PC), ethanolamine (forming phosphatidylethanolamine, PE), serine (forming phosphatidylserine, PS), or inositol (forming phosphatidylinositol, PI). Each of these groups is small and hydrophilic and, together with the negatively charged phosphate to which it is attached, forms a highly water-soluble domain at one end of the molecule, called the head group. In contrast, the fatty acyl chains are hydrophobic, unbranched hydrocarbons approximately 16 to 22 carbons in length. A membrane fatty acid may be fully saturated (i.e., lack double bonds), monounsaturated (i.e., possess one double bond), or polyunsaturated. Phosphoglycerides often contain one unsaturated and one saturated fatty acyl chain. With nonpolar fatty acid chains at one end of the molecule and a polar head group at the other end, all of the phosphoglycerides exhibit a distinct amphipathic character. ii. Sphingolipids: A less abundant class of membrane lipids, called sphingolipids, are derivatives of sphingosine, an amino alcohol that contains a long hydrocarbon chain. Sphingolipids consist of sphingosine Cell Architecture 12  Cell Biology linked to a fatty acid by its amino group. The various sphingosine-based lipids have additional groups esterified to the terminal alcohol of the sphingosine moiety. If the substitution is phosphorylcholine, the molecule is sphingomyelin, which is the only phospholipid of the membrane that is not built with a glycerol backbone. If the substitution is a carbohydrate, the molecule is a glycolipid. Since all sphingolipids have two long, hydrophobic hydrocarbon chains at one end and a hydrophilic region at the other, they are amphipathic and basically similar to phosphoglycerides. iii. Sterols: Cholesterol constitutes up to 50 % of the lipid molecules in the plasma membrane of animal cells. Cholesterol molecules are oriented with their small hydrophilic hydroxyl group toward the membrane surface and the remainder of the molecule embedded in the lipid bilayer. FIGURE 6: Three classes of membrane lipids. (a) phosphoglycerides, (b) sphingolipids, (c) cholesterol. Cell Architecture 13  Cell Biology 2) Intracellular Components a) Cytoplasm  The cytosol is the organelle-free internal fluid of the cell, where a portion of cell metabolism occurs here.  The cytosol contains water, dissolved ions, small molecules, and proteins.  In eukaryotes, the cytosol contains the cell organelles; this is collectively called cytoplasm. b) The Nucleus (Figure 7)  The nucleus is found in all animal cells except erythrocytes. Nucleus, the largest organelle in animal cells, is surrounded by two membranes, each one a phospholipid bilayer containing many different types of proteins. The inner nuclear membrane defines the nucleus itself. In most cells, the outer nuclear membrane is continuous with the endoplasmic reticulum, and the space between the inner and outer nuclear membranes is continuous with the lumen of the endoplasmic reticulum.  The two nuclear membranes appear to fuse at nuclear pore complexes; ring-like structures composed of specific membrane proteins through which material moves between the nucleus and the cytosol.  Intermediate-filament proteins called lamins form a two-dimensional network, called the nuclear lamina, along the inner surface of the inner membrane, giving it shape and rigidity. The breakdown of the lamina occurs early in cell division.  In a growing or differentiating cell, the nucleus is metabolically active, as it is the site of DNA replication and the synthesis of ribosomal RNA, mRNA, and a large variety of noncoding RNAs. Inside the nucleus one can often see a dense subcompartment, termed the nucleolus, where ribosomal RNA is synthesized and ribosomes are assembled. Cell Architecture 14  Cell Biology FIGURE 7: Eukaryotic cell showing Structure of the Nucleus. c) Endomembrane System Most eukaryotic cells contain extensive internal membranes that enclose specific subcellular compartments, termed organelles, to compartmentalize different molecules and allow cell to remain organized. Organelles are connected directly by continuous membranes or indirectly by transport vesicles. Here we review different organelles and their functions: i. Endoplasmic Reticulum (Figure 8)  Generally the largest membrane in an eukaryotic cell encloses the organelle termed the endoplasmic reticulum (ER)—an extensive network of closed, flattened membrane-bounded sacs called cisternae.  It accounts for more than half the total membrane in many eukaryotic cells. Cell Architecture 15  Cell Biology  ER has a number of functions in the cell but is particularly important in the synthesis of lipids, secreted proteins, and many types of membrane proteins.  The smooth ER is smooth because it lacks ribosomes; it is the site of synthesis of fatty acids and phospholipids.  In contrast, the cytosolic side of the rough ER is studded with ribosomes; these ribosomes synthesize certain membrane and organelle proteins and virtually all proteins that are to be secreted from the cell.  As a growing polypeptide emerges from a ribosome, it passes through the rough ER membrane with the help of specific transport proteins that are embedded in the membrane. Newly made membrane proteins remain associated with the rough ER membrane, and proteins to be secreted accumulate in the lumen, the aqueous interior of the organelle.  Several minutes after proteins are synthesized in the rough ER, most of them leave the organelle within small membrane-bounded transport vesicles. These vesicles, which bud from regions of the rough ER not coated with ribosomes, carry the proteins to another membrane-bounded organelle, the Golgi complex. FIGURE 8: Rough and smooth Endoplasmic reticulum. Cell Architecture 16  Cell Biology ii. Golgi Apparatus (Figure 9)  Secreted and membrane proteins undergo a series of enzyme–catalyzed chemical modifications in the Golgi complex that are essential for these proteins to function normally.  After proteins to be secreted and membrane proteins are modified in the Golgi complex, they are transported out of the complex by a second set of vesicles, which bud from one side of the Golgi complex. FIGURE 9: Structure of Golgi bodies. iii. Endosomes  Although transport proteins in the plasma membrane mediate the movement of ions and small molecules into the cell across the lipid bilayer, proteins and some other soluble macromolecules in the extracellular milieu are internalized by endocytosis.  In this process, a segment of the plasma membrane invaginates into a coated pit, whose cytosolic face is lined by a specific set of proteins that cause vesicles to form. The pit pinches from the membrane into a small membrane-bounded vesicle that contains the extracellular material.  The vesicle is delivered to and fuses with an endosome, a sorting station of membrane-limited tubules and vesicles (Figure 10). From this compartment, some membrane proteins are recycled back to the plasma membrane; other Cell Architecture 17  Cell Biology membrane proteins are transported in vesicles that eventually fuse with lysosomes for degradation. iv. Lysosomes  Lysosomes provide an excellent example of the ability of intracellular membranes to form closed compartments in which the composition of the lumen (the aqueous interior of the compartment) differs substantially from that of the surrounding cytosol.  Found exclusively in animal cells, lysosomes are responsible for degrading many components that have become obsolete for the cell or organism.  The process by which an aged organelle is degraded in a lysosome is called autophagy (“eating oneself”).  Materials taken into a cell by endocytosis or phagocytosis may also be degraded in lysosomes (Figure 10). In phagocytosis, large, insoluble particles (e.g., bacteria) are enveloped by the plasma membrane and internalized. Endosomes and other cellular structures deliver materials to lysosomes.  Lysosome contain a group of enzymes collectively termed acid lysosomal hydrolases, that degrade polymers into their monomeric subunits, and work most efficiently at acidic pH values that helps to denature proteins making them accessible to the action of the lysosomal hydrolases.  These enzymes (e.g. proteases, nucleases, phosphatases, and enzymes for degrade complex polysaccharides and glycolipids) are less active at the neutral pH of cells and most extracellular fluids. Thus if a lysosome releases its enzymes into the cytosol, where the pH is between 7.0 and 7.3, they cause little degradation of cytosolic components.  Cytosolic and nuclear proteins generally are not degraded in lysosomes, but rather in proteasomes, large multi-protein complexes in the cytosol. Cell Architecture 18  Cell Biology FIGURE 10: Schematic overview of three pathway by which materials are moved to lysosomes. v. Peroxisomes (Microbodies)  A class of roughly spherical organelles containing several oxidases and catalases (0.2–1.0 μm in diameter) that is present in all animal cells, except erythrocytes.  Oxidase enzymes: use molecular oxygen to oxidize organic substances (lipids) and in the process hydrogen peroxide is formed (H2O2, a corrosive substance). Catalase enzymes: degrades hydrogen peroxide to yield water and oxygen.  They resemble a lysosome, however, they are not formed in the Golgi complex. They are self-replicating, like the mitochondria.  They function to rid the body of toxic substances like hydrogen peroxide, or other metabolites. vi. Mitochondria (Energy-related organelle) (Figure 11)  Are the principal sites of ATP Production in aerobic cells. Most eukaryotic cells contain many mitochondria, which occupy up to 25 % of the volume of the cytoplasm. Cell Architecture 19  Cell Biology  The two membranes that bound a mitochondrion differ in composition and function. The outer mitochondrial membrane contains proteins that allow many molecules to move from the cytosol to the intermembrane space between the inner and outer membrane. The inner mitochondrial membrane, which is much less permeable, is about 20 % lipid and 80 % protein—a proportion of protein that is higher than those in other cellular membranes. The surface area of the inner membrane is greatly increased by a large number of infoldings, or cristae, that protrude into the matrix, or central aqueous space.  Mitochondria contain small DNA molecules that encode a small number of mitochondrial proteins; the majority of mitochondrial proteins are encoded by nuclear DNA.  Mitochondria can be regarded as the “power plants” of the cell: - In eukaryotic cells, the initial stages of glucose degradation take place in the cytosol, where 2 ATP molecules per glucose molecule are generated. The terminal stages of oxidation and ATP synthesis are carried out by enzymes in the mitochondrial matrix and inner membrane; as many as 28 ATP molecules per glucose molecule are generated in mitochondria. - Similarly, virtually all the ATP formed in the oxidation of fatty acids to carbon dioxide is generated in mitochondria. FIGURE 11: Structure of mitochondrion. Cell Architecture 20  Cell Biology d) Cytoskeleton (Figure 12)  Three types of filaments make up the animal-cell cytoskeleton: microfilaments, microtubules, and intermediate filaments. Each of the three types of cytoskeletal filaments is a polymer of protein subunits held together by weak, non-covalent bonds. This type of construction lends itself to rapid assembly and disassembly, which is dependent on complex cellular regulation. i. Microtubules: are stiff, long, hollow, unbranched tubes composed of subunits of the protein tubulin, that can exist as a single structure, or in bundled arrangements such as those seen in specialized cell-surface structures (e.g. cilia and flagella), or in cell division (the fibers of the mitotic spindle and cytokinesis). ii. Microfilaments: are solid, thinner structures, often organized into a branching network, and composed of the protein actin to form flexible and dynamic structures. iii. Intermediate filaments: are strong, flexible, rope-like fibers composed of a variety of related proteins that provide mechanical strength to cells that are subjected to physical stress (including neurons, muscle cells, and the epithelial cells that line the body’s cavities).  Function: - Maintain cell shape (by Microfilaments). - An internal framework responsible for fixing and positioning the various organelles within the interior of the cell (by Intermediate filaments). - A network of tracks that direct the movement of materials and organelles within cells (by Microtubules). - The force-generating apparatus that moves cells from one place to another (i.e. locomotion, by Microtubules) (Figure 13). - An essential component of the cell’s division machinery. Cytoskeletal elements make up the apparatus responsible for separating the Cell Architecture 21  Cell Biology chromosomes during mitosis and meiosis (mitotic spindle) and for splitting the parent cell into two daughter cells during cytokinesis (by Microtubules). Made up of tubulin Make up microfilaments FIGURE 12: Different types of animal cytoskeleton. FIGURE 13: Overview of the cytoskeleton of a migrating cell, such as a fibroblast or a macrophage, having morphologically distinct domains, with a leading edge at the front. Cell Architecture 22  Cell Biology 3) Surface Appendages  Cilia and flagella are extensions of the plasma membrane (Figure 14).  They contain a bundle of microtubules that gives them shape and, together with motor proteins, allows them to beat rhythmically.  They push materials across epithelial surfaces, enable sperm to swim, and push eggs through the oviduct.  Flagella: is a long hair-like motile organelle that project from the surface of a variety of eukaryotic cells in few numbers showing rolling motion that generates force in the same direction as the flagellum’s axis.  Cilia: are short hair-like motile organelles that project from the surface of a variety of eukaryotic cells. Cilia tend to occur in large numbers on a cell’s surface with alternating power generating force in a direction perpendicular to the cilium’s axis. (a) (b) FIGURE 14: Surface appendages of (a) ciliated epithelium lining a mammalian trachea viewed in a scanning electron microscope (SEM), (b) Flagella of sperms. Cell Architecture 23  Cell Biology Extracellular matrix (ECM) (Figure 15) An organized network of extracellular materials that is present beyond the plasma membrane in the extracellular space. In animals, ECM has multiple functions: - The ECM helps organize cells into tissues and coordinates their cellular functions by activating intracellular signaling pathways that control cell growth, proliferation, and gene expression. - The ECM can directly influence cell and tissue structure and function. - In addition, it can serve as a repository for inactive or inaccessible signaling molecules (e.g., growth factors) that are released to function when the ECM is disassembled or remodeled by hydrolyases, such as proteases. FIGURE 15: Extracellular Matrix. Cell Architecture 24  Cell Biology Membranes Transport Cellular Membrane is a selectively permeable barrier All cellular membranes (plasma and organelle membranes) must serve not only as barriers, but also as conduits, selectively transporting molecules and ions from one side of the membrane to the other maintaining a constant internal environment. For example, the plasma membrane is a selectively permeable barrier between the cell and the extracellular environment ensuring that essential molecules such as ions, glucose, amino acids, and lipids readily enter the cell, metabolic intermediates remain in the cell, and waste compounds leave the cell. Transport through a cell membrane can be: 1) Passive (no energy required) 2) Active (requires energy) 3) Vesicle transport 1) Passive Diffusion a) Simple Diffusion Gases, such as O2 and CO2, and few small, uncharged polar molecules, such as urea and ethanol, can readily move by passive (simple) diffusion across an artificial membrane composed of pure phospholipid or of phospholipid and cholesterol (Figure 16). No metabolic energy is expended because movement is from a high to a low concentration of the molecule, down its chemical concentration gradient. Such transport reactions are spontaneous. Membrane Transport 25  Cell Biology FIGURE 16: Relative permeability of a pure phospholipid bilayer to various molecules. A bilayer is permeable to small hydrophobic molecules and small uncharged polar molecules, slightly permeable to water and urea, and essentially impermeable to ions and to large polar molecules. Factors Affecting Diffusion Rates: Diffusion rate of any substance across a pure phospholipid bilayer is proportional to: 1) Concentration gradient: difference between high and low concentration 2) Molecule Size: Smaller are faster. 3) Molecule Hydrophobicity: The first and rate-limiting step in transport by passive diffusion is movement of a molecule from the aqueous solution Membrane Transport 26  Cell Biology into the hydrophobic interior of the phospholipid bilayer, which resembles oil in its chemical properties. So, the more hydrophobic a molecule is, the faster it diffuses across a pure phospholipid bilayer. 4) Membrane potential: affects transport of charged molecules. The electric potential (voltage) that exists across most cellular membranes results from a small imbalance in the concentration of positively and negatively charged ions on the two sides of the membrane. The combination of the forces of concentration gradient and the membrane potential, called the electrochemical gradient, determines the energetically favorable direction of transport of a charged molecule across a membrane. b) Facilitated Diffusion via Transport Proteins Movement of a wide variety of molecules and ions across cellular membranes is mediated by selective membrane transport proteins embedded in the phospholipid bilayer. Transport proteins are thought to allow movement of hydrophilic substances without their coming into contact with the hydrophobic interior of the membrane. There are two types of transport proteins: (Figure 17) 1) Channel proteins transport water or specific types of ions and small hydrophilic molecules down their concentration or electric potential gradients through a hydrophilic “tube” across the membrane. 2) Transporters (also called carriers) There are two types of transporters: i- Uniporters: transport a single type of molecule down its concentration gradient via facilitated diffusion (e.g. glucose and amino acids). ii- Cotransporters (antiporters and symporters) transport two or more different solutes simultaneously. They couple the movement of one type of ion or molecule against its concentration gradient with the movement of one or more different ions down its concentration gradient. Membrane Transport 27  Cell Biology FIGURE 17: Different membrane transport proteins. Because different cell and organelle types require different mixtures of low molecular weight compounds, the cell membrane of each type contains a specific set of transport proteins that allow only certain ions and molecules to cross, creating a different internal environment. Several features distinguish uniport from simple diffusion: 1) The rate of substrate movement by uniporters is far higher than simple diffusion through a pure phospholipid bilayer. 2) Because the transported molecule never enters the hydrophobic core of the phospholipid bilayer, its hydrophobicity is irrelevant. 3) Transport occurs via a limited number of uniporter molecules. Consequently, there is a maximum transport rate, Vmax, which depends on the number of uniporters in the membrane. 4) Transport is reversible, and the direction of transport will change if the direction of the concentration gradient changes. 5) Transport is specific. Each uniporter transports only a single type of molecule or a single group of closely related molecules. A measure of the affinity of a transporter for its substrate is the Michaelis constant, Km, which is the concentration of substrate at which transport is half Vmax. Membrane Transport 28  Cell Biology 2) Active Transport ATP-powered pumps (or simply pumps) are ATPases that use the energy of ATP hydrolysis to move ions or small molecules across a membrane against a chemical concentration gradient, an electric potential, or both. Mechanism of Na-K pump: (Figure 18) 1) The pump, with bound ATP, binds 3 intracellular Na+ ions. 2) ATP is hydrolyzed, leading to phosphorylation of the pump and subsequent release of ADP. 3) A conformational change in the pump exposes the Na+ ions to the outside. In other words, the phosphorylated form of the pump has a low affinity for Na+ ions, so they are released. 4) The pump binds 2 extracellular K+ ions. This causes the dephosphorylation of the pump, reverting it to its previous conformational state, transporting the K+ ions into the cell. 5) The unphosphorylated form of the pump has a higher affinity for Na+ ions than K+ ions, so the two bound K+ ions are released inside the cell. ATP binds, and the process starts again. Membrane Transport 29  Cell Biology FIGURE 18: Sodium-Potassium Exchange Pump. 3) Vesicular Transport How are cells able to take in materials that are too large to penetrate this membrane regardless of its permeability properties? And how are proteins that reside in the plasma membrane recycled to the cell’s internal compartments? Both of these requirements are met by the endocytic pathway, in which segments of the plasma membrane invaginate to form cytoplasmic vesicles that are transported into the cell interior. The uptake of molecules or liquid is called receptor-mediated or fluid-phase endocytosis respectively, and the uptake of large particles is called phagocytosis. Membrane Transport 30  Cell Biology Phagocytosis (Figure 19 a) A few cell types (e.g., macrophages) can take up whole bacteria and other large particles by phagocytosis, a nonselective actin-mediated process in which extensions of the plasma membrane (pseudopodia) envelop the ingested material, forming large vesicles called phagosomes. In most animals, phagocytosis is a protective mechanism rather than a mode of feeding. Once inside the phagocyte, microorganisms may be killed by lysosomal enzymes or by oxygen free radicals generated within the lumen of the phagosome. Endocytosis (Figure 19 b) Endocytosis can be divided broadly into two categories: 1) Pinocytosis (also known as bulk-phase endocytosis) is the nonspecific uptake of small droplets of extracellular fluids. Any molecules, large or small, that is present in the enclosed fluid also gain entry into the cell. 2) Receptor-mediated endocytosis (RME), in contrast, brings about the uptake of specific extracellular macromolecules (ligands) following their binding to receptors on the external surface of the plasma membrane. This allows for the selective and efficient uptake of macromolecules that may be present at relatively low concentrations in the extracellular fluid. Cells have receptors for the uptake of many different types of ligands, including hormones, growth factors, enzymes, and blood-borne proteins carrying certain nutrients. The plasma-membrane region containing the receptor- ligand complex buds inward to form a pit and then pinches off, becoming a transport vesicle. (a) (b) FIGURE 19: Vesicular transport. (a)phagocytosis, (b) Receptor-mediated endocytosis. Membrane Transport 31  Cell Biology The Genetic Material Genome Packaging An average human cell contains totally about 6.4 billion base pairs of DNA divided among 46 chromosomes (the value for a diploid number of chromosomes). Each chromosome contains a single, continuous double-stranded DNA molecule; the larger the chromosome, the more DNA it contains. The largest human chromosome contains about 0.3 billion base pairs. Given that each base pair is 0.34 nm in length, 0.3 billion base pairs would constitute a DNA molecule from just one chromosome of greater than 10 cm long. How is it possible to fit all 46 chromosomes into a nucleus that is only 10 µm (1 x 10-5 m) in diameter and, at the same time, maintain the DNA in a state that is accessible to enzymes and regulatory proteins? Just as important, how is the single DNA molecule of each chromosome organized so that it does not become hopelessly tangled with other chromosomes? The answers lie in the remarkable manner in which a DNA molecule is packaged in eukaryotic cells (Figure 20). FIGURE 20: Levels of organization of chromatin. Genetic Material 32  Cell Biology During interphase, when cells are not dividing, the genetic material exists as a nucleoprotein complex called chromatin, which is uncondensed, dispersed through much of the nucleus (Figure 21A). During mitosis, further folding and compaction of chromatin produces the visible condensed metaphase chromosomes (Figure 21B). The most abundant proteins associated with eukaryotic DNA are histones, a family of small, basic proteins present in all eukaryotic nuclei. A B FIGURE 21: (A) chromatin, (B) Chromosome. 1- Structure of Nucleosomes. Isolated chromatin resembles “beads on a string”. In this extended form, the string is composed of free DNA called “linker” DNA connecting the bead-like structures termed nucleosomes. Each nucleosome contains a nucleosome core particle consisting of 146 base pairs of supercoiled DNA wrapped almost twice around a disk-shaped complex of eight histone molecules (octamer) (Figure 22) like thread around a spool. Histones are a remarkable group of small proteins that possess an unusually high content of the basic amino acids arginine and lysine. Histones are divided into five classes, which can be distinguished by their Genetic Material 33  Cell Biology arginine/lysine ratio. The amino acid sequences of histones have undergone very little change over long periods of evolutionary time between distantly related species. The DNA component of nucleosomes is much less susceptible to nuclease digestion than is the linker DNA between them. If nuclease treatment is carefully controlled, all the linker DNA can be digested, releasing individual nucleosomes with their DNA component. FIGURE 22: Nucleosomal organization of chromatin. Why are histones highly conserved? - One reason is the positively charged histones interact with the negatively charged backbone of the DNA molecule, which is identical in all organisms. - In addition, nearly all of the amino acids in a histone molecule are engaged in an interaction with another molecule, either DNA or another histone. As a result, very few amino acids in a histone can be replaced with other amino acids without severely affecting the function of the protein. Genetic Material 34  Cell Biology 2- Structure of the 30-nm Fiber Nucleosomes are folded in a helical conformation form (Figure 23). The chromatin in chromosomal regions that are not being transcribed or replicated exists predominantly in the condensed 30-nm fiber form. FIGURE 23: 30-nm Fiber. Non-histone proteins Other low-abundance non-histone proteins associated with chromatin regulate DNA replication during the eukaryotic cell cycle. Although histones are the predominant proteins in chromosomes, histone- depleted metaphase chromosomes reveal long loops of DNA anchored to a chromosome scaffold composed of non-histone proteins. This scaffold has the shape of the metaphase chromosome and persists even when the DNA is digested by nucleases (Figure 24). Non-histone Proteins Provide a Structural Scaffold for Long Chromatin Loops. Genes are located primarily within chromatin loops, which are attached at their bases to the chromosome scaffold. Genetic Material 35  Cell Biology FIGURE 24: Non-histone Proteins Providing a Structural Scaffold for Chromatin loops. Types of Chromatin After mitosis has been completed, most of the chromatin in highly compacted mitotic chromosomes returns to its diffuse interphase condition, called lightly packed euchromatin (the only form of chromatin present in prokaryotes). However, approximately 10% of the chromatin, generally remains in a condensed, compacted form throughout interphase. This compacted, densely stained chromatin is typically concentrated near the periphery of the nucleus. Chromatin that remains compacted during interphase is called heterochromatin (present only in eukaryotes) to distinguish it from euchromatin, which returns to a dispersed, uncondensed, active state. Heterochromatin is divided into two classes: 1- Constitutive heterochromatin remains in the compacted state in all cells at all times and, thus, represents DNA that is permanently silenced. 2- Facultative heterochromatin is chromatin that has been specifically inactivated during certain stages of an organism’s life or in certain types of differentiated cells. Genetic Material 36  Cell Biology 3- Structure of Metaphase Chromosomes (Figure 25) Centromere region of chromosome Kinetochore Kinetochore microtubules Sister Chromatids FIGURE 25: Metaphase chromosome Chromosomes are seen to be composed of two chromatids attached together at the centromere. The centromere site is marked by a distinct constriction in the chromosome. The centromere contains highly repetitive DNA sequences and a protein- containing structure called the kinetochore. Kinetochore serves as a site for the attachment of spindle microtubules that move and separate the chromosomes during cell division. Genetic Material 37  Cell Biology The Cell Cycle Phases of Cell Cycle (Interphase and M phase) The term cell cycle refers to the ordered series of events that lead to cell division and the production of two daughter cells, each containing chromosomes identical to those of the parent cell. Two main molecular processes take place during the cell cycle, with resting intervals in between: during the S phase of the cycle, each parent chromosome is duplicated to form two identical sister chromatids; and in mitosis, the resulting sister chromatids are distributed to each daughter cell (Figure 26). FIGURE 26: Eukaryotic cell cycle Cell Cycle 38  Cell Biology 1- Interphase (Growth Phase): is the period between cell divisions, when the cell grows and engages in diverse metabolic activities. Interphase may extend for days, weeks, or longer, depending on the cell type and the conditions. Interphase is divided into 3 phases (Figure 27): a) G1 (first gap) phase: the period in which cycling (replicating) somatic cells grow in size and synthesize the RNAs and proteins required for DNA synthesis. When cells have reached the appropriate size and have synthesized the required proteins, they enter the cell cycle, by traversing a point in G1 known as the restriction point in mammals. b) S (synthesis) phase: is the period in which cells actively replicate their chromosomes. S phase is also the period when the cell synthesizes the additional histones that will be needed as the cell doubles the number of nucleosomes in its chromosomes. Once the individual chromosomes have been duplicated, they are held together by proteins called cohesins. Also, the centrosomes; a cytoplasm special microtubule-organizing structure containing two centrioles situated at right angles to one another, is duplicated. Once the restriction point has been crossed, cells are committed to cell division, and the first step toward successful cell division is the entry into this S phase. c) G2 (second gap) phase: the resting period between the completion of nuclear DNA replication and mitosis. FIGURE 27: Interphase Cell Cycle 39  Cell Biology 2- M Phase (Cell Division) is the period when the contents of a cell are actually divided, producing two cells. It usually lasts only an hour or so in mammalian cells (Figure 28). Once S phase has been completed and the entire genome has been duplicated, the pairs of duplicated DNA chromosomes—the sister chromatids—are segregated to the future daughter cells. This process requires not only the formation of the apparatus that facilitates this segregation—the mitotic spindle—but essentially a complete remodeling of the cell. Mitosis is a stage of the cell cycle when the cell devotes virtually all of its energy to a single activity—chromosome segregation. As a result, most metabolic activities of the cell, including transcription and translation, are curtailed during mitosis, and the cell becomes relatively unresponsive to external stimuli. FIGURE 28: M-phase Cell Cycle 40  Cell Biology a) Mitotic phase: is a process of nuclear division in which the replicated DNA molecules of each chromosome are faithfully segregated into two nuclei. Mitosis is divided into five stages (Figure 28, 29, 30): i- Prophase: In general, chromosomal material condenses to form compact mitotic chromosomes. Cytoskeleton is disassembled, and mitotic spindle is assembled. Different organelles and nuclear envelope disappear (last step). - Breakdown of the interphase microtubules array occurs, as the duplicated centrosomes become more active in microtubule nucleation. This activity provides two sites of assembly for dynamic microtubules, forming the mitotic asters (sunburst arrangement). - Additionally, the dynamics of the growing microtubules themselves increase. The two asters are then moved to opposite sides of the nucleus. The separated centrosomes will become the two poles of the mitotic spindle; the microtubule-based structure that separates chromosomes (Figure 29). FIGURE 29: Mitotic spindle of an animal cell. Cell Cycle 41  Cell Biology - In addition, endocytosis and exocytosis are ceased, and the microfilaments are generally rearranged to give rise to a rounded cell. - In the nucleus: the nucleolus breaks down and chromosomes begin to condense. The cohesins proteins, holding together each pair of duplicated chromosomes, or sister chromatids as they are called at this stage (each is a single DNA complex), are degraded except at the centromeric region, where the two sister chromatids remain linked by intact cohesins (Figure 18-37b). Also during prophase, specialized structures called kinetochores, which will become sites of microtubule attachment, assemble at the centromeric region of each sister chromatid. Each chromatid has a kinetochore, so a sister chromatid pair has two kinetochores. ii- Prometaphase: Chromosomes are captured and oriented into position at the center of the cell by means of microtubules. As the microtubules of the mitotic spindle penetrate into the central region of the cell, the free ends of the microtubules are seen to grow and shrink in a dynamic fashion, as if they were “searching” for a chromosome. Each kinetochore on the sister chromatid pair typically makes initial contact with the microtubule. Regardless of how it occurs, the two sister chromatids of each mitotic chromosome ultimately become connected by their kinetochores to microtubules that extend from opposite poles, then chromosomes are moved to spindle equator by a process called congression toward the center of the mitotic spindle, midway between the poles. iii- Metaphase: Chromosomes are aligned on the metaphase plate. The spindle assembly checkpoint pathway monitors unattached kinetochores and delays anaphase until all chromosomes are attached. iv- Anaphase: Centromeres split, and chromatids separate so that each chromatid can be pulled to its respective spindle pole by the microtubules Cell Cycle 42  Cell Biology attached to its kinetochore (anaphase a). Spindle poles move farther apart (anaphase b). v- Telophase: Chromosomes cluster at opposite spindle poles. Chromosomes become dispersed. Nuclear envelope assembles around chromosome clusters. Golgi complex and endoplasmic reticulum reform. FIGURE 30: Five stages of mitosis in an animal cell b) Cytokinesis phase: is a process by which a dividing cell splits in two, partitioning the cytoplasm into two cellular packages and producing two daughter cells, via assembling a microfilament-based contractile ring attached to the plasma membrane that will eventually contract and pinch the cell into two cellular packages (Figure 28). The first hint of cytokinesis in most animal cells appears during anaphase as an indentation of the cell surface in a narrow band around the cell. As time progresses, the indentation deepens to form a furrow that moves inward toward the center of the cell. The plane of the furrow lies in the same plane previously occupied by the chromosomes of the metaphase plate, so that the two sets of chromosomes are ultimately partitioned into different cells (Figure 31). Cell Cycle 43  Cell Biology As one cell becomes two cells, additional plasma membrane is delivered to the cell surface via cytoplasmic vesicles that fuse with the advancing to the plasma membrane that will eventually contract and pinch the cell into two. Two aspects of the contractile ring are essential to its function. First, it has to be appropriately placed in the cell. It is known that this placement is determined by a signal provided by the spindle, so that the ring forms equidistant between the two spindle poles. Second, the timing of its contraction: if it were to contract before all chromosomes had moved to their respective poles, disastrous genetic consequences would ensue. 100µm Cleavage furrow Contractile ring of microfilaments FIGURE 31: Cytokinesis phase of mitosis. Cell Cycle 44  Cell Biology Meiosis: A Special Type of Cell Division In nearly all diploid eukaryotes, meiosis generates haploid germ cells (eggs and sperms), which can then fuse with a germ cell from another individual to generate a diploid zygote that develops into a new individual. Meiosis is a fundamental aspect of the biology and evolution of all eukaryotes because it results in the re- assortment of the chromosome sets received from an individual’s two parents. Chromosome re-assortment and homologous recombination between parent DNA molecules during meiosis guarantee that each haploid germ cell generated will receive a unique combination of alleles that is distinct from each parent as well as from every other haploid (1n) germ cell formed. The mechanisms of meiosis are analogous to those of mitosis, however, several key features of meiosis allow this process to generate genetically diverse haploid cells as summarized in Figure (32). In the mitotic cell cycle, each S phase is followed by chromosome segregation and cell division. In contrast, during meiotic cell division, one round of DNA replication is followed by two consecutive chromosome segregation phases. This process leads to the formation of haploid, rather than diploid, daughter cells. During the two divisions, maternal and paternal chromosomes are shuffled and divided so that the daughter cells are different in genetic makeup from the parent cell. In this section, we discuss the similarities between mitosis and meiosis as well as the meiosis-specific mechanisms that transform the canonical mitotic cell cycle machinery so that it brings about the unusual cell division that leads to the formation of haploid daughter cells. Cell Cycle 45  Cell Biology FIGURE 32: Comparison of the main features of mitosis and meiosis. As shown in Figure (33), meiosis shows 2 stages; meiosis I and meiosis II. Step 1: All chromosomes are replicated during S phase before the first meiotic division, giving a 4n chromosomal complement. Sister chromatids are formed. Step 2: As chromosomes condense during the first meiotic prophase, the replicated homologs pair and undergo homologous recombination, leading to at least one crossover event. At metaphase I, both chromatids of one chromosome associate with microtubules emanating from one spindle pole. Step 3: During anaphase I, the homologous chromosomes, each consisting of two chromatids, are pulled to opposite spindle poles. Cell Cycle 46  Cell Biology Step 4: Cytokinesis yields two daughter cells (now 2n), which enter meiosis II without undergoing DNA replication. At metaphase of meiosis II, sister chromatids associate with spindle microtubules from opposite spindle poles, as in mitosis. Steps 5 and 6: Segregation of sister chromatids to opposite spindle poles during anaphase of meiosis II, followed by cytokinesis, generates haploid gametes. Metaphase I Meiosis I Anaphase I Meiosis II Anaphase II FIGURE 33: Meiosis. Cell Cycle 47  Cell Biology a) Meiosis I: (Figure 33, 36) i- Prophase I: It occupies more than 90% of the time required for meiosis, during which the chromosomes become compacted, and compose paired chromatids. A process of chromosome pairing occurs that is called synapsis (association of homologues with one another), and is an interesting event with many important unanswered questions. Chromosome synapsis is accompanied by the formation of a complex structure called the synaptonemal complex (SC); a ladder-like structure with transverse protein filaments connecting the two lateral elements, binding together the chromatin of the sister chromatids. The complex formed by a pair of synapsed homologous chromosomes is called a bivalent or a tetrad and are held closely together along their length by SC. A number of electron-dense bodies, namely recombination nodules correspond to the sites where crossing-over between interacting chromatids is taking place, contain the enzymatic machinery that facilitates genetic recombination, are seen within the center of the SC. Then the dissolution of the SC occurs leaving the chromosomes attached to one another at specific points by X-shaped structures, termed chiasmata (singular chiasma) (Figure 34). Chiasmata are located at sites on the chromosomes where crossing-over between DNA molecules from the two chromosomes had previously occurred. Chiasmata are formed by covalent junctions between a chromatid from one homologue and a non-sister chromatid from the other homologue. Later, the meiotic spindle is assembled and the chromosomes are prepared for separation. The nucleolus disappears, the nuclear envelope breaks down, and the tetrads move to the metaphase plate. Chiasmata (crossing over site) FIGURE 34: Crossing over. Cell Cycle 48  Cell Biology ii- Metaphase I: The two homologous chromosomes of each bivalent are connected to the spindle fibers from opposite poles. In contrast, sister chromatids are connected to microtubules from the same spindle pole, which is made possible by the side-by-side arrangement of their kinetochores (Figure 35a). The orientation of the maternal and paternal chromosomes of each bivalent on the metaphase I plate is random; the maternal member of a particular bivalent has an equal likelihood of facing either pole. Consequently, when homologous chromosomes separate during anaphase I, each pole receives a random assortment of maternal and paternal chromosomes (Figure 35b). iii- Anaphase I: Separation of homologous chromosomes at anaphase I requires the dissolution of the chiasmata that hold the bivalents together (Figure 35b). The chiasmata disappear at the metaphase I–anaphase I transition, as the arms of the chromatids of each bivalent lose cohesion. In contrast, cohesion between the joined centromeres of sister chromatids remains strong, as a result, sister chromatids remain firmly attached to one another as they move together toward a spindle pole during anaphase I. Thus, anaphase I is the cytological event that corresponds to Mendel’s law of independent assortment. As a result of independent assortment, organisms are capable of generating a nearly unlimited variety of gametes. FIGURE 35: Separation of homologous chromosomes during meiosis I and separation of chromatids during meiosis II. Cell Cycle 49  Cell Biology iv- Telophase I: produces less dramatic changes than telophase of mitosis. Although chromosomes often undergo some dispersion, they do not reach the extremely extended state of the interphase nucleus. The nuclear envelope may or may not reform during telophase I. b) Interkinesis: is the stage between the two meiotic divisions and is generally short-lived. Cells in this stage are characterized as being haploid because they contain only one member of each pair of homologous chromosomes. Even though they are haploid, they have twice as much DNA as a haploid gamete because each chromosome is still represented by a pair of attached chromatids. c) Meiosis II: i- Prophase II: a much simpler prophase than its prophase I. If the nuclear envelope had reformed in telophase I, it is broken down and the chromosomes become recompacted again. ii- Metaphase II: Chromosomes line up at the metaphase plate. Unlike metaphase I, the kinetochores of sister chromatids of metaphase II face opposite poles and become attached to opposing sets of chromosomal spindle fibers (Figure 35c). The progression of meiosis in vertebrate oocytes stops at metaphase II. Metaphase II arrest is released only when the oocyte (now called an egg) is fertilized. The fertilized egg responds to these changes by completing the second meiotic division. iii- Anaphase II: begins with the synchronous splitting of the centromeres, which had held the sister chromatids together, allowing them to move toward opposite poles of the cell (Figure 35d). iv- Telophase II: in which the chromosomes are once again enclosed by a nuclear envelope. The products of meiosis are haploid cells with a 1C amount of nuclear DNA. Cell Cycle 50  Cell Biology (a) (b) FIGURE 36: Main phases of (a) meiosis I and (b) meiosis II. Cell Cycle 51  Cell Biology Enzymes Definition Enzymes are organic catalysts in cells that speed up chemical reactions without getting changed or consumed themselves. Enzymes are typically proteins. Certain types of RNA can also serve as catalysts (e.g. ribozymes). Even though enzymes are proteins, many of them are conjugated proteins; that is, they contain non- protein components, called cofactors, which may be inorganic (metals) or organic (coenzymes). Vitamins and their derivatives often function as coenzymes. The Properties of Enzymes As is true of all catalysts, enzymes exhibit the following properties: 1) They are required only in small amounts. 2) They are not altered irreversibly during the course of the reaction, and therefore each enzyme molecule can participate repeatedly in individual reactions. 3) They have no effect on the thermodynamics of the reaction. The Active Site As catalysts, enzymes accelerate bond-breaking and bond-forming processes. To accomplish this task, enzymes become intimately involved in the activities that are taking place among the reactants. Enzymes do this by forming a complex with reactants, called an enzyme-substrate (ES) complex. In most cases, the association between enzyme and substrate is non-covalent, though many examples are known in which a transient covalent bond is formed. That part of the enzyme molecule that is directly involved in binding the substrate is termed the active site. The active site and the substrate(s) have complementary shapes, enabling them to bind together with a high degree of precision. The binding of substrate to enzyme is accomplished by the same types of non-covalent interactions (ionic bonds, hydrogen bonds, hydrophobic interactions) that determine the structure of the protein itself. Enzymes 52  Cell Biology The structure of the active site accounts not only for the catalytic activity of the enzyme, but also for its specificity. As noted above, most enzymes are capable of binding only one or a small number of closely related biological molecules. Classifications of Enzymes  Exoenzymes: extracellular; break down large food molecules or harmful agents e.g. Cellulase, amylase, penicillinase.  Endoenzymes: intracellular enzymes; varied functions e.g. Metabolic enzymes.  Constitutive enzymes: always present and in relatively constant amounts.  Induced enzymes: produced only when the substrate is present. Factors affecting the rate of the reaction 1) Temperature: Higher temperature generally causes more collisions among the molecules and therefore increases the rate of a reaction. Above a certain temperature, activity begins to decline because the enzyme begins to denature. The rate of the reaction therefore increases with temperature but then decreases. 2) pH: Each enzyme has optimal pH that maintains its normal configuration. A change in pH alters ionization of side chains, eventually resulting in denaturation. 3) Enzyme Concentration: Increasing the enzyme concentration will increase the speed of reaction (Figure 37). 4) Substrate Concentration: Increasing the substrate concentration will increase the reaction rate until reaching the saturation of all active sites. FIGURE 37: Factors affecting enzyme activity (a) Enzyme conc. (b) Substrate Conc. Enzymes 53  Cell Biology Mechanisms of Enzyme Catalysis 1) Substrates bound to the surface of an enzyme are brought very close together in precisely the correct orientation to undergo reaction. In contrast, when reactants are present in solution, they are free to undergo translational and rotational movements, and even those possessing sufficient energy do not necessarily undergo a collision that results in the formation of a transition-state complex. 2) Changing Substrate Reactivity: Enzymes are composed of amino acids having a variety of different types of side chains, from fully charged to highly nonpolar. When a substrate is bound to the surface of an enzyme, the distribution of electrons within that substrate molecule is influenced by the neighboring side chains of the enzyme. This influence increases the reactivity of the substrate and stabilizes the transition-state complex formed during the reaction. These effects are accomplished without the input of external energy, such as heat. 3) Inducing Strain in the Substrate (induced-fit model): Although the active site of an enzyme may be complementary to its substrate(s), various studies reveal a shift in the relative positions of certain atoms of the enzyme once the substrate has bound. In many cases, the conformation shifts so that the complementary fit between the enzyme and reactants is improved (an induced-fit), and the proper reactive groups of the enzyme move into place. As these conformational changes occur, mechanical work is performed, allowing the enzyme to exert a physical force on certain bonds within a substrate molecule. This has the effect of destabilizing the substrate, causing it to adopt the transition state in which the strain is relieved (Figure 38). Enzymes 54  Cell Biology FIGURE 38: Induced-fit model Enzyme. Enzyme Inhibitors: Enzyme inhibitors are molecules that are able to bind to an enzyme and decrease its activity. Enzyme inhibitors can be divided into two types: a) Irreversible inhibitors: are those that bind very tightly to an enzyme, often by forming a covalent bond to one of its amino acid residues. b) Reversible inhibitors: on the other hand, reversible inhibitors bind only loosely to an enzyme, and thus are readily displaced. The simplest cases of reversible inhibitors are (Figure 39): i. Competitive inhibitors: are reversible inhibitors that compete with a substrate for access to the active site of an enzyme. Since substrates have a complementary structure to the active site to which they bind, competitive inhibitors must resemble the substrate to compete for the same binding site, but differ in a way that prevents them from being transformed into product. ii. Non-competitive inhibitors: the substrate and inhibitor do not compete for the same binding site; generally, the inhibitor acts at a site other than the enzyme’s active site. The level of inhibition depends only on the concentration of the inhibitor, and increasing the concentration of the substrate cannot overcome it. Enzymes 55  Cell Biology (C) Non Competitive Inhibitor FIGURE 39: Two types of Reversible inhibitors. Enzymes 56  Cell Biology Cell Death Most Programmed Cell Death Occurs Through Apoptosis Dying cells shrink, condense, and then fragment, releasing small membrane- bound apoptotic bodies, which are then engulfed by other cells (Figure 40). Within these apoptotic cells, nuclei condense, and the DNA is fragmented. Importantly, the intracellular constituents are not released into the extracellular milieu, where they would probably have deleterious effects on neighboring cells, but instead are phagocytosed by neighboring cells. The stereotypical changes that occur during apoptosis, such as condensation of the nucleus and phagocytosis by surrounding cells, suggested to early scientists that this type of cell death was regulated and under the control of a strict program. This program is critical during both embryonic and adult life to maintain normal cell number and composition. FIGURE 40: Features of cell apoptosis. Cell Death 57  Cell Biology References 1. Alberts, B., Bray, D., Hopkin, K., Johnson, A.D., Lewis, J., Raff, M., Roberts, K. and Walter, P., 2013. Essential cell biology. Garland Science 2. Banfalvi, G., 2017. Cell cycle synchronization. Springer New York. 3. Cooper, G.M., Hausman, R.E. and Hausman, R.E., 2000. The cell: a molecular approach (Vol. 10). Washington, DC: ASM press. 4. DePamphilis, M.L., 1996. DNA replication in eukaryotic cells. Cold Spring Harbor Laboratory. 5. Karp, G., 2009. Cell and molecular biology: concepts and experiments. John Wiley & Sons. 6. Lodish, H., Berk, A., Kaiser, C.A., Krieger, M., Scott, M.P., Bretscher, A., Ploegh, H. and Matsudaira, P., 2008. Molecular cell biology. Macmillan. 7. Stauffer, S., Gardner, A., Ungu, D.A.K., López-Córdoba, A. and Heim, M., 2018. Mitosis. In Labster Virtual Lab Experiments: Basic Biology (pp. 11- 26). Springer Spektrum, Berlin, Heidelberg. References 58