Essential Cell Biology 3rd: Ch 4 Protein Structure and Function

INTRO

• Not only do proteins make up the majority of the dry weight of the cell, but they execute almost all of its myriad functions.

• All enzymes are proteins, but not all proteins are enzymes.

• The many functions that proteins carry out are possible do to the huge number of different shapes they are capable of adopting.

THE SHAPE AND STRUCTURE OF PROTEINS

• By understanding the structure at the atomic level allows us to describe how the exact shape of each protein determines its function in the cell.

• The shape of a protein is specified by its amino acid sequence.

• Proteins are composed of a combination of the 20 different amino acids linked together by covalent peptide bonds. Because of this proteins are referred to as either polypeptides or polypeptide chains. 

 

• Each protein's amino acids are present in a specific unique order, called the amino acid sequence. This sequence is identical from one molecule of the protein to the next.

• The side chains of the amino acid are what give it its unique properties  

 

• Each polypeptide contains a polypeptide backbone that supports the amino acid side chains. This backbone is made from the repeated core sequences of the amino acids.

• The shape of the protein chain is constrained by different sets of noncovalent bonds that involve not only the polypeptide chain but also the amino acid side chains.

• Hydrogen bonds, electrostatic attractions, and van der Waals attraction.

• Hydrophobic interactions also play a role in the shape of proteins. In the aqueous environment of the cell, hydrophobic nonpolar side chains of particular amino acids tent to be forced together to minimize the disruptive effect on the hydrogen bonded network of the surrounding water molecules.

• These amino acids are phenylalanine, leucine, valine, and tryptophan tend to cluster in the interior of the folds of the protein chains and tend to form hydrogen bonds with other amino acid side chains or the polypeptide backbone.

• Proteins fold into a conformation of lowest energy

• The conformation of a protein is determined by the order of the amino acid chain. The structure can be denatured if treated with solvents that disrupt the noncovalent interactions, but when the denaturing solvent is removed the protein will return, or renature, into its original conformation.

• This shows that the information needed to determine the three dimensional structure of a protein is determined by its amino acid sequence.

• Each protein normally folds into one conformation, which minimizes the free energy of the structure (G). The proper folding of proteins can usually happen on its own, but in cells its generally assisted by special proteins called molecular chaperones.

• This structure of the protein may change slightly though when it interacts with other molecules, and is crucial to it's function.

• If the protein folds incorrectly they can form what is called Aggregated proteins, which can damage the cell or even whole tissues. Prion diseases, Alzheimer's, and Huntington's diseases are examples of these. And in some cases aggregated proteins can cause normal proteins to change into aggregated proteins.

• The reasons the protein chaperones are so important is because they prevent the newly synthesized protein chains from associated with the wrong proteins in the crowded cytoplasm. But the overall structure of the protein is still determined by the amino acid sequence.

• Proteins come in a wide variety of complicated shaped

• Proteins are the most structurally diverse macromolecules in the cell.

• On average they are between 50 and 2000 amino acids, but they can range anywhere from 30 to 13000. 
• these are just some of the proteins that we will use in this course... 

 

• We can now easily determine the amino acid sequence of proteins by sequencing the gene that encodes it and using the universal code to determine the order of the amino acids in the polypeptide chain.

• Even though we know the sequence of proteins we have not yet figured out how to reliably predict the three dimensional conformation of proteins from only the sequence of the amino acids. The only way to currently determine this shape is by x-ray crystallography or nuclear magnetic resonance (NMR)

• There are four different ways to model protein's three dimensional structure

• Polypeptide backbone model

• Ribbon model 

• Wire model that includes the amino acid side chains

 

• Space filling model

• The Alpha Helix and the Beta sheet are common folding patterns

• Even though each protein folds in its own way, there are two common folding patters.

• Alpha helix

• Beta (pleated) sheet 

 

• These two patterns are common because they result from hydrogen bonds that form between the N-H and C=O groups in the backbone. Since the backbone is involved in this and not the side chains, these two forms can be generated by many different amino sequences.

• Helices form readily in biological structures

• A helix is made by placing similar subunits next to each other in the same repeated relationship to the one before, and it can be either right handed or left handed.

• And Alpha helix is made from a structurally rigid cylinder, where there is a hydrogen bond between every fourth amino acid linking the C=O of one peptide bond to the N-H of another.

• Sometimes two or three Alpha helices will wrap around one another to form a particularly stable coiled-coil structure. This structure forms when Alpha helices have more of their nonpolar (hydrophobic) side chains on one side so they can twist around each other to minimize their contact with the cytosol.

• Beta sheets form rigid structures at the core of many proteins.

• Beta sheets are made when hydrogen bonds form between segments of polypeptide chains lying side by side.

 

• Parallel beta sheets are formed when the structure consists of neighboring polypeptide chains that run in the same orientation, from the N-terminus to the C-terminus. When the sheet is folded back and forth over itself, it forms an antiparallel beta sheet.

• Both sheets form a rigid pleated structure that forms the core of many proteins.

• Proteins have several levels of organization

• The Alpha helix and Beta sheets are only the first level of protein structure, there are other levels of structure build upon structures forming the final protein's conformational structure.

• Primary structure - the amino acid sequence.

• Secondary structure - the Alpha helix and the Beta sheet

• Tertiary structure - the full three dimensional conformation of the entire polypeptide chain.

• Quaternary structure - the complex of more than one polypeptide chain interacting with each other.

• Protein domain - any segment of a polypeptide chain that can fold independently into a compact, stable structure.

• A domain usually consists of about 100-250 amino acids, and are often associated with different function. Ie. the polymerase involved in DNA replication.

• Few of the many possible polypeptide chains will be useful.

• While the possible number of combinations being 20^n, the actual stable, biologically useful combinations are much less than this number.

• Proteins have adapted over time, and some are even conserved across species.

• A change in even a few amino acids in a protein can disrupt the structure of the protein and eliminate its function.

• Proteins can be classified into families

• Many proteins can be grouped into families, showing us the evolution of proteins through time.

• Protein families - each family member has an amino acid sequence and a three-dimensional conformation that closely resembles that of the other family members.

• Large protein molecules often contain more than one polypeptide chain.

• The noncovalent bonds that help to hold the protein in its conformation, can also allow proteins to bind to each other to produce larger structures.

• Binding site- any region on a proteins surface that can interact with another molecule through sets of noncovalent bonds.

• Subunit- each individual protein that makes up a larger protein through noncovalent binding.

• DIMER- a molecule or molecular complex consisting of two identical molecules linked together

• Proteins can assemble into filaments, sheets, or spheres

• It is possible to dissociate structures into their constituent macromolecules. If we then mix these molecules back together, they will reassemble spontaneously into the original structure.

• This shows that the cell structure is self-organizing.

• Some types of proteins have elongated fibrous shapes

• Most proteins previously mentioned are globular proteins - more of a rounded compact shape. But others are simple, elongated three dimensional structures referred to as fibrous proteins.

• Examples would be Keratin, the extracellular matrix, Collagen, and Elastin. 

 

• Extracellular proteins are often stabilized by covalent cross-linkages

• Many protein molecules are either attached to the outside of a cell's plasma membrane or secreted as part of the extracellular matrix.

• Proteins are often strengthened by covalent cross-linkages to help prevent their deformation do to extracellular conditions.

• The most common cross-linkage bond is the Sulfur-Sulfur, disulfide bond.

 

• These bonds are catalyzed during formation by an enzyme in the ER. 

• S-S bonds do not change the shape of the protein, but act more like a staple, to help reinforce the favored conformation.

• These bonds are not formed in the cytosol, as the extra reinforcement is not needed in the mild environment of the cell.

HOW PROTEINS WORK

• A protein's function depends on its ability to bind specifically to other molecules. This can allow them to act as catalysts, structural supports, signal receptors, and tiny motors.

• All proteins bind to other molecules

• Regardless of the strength of the bond between molecules, the selectivity for the bond is always very specific.

• Each protein molecule can bind to just one, or a few, molecules out of the many thousands of different molecules it may encounter.

• Ligand- any substance that is bound to a protein, whether it is an ion, a small molecule, or a macromolecule.

• An encounter that will result in a bond will only occur if the surface contours of the ligand molecule fit very closely to the protein.

• Binding site- the region of the protein that associates with a ligand. And is usually formed by a cavity in the protein surface, formed by a particular arrangement of amino acids.

• But changes to interior amino acids that do not provide binding sites matter as well, these can distort the shape of the binding sites and proteins resulting in loss of function.

• The binding sites of antibodies are especially versatile.

• Antibodies - immunoglobulins, are proteins produced by the immune system in response to foreign molecules. 

• These will bind very tightly to the target either inactivating it or marking it for destruction.

• Antigen - the antibodies target

• Antibodies are Y shaped with two identical binding sites that complement a small portion of the surface of the antigen molecule. 

 

• The amino acid sequence in these loops can be changed by mutation without altering the basic structure of the antibody.

• Enzymes are powerful and highly specific Catalysts.

• Many proteins just bind to other molecules and their job is done, but for enzymes, this is only the first step.

• Enzymes can be grouped into functional classes based on the chemical reactions they catalyze.

Enzyme Class	Biochemical Function
Hydrolase	General term for enzymes that catalyze a hydrolytic cleavage reaction.
Nuclease	Breaks down nucleic acids by hydrolyzing bonds between nucleotides
Protease	Breaks down proteins by hydrolyzing peptide bonds between amino acids.
Synthase	General name used for enzymes that synthesize molecules in anabolic reactions by condensing two molecules together.
Isomerase	Catalyzes the rearrangement of bonds within a single molecule.
Polymerase	Catalyzes polymerization reactions such as the synthesis of DNA and RNA.
Kinase	Catalyze the addition of phosphate groups to molecules. Protein kinases are an important group of kinases that attach phosphate groups to proteins.
Phosphatase	Catalyzes the hydrolytic removal of a phosphate group from a molecule.
Oxido-reductase	General name for enzymes that catalyze reactions in which one molecule is oxidized while the other is reduced. Enzymes of this type are often called oxidases, reductases, or dehydrogenases.
ATPase	Hydrolyzes ATP. Many proteins with a wide range of roles have an energy-harnessing ATPase activity as part of their function, including motor proteins such as myosin and membrane transport proteins such as the sodium-potassium pump.

• Lysozyme illustrates how and enzyme works.

• Lysozyme severs the polysaccharide chains that form the cell walls of bacteria through hydrolysis, causing the cell wall to rupture.

• They lysozyme holds six sugars with its active site distorting only one of the sugar-sugar bonds, then catalyzes the addition of a water molecule to the distorted bond, breaking it.

• This does not happen on its own partially because of the activation energy required for this to occur, but also because the molecule would have to be distorted into a particular shape, the transition state, for the water to interact with the bond.

• Step by step

1. The enzyme stresses its bound substrate by bending some critical chemical bonds in one sugar, so that the shape of the sugar more closely resembles the shape of high-energy transition states formed during the reaction. 
2. The negatively charged aspartic acid reacts wit the C1 carbon atom on the distorted sugar, breaking this sugar-sugar bond and leaving the aspartic acid covalently linked to the site of the bond cleavage.  
3. Aided by the negatively charged glutamic acid, a water molecule reacts with the C1 carbon atom, displacing the aspartic acid and completing the process of hydrolysis.

 

• Most drugs inhibit enzymes

• Pharmaceutical companies often target enzymes of interest to develop medications to help treat or prevent illness.

• Tightly bound small molecules add extra function to proteins.

• Proteins often use non-protein molecules to preform functions that would be difficult of impossible using amino acids alone.

• Ex. Retinal is attached via a lysine side chain to rhodopsin, the purple light sensitive pigment made by the rod cells. The retinal will change its shape when it absorbs a photon, which is then amplified by the rhodopsin, triggering a network of enzymatic reactions that lead to an electrical signal being sent to the brain.

• Enzymes, like other proteins, usually have a small molecule or metal atom associated with their active site that assists with their catalytic functions.

HOW PROTEINS ARE CONTROLLED

• Protein and enzyme activity are controlled at many levels.

• Gene expression

• Confining sets of enzymes in subcellular compartments

• The proteins shape can be altered, effectively altering its function.

• The catalytic activities of enzymes are often regulated by other molecules.

• Feedback inhibition- an enzyme acting early in a reaction pathway is inhibited by a late product of that pathway.

• This occurs when a molecule other than a substrate specifically binds to an enzyme at a regulatory site outside of the active site, which can alter the rate at which the enzyme works.

• This works almost instantaneously, and is rapidly reversed when product levels fall.

• Feedback inhibition is a negative regulation, it prevents an enzyme from acting.

• Positive regulation - the enzyme's activity is stimulated by a regulatory molecule rather than being shut down.

• This tends to occur when one metabolic branch stimulates the activity of an enzyme in another pathway.

• Allosteric enzymes have binding sites that influence one another.

• Enzymes have active sites and at one or more sites that recognize regulatory molecules, and the substrate and the regulatory sites must somehow "communicate" in a way that allows the catalytic events at the active site to be influenced by the binding of the regularity molecule at the separate site.

• This "communication" is accomplished through conformational changes that take place in the enzyme. The binding of the regulatory molecule causes a conformational change, shifting the active binding site.

• Allosteric - they (proteins) can adopt two or more slightly different conformations. By shifting from one to the other, activity can be regulated.

• This is true for enzymes as well as many proteins including receptors, structural proteins, and motor proteins.

• Phosphorylation can control protein activity by triggering a conformational change.

• Eucaryotic cells can also regulate protein activity by adding a phosphate to one of the amino acid chains.

• Protein phosphorylation - This will cause a major conformational change because the phosphate group carries two negative charges attracting a cluster of positively charged amino acid side chains.

• More than 1/3 of the proteins in mammals appear to use this at some point, and can either inhibit or stimulate protein activity. Inhibition or stimulation is dependent on the protein involved AND the site where it is being phosphorylated. 

 

• Protein phosphorylation involves the enzyme- catalyzed transfer of the terminal phosphate group of ATP to the hydroxyl group on a serine, threonine, or tyrosine and is catalyzed by a protein kinase. The removal, or dephosphorylation, is catalyzed by a protein phosphatase.

• The removal of the phosphate groups occurs in response to signals given when the state of the cell changes.

• GTP -binging proteins are also regulated by the cyclic gain and loss of a phosphate group

• This form of regulation uses either guanosine triphosphate (GTP) or guanosine diphosphate (GTP). The proteins are in their active states when GTP is bound to the protein. Then protein itself can then hydrolyzes the GTP to GDP (releasing a phosphate) and change to an inactive conformation. 

 

• Nucleotide hydrolysis allows motor proteins to produce large movements in cells

• Motor proteins - proteins whose major function is to move other molecules. 

• For the motor proteins to move backwards, the hydrolysis of ATP would have to be reversed as well. And since this is not thermodynamically favorable, it does not occur, and the motor protein is moved rapidly forward. 

 

• Proteins often form large complexes that function as protein machines.

• Larger proteins with many domains tend to have more elaborate functions compared to single domain proteins.

• Protein machines - a highly coordinated, linked set of 10 or more proteins

• This is driven by hydrolysis or bound ATP or GTP

• Covalent modification controls the location and assembly of protein machines

• Most protein machines form a specific sites in the cell and are activated only when and where they are needed, and is generally accomplished by the covalent addition of a modifying group to one or more specific amino acid side chains on the participating proteins.

• There are more than 200 types of covalent modifications that can occur in the cell, each helping to regulate protein function.

• Phosphorylation can not only inhibit or increase protein activity, but it can also promote the assembly of  proteins into larger complexes.

• Regulatory protein code - a set of covalent modifications that a protein contains at any moment.

• The attachment or removal of these modifying groups controls the behavior of a protein, changing its activity or stability, its binding partners, or its location inside the cell.

HOW PROTEINS ARE STUDIED

• Cells can be grown in a culture dish

• In vitro - reactions carried out in a test tube in the absence of cells.

• In vivo - any reaction taking place inside a living cell, even cells that are growing in culture.

• Cultured cells are accessible to study in ways that are often not possible in intact tissues.

• The cultured cells can also provide a ready source of raw materials.

• Purification techniques allow homogeneous protein preparations to be obtained from cell homogenates.

• Steps to purification of proteins through chromatography. 

 

1. Break open the cells to release their contents. The contents is called a cell homogenate.
2. Initial fractionation to separate out the class of molecules of interest.
3. Chromatography. This can be used to separate the individual components into different portions, which can then be put through an assay to determine which fractions contain the protein of interest.
4. Additional chromatography can then be used to reach the pure form of the protein.

• The most common forms of chromatography separate polypeptides on the basis of their size, charge, or their ability to bind to a particular chemical group.

• Antibodies can sometimes be used to help extract proteins of interest. 

• This can also be used to isolate proteins that interact physically with the protein as well. To do this the purified protein is attached to the matrix of the chromatography column, and the proteins that bind to this protein will collect in the column and can then be eluted by changing the composition of the washing solution.

• Electrophoresis can also be used to separate proteins. 

 

1. A mixture of proteins are loaded into a polymer gel 
2. This is then subjected to an electric field. The proteins will them migrate through the gel at different speeds depending on their size and net charge.   
3. If the proteins are too similar, or the number too great, a two dimensional gel electrophoresis can be used.  
4. The bands or spots can be visualized by staining

• Large amounts of almost any protein can be produced by genetic engineering techniques.

• Advances in genetic engineering now allows us to produce a large amount of almost any desired protein

• We can use these proteins in research, medicine, and other types of therapy.

• We can also genetically engineer techniques to design proteins that preform novel tasks.

• Automated studies of protein structure and function are increasing the pace of discovery.

• Proteomics - the large scale study of cellular proteins in which the activities or structures or hundreds, even thousands, of proteins are analyzed by highly sensitive, automated techniques.

Essential Cell Biology 3rd ed: Ch3 Energy, Catalysis, and Biosynthesis

INTRO  

• To carry out the many chemical reaction needed to sustain a cell, a living organism requires both a source or atoms in the form of food molecules and a source of energy. Both of these things must come, ultimately, from the nonliving environment.

• Most of the chemical reactions that cells preform would normally occur only at temperatures that are much higher than those inside a cell. Because of this, each reaction requires a large boost in chemical reactivity for the reaction to occur within the cell. This boost is provided by enzymes, which will catalyze the chemical reaction to push the reaction forward, and tend to be connected in series to other reactions so that the product of one reaction becomes the starting material of the next. These long chemical reaction sequences are referred to as metabolic pathways.

• There are two opposing streams of chemical reactions that occur in cells

• Catabolic pathways- these break down foodstuffs into smaller molecules, gathering energy and some small building blocks that are needed for the cell.

• Anabolic pathways- (biosynthetic) use the energy harnessed by catabolism to drive the synthesis of the many molecules that form the cell.

THE USE OF ENERGY BY CELLS

• An organism's ability to maintain order is made possible by elaborate cellular mechanisms that extract energy from the environment and convert it into the energy stored in chemical bonds.

• Biological order is made possible by the release of heat energy from cells

• Entropy- the measure of a system's disorder.  

• Living cells are generating order and may appear to defy the second law of thermodynamics, but because a cell is not an isolated system, taking in energy from its environment to preform chemical reactions that release heat, they are still in compliance with the law

• This heat dispersed into the cell's surroundings is increasing the intensity of the thermal motions of the resident molecules, increasing the entropy of the system as a whole.

• Heat is energy in its most disordered form

• It is the tight coupling of heat production to an increase in order that distinguishes the metabolism of a cell form the wasteful burning of fuel in a fire.

• By directly linking the "burning" of food molecules to the generation of biological order- cells are able to create and maintain an island of order in a universe tending toward chaos. 

• The percentage of the different forms of energy will change as a result of the chemical reactions in the cell, as the overall amount of energy must still be the same, per the first law of thermodynamics. This conversion of chemical energy into heat energy is essential if the reactions inside the cell are to cause the universe as a whole to become more disordered- as required by the second law.

• Photosynthetic organisms use sunlight to synthesize organic molecules.

• Photosynthesis- a process that converts the electromagnetic energy in sunlight into chemical bond energy in cells. This is how solar energy enters the living world.

• Photosynthetic organisms are able to obtain all of the atoms they need from inorganic sources, such as the dirt or other non-living things around them. They then use the energy the derive from the sunlight to form chemical bonds between atoms, linking them into small chemical building blocks that will make up the plant.

• The reactions of photosynthesis will occur in two stages, one that is dependent on light and one that is not.

• In the light dependent stage, energy from sunlight is captured and transiently stored as a chemical bond energy in specialized small molecules that carry energy in their chemical groups. Molecular oxygen (O2) derived from the splitting of water by light is released as a byproduct of the first stage.

• In the second stage, the molecules that serve as energy carriers are used to help drive carbon fixation, in which sugars are manufactured from carbon dioxide gas (CO2) and water. This provides a stored chemical source of energy. 
• the over all reaction can be simplified as...

• Cells obtain energy by the oxidation of organic molecules

• In both plants and animals, energy is extracted from food molecules by a process of gradual oxidation, or controlled burning. To do this, the carbon and hydrogen atoms in the sugar and other organic molecules combine with oxygen (oxidation) to produce CO2 and H2O in a process known as cellular respiration.

• Photosynthesis and respiration are complementary processes. 

 

• Oxidation and reduction involve electron transfers

• The cell does not oxidize organic molecules in one step, through he use of enzyme catalysts metabolism carries the molecules through a large number of reactions that only rarely involve the direct addition of oxygen.

• Oxidation- this literally means the addition of an oxygen to a molecule, but generally, it means any case where electrons are removed from one atom and given to another. 

• Reduction-  is the addition of electrons to an atom.

• These two reactions will occur simultaneously.

• Oxidation/reduction reactions may even occur when there is only a partial shift in electrons. Ex. Polar covalent bonds.

• Hydrogenation reactions are reductions, and dehydrogenation reactions are oxidations.

• If the number of C-H bonds increases, its reduction. If the number of C-H bonds decreases, its oxidation.

FREE CATALYSIS

• Enzymes can speed up energetically favorable reactions- those that will later produce disorder- but they themselves can not force energetically unfavorable reactions.

• Enzymes lower the energy barriers that prevent chemical reactions from occurring.

• Free energy- (ΔG) energy that can be harnessed to do work or drive chemical reactions, and reflects a loss of orderliness.

• Chemical reactions proceed only in the direction that leads to a loss of free energy.

• Activation energy- the energy required to be added to the system to overcome an energy barrier before the chemical reaction can occur moving it to a lower energy (more stable) state. <-- this is what enzymes lower.

• Each enzyme binds tightly to one or two molecules, called substrates, and holds them in a way that greatly reduces the activation energy needed to facilitate a specific chemical interaction between them.

• Catalyst - a substance that can lower the activation energy of a reaction.

• These increase the rate of chemical reactions because they allow a larger percent of the random collisions with surrounding molecules to kick the substrates over the energy barrier.

• Enzymes are among the most effective catalysts know. Life could not exist without enzymes. 

• Every enzyme is a catalyst, but not every catalyst is an enzyme.

• Enzymes are highly selective, and usually only speed up one reaction out of the several possible reactions that could occur with the available substrate molecules.

•  Its selectivity comes from uniquely shaped active sites, so that only the right substrates will fit.

• After the reaction ALL catalysts -including enzymes- remain unchanged, and can function over and over again.

• The free-energy change for a reaction determines whether it can occur

• The change in free energy (ΔG) measures the amount of disorder created in the universe when a reaction takes place.

• If ΔG is negative;  then the reaction is energetically favorable, creating disorder by lowering the free energy of the system. 

• A reaction can only occur spontaneously if ΔG is negative.

• If ΔG is positive; then the reaction is energetically unfavorable, this reaction by itself would create more order, and therefor can not occur spontaneously.

• These reactions can only take place if they are coupled with a second reaction that has a negative ΔG large enough that the net ΔG of the entire process is negative.

• Enzymes also help with the coupling of these reactions

• The concentration of reactants influences the free-energy change and a reaction's direction.

• ΔG does not just depend on the energy stored in each molecule involved in the reaction, but also on the concentration of the molecules.

• Has to do with ratio percentages.

• The standard free energy change makes it possible to compare the energetics of different reactions.

• ΔG⁰- is the standard free energy change of a reaction, and is independent of concentration, instead it depends on the molecules behavior under ideal conditions where the concentrations of all reactants are set to 1mol/liter.

• ΔG =  ΔG⁰ + RT ln [x]/[Y]

• Where  ΔG is in kilocalories per mol, [Y] and [X] denote the concentrations of Y and X in moles per liter, and RT is the product of the gas constant and the absolute temperature.

• Cells exist in a state of chemical disequilibrium.

• Equilibrium - the point where the rates of the forward and reverse reactions are equal, and no further net change in the concentrations of the substrate or product is occurring.

• At equilibrium  ΔG is zero. At this point no reaction will occur and any cell that reaches the point of chemical equilibrium would be dead. This is because the maintenance of order within the cell requires continuous input of energy. To avoid this the cell is constantly exchanging materials with their environment.

• The equilibrium constant is directly proportional to  ΔG⁰.

• Equilibrium constant, K.

• K=[X]/[Y] 

• Where [X] is the concentration of the product and [Y] is the concentration of the reactant at equilibrium.

• In complex reaction, the equilibrium constant depends on the concentrations of all reactants and products.

• The same principle that is used above is used when two reactants combine to form a single product, except  the equilibrium constant now depends on the concentrations of both reactants and that of the product.

• K=[AB]/[A][B]

• The equilibrium constant indicates the strength of molecular interactions. 

• This free energy concept does not just apply to the breaking and forming of covalent bonds, it also applies to non-covalent bonds. Which are also very important to the cell, and include the binding of enzymes to the substrate

• Two molecules will bind to each other if the ΔG⁰ of the interaction is negative, resulting in a a complex that has a lower free energy that the sum of the free energies of the two partners when unbound.

• Because  ΔG⁰ is related directly to K, K is commonly used as a measure of the binding strength of a noncovalent interaction between two molecules, and can indicate how specific the interaction is between the two molecules.

• Binding energy - the energy released in the binding interaction.

• This will increase as K increases, so the larger the K is, the greater the drop in free energy between dissociated and associated states.

• For sequential reactions, the changes in free energy are additive

• A reaction with a positive  ΔG is not favorable, and will not happen unless an enzyme is used and the reaction is followed by another reaction that makes the sum of the  ΔG negative.

• Rapid diffusion allows enzymes to find their substrates.

• Rapid binding is possible because motions are enormously fast at the molecular level, due to heat energy.

• Diffusion- the idea that molecules are in constant motion and will explore the confined area by wandering randomly through it.

• The average distance that it travels from the starting point is proportional to the square root of the time it takes. So diffusion only works well for very short distances.

• Enzymes and other macromolecules do not move very easily through the cell though, and some are even held near where they are needed by scaffold proteins. And even if they are not held in place, they move so slow relative to the other small molecules in the cell that we view them as  stationary, and their effectiveness is dependent on the concentration of the substrate.

• When an enzyme and substrate have collided and snuggled together properly at the active site, they form multiple weak bonds with each other that persist until random thermal motion causes the molecules to dissociate again.

• These can include; hydrogen bonds, Vander Waals, and electrostatic attractions.

• Vmax and Km Measure enzyme performance

• The rate at which each enzyme functions varies greatly from one enzyme to another.

• This rate can be measured by mixing purified enzymes and substrates together under carefully defined conditions.

• Vmax- the point where the active sites of all enzymes molecules in the sample are fully occupied by substrate.

• At this point the rate of product formation only depends only on how rapidly the substrate molecule can be processed. This is referred to as the turnover number.

• Km- the concentration of the substrate needed to make the enzyme work efficiently is measured by Michaellis' constant, and referrers to the concentration of substrate at which the enzyme works at half its maximum speed (0.5Vmax).

• A low Km value will indicate that an enzyme binds very tightly to the substrate and a high value will show a weaker bond.

• When an enzyme lowers the activation energy of the reaction, it also lowers the activation energy of the reverse reaction by the same amount.

• Because of this the equilibrium point, and ΔG⁰, of the reaction will remain unchanged.

ACTIVATED CARRIER MOLECULES AND BIOSYNTHESIS

• The energy released by the oxidation of food molecules has to be temporarily stored before it can be used by the cell, and is usually stored in the form of a chemical bond in a small set of activated "carrier molecules".

• Active carriers store energy either as a readily transferable chemical group or as high-energy electrons. ATP, NADH, and NADPH are common examples of these.

• The formation of an activated carrier is coupled to an energetically favorable reaction.

• When a fuel molecule like glucose is oxidized in the cell, enzyme-catalyzed reactions ensure the a large part of the free energy released is stored in a chemically useful form. If it is not stored its released as heat.

• Coupled reaction- an energetically favorable reaction is used to drive an energetically unfavorable one, which produces and activated carrier or some other useful molecule.

• The amount of heat released by the oxidation of foodstuffs decreases by exactly the same about as the energy stored in covalent bonds.

• ATP is the most widely used activated carrier molecule

• This is the most important and versatile of the activated carriers.

• ATP is synthesized in an energetically unfavorable phosphorylation reaction, where a phosphate group is added to ADP (Adenosine 5'-diphosphate). ATP can then give up its stored energy through a hydrolysis reaction where the terminal phosphate is lost, converting it back to ADP.

 

• Phosphorylation is any reaction that involves the transfer of a phosphate group into a molecule, and is an example of condensation reactions. 

• This favorable release of energy is coupled with many unfavorable reactions, that would otherwise not occur.

• It can be used to supply energy for many of the pumps in the membrane, to power molecular motors, as well as many other uses.

• Energy stored in ATP is often harnessed to join two molecules together

• There can also be intermediate steps before getting to the desired product.

• The hydrolysis of ATP can be couple indirectly to push the reaction forward, by first forming an unfavorable intermediate product that can then undergo a favorable reaction to result in the desired end product.

• NADH and NADPH are important electron carriers

• Two common activated carriers that carry both high energy electrons and hydrogen atoms are NAD+ (nicotinamide adenine dinucleotide) and NADP+ (nicotinamide adenine dinucleotide phosphate). 

 

• Both of these pick up energy in the form of two high energy electrons plus a proton, becoming NADH (reduced nicotinamide adenine dinucleotide) and NADPH (reduced nicotinamide adenine dinucleotide phosphate). 
• the extra hydrogen will be added to the carbon directly across from the ringed nitrogen in the high lighted area shown above.

• NADPH is formed during a special set of energy yielding catabolic reactions, where a hydrogen atom and two electrons are removed from the substrate molecule and added to the nicotinamide ring of the NAPD+. This occurs through a typical redox reaction. 

 

• The hydride ion can then be readily given up in a similar redox reaction since the ring is actually more stable without the extra electrons.

• The extra phosphate group on NADPH is located far from the region involved in the transfer of electrons, but it gives the molecule a different shape (compared to NADH), making it possible for NADH and NADPH to bind as substrates to different sets of enzymes.

• The division of labor among the two carriers is because of the need to regulate two independent reactions.

• NADH is an intermediate is catabolic system of reactions that generate ATP. And NADPH is used with enzymes that catalyze anabolic reactions for syntheses of biological molecules.

• These two carriers are made through and regulated by different pathways

• The ratio of NAD+ to NADH is kept high, and the ratio of NADP+ to NADPH is kept low. So there is plenty of NADPH to act as a reducing agent, and NAD+ to act as an oxidizing agent.

• Cells make use of many other activated carrier molecules

• FADH2, like NADH also carries hydrogen and high energy electrons.

 

• Coenzyme A can carry an acetyl group in a transferable linkage (acetyl CoA) and can add two carbon units during the biosynthesis of the hydrocarbon tails of fatty acids.

Activated carrier	Group carried in linkage
ATP	Phosphate
NADH, NADPH, FADH2	Electrons and hydrogens
Acetyl CoA	Acetyl group
Carboxylated biotin	Carboxyl group
S-adenosylmethionine	Methyl group
Uridine diphosphate glucose	Glucose

• The synthesis of biological polymers requires an energy input

• Macromolecules make up the majority of the cells dry weight, and are made from monomers that are linked together during enzym-catalyzed condensation reactions.

• If none of these pathways provides a low enough  ΔG (ATP gives about -13kcal/mol) then AMP can be used at about -26kcal/mol