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BIOCHEMISTRY CHARACTERISTIC BIO-MEMBRANE AND ORGANELLES MODULE 1.1: THE CELL & THE BIOMOLECULES Mitochondria CELL:...

BIOCHEMISTRY CHARACTERISTIC BIO-MEMBRANE AND ORGANELLES MODULE 1.1: THE CELL & THE BIOMOLECULES Mitochondria CELL: Chloroplasts (plastids) basic building blocks of life Rough Endoplasmic Reticulum (RER) smallest living unit in an organism Ribosome grow, reproduce, use energy, adapt, respond Smooth Endoplasmic Reticulum (SER) to their environment Golgi Apparatus may be an entire organism or one of billions Lysosomes that make up the organism PROKARYOTES: bacteria & lacks a nucleus or membrane-bound structures (organelles) EUKARYOTES: most other cells & have a nucleus and organelles (plants, fungi & animals) FOUR (4) BIOMOLECULES 1. Protein 2. Carbohydrates 3. Lipids and fats 4. Nucleic acid 1. PROTEIN- folding to its 3D structure is dictated by a sequence of amino acids that constitute the protein. Membrane proteins embedded (yellow) in membranes and attached (blue) to them, permit the exchange of material and information with the environment. 2. CARBOHYDRATES- fuels and information Fig. nucleotide (in this case, adenosine triphosphate) molecules. Glucose is the most common fuel sugars consists of a base (shown in blue), a five-carbon and glycogen is the stored glucose in animals. sugar (black), and at least one phosphoryl group (red). ENERGY OF THE CELL Living cells are inherently unstable. Constant flow of energy prevents them from becoming disorganized. Cells obtain energy mainly by the oxidation of biomolecules (electrons transferred from 1 molecule to another & in doing so they lose energy) This energy captured by cells & used to maintain highly organized cellular structure & functions. 3. LIPIDS AND FATS- Storage form of fuel and also serve as cell barrier. (One part of a lipid molecule is HOW DO COMPLEX STRUCTURES OF CELLS hydrophilic; the other part is hydrophobic. In water, MAINTAIN HIGH INTERNAL ORDER? lipids can form a bilayer, constituting a barrier that separates two aqueous components. 1. Synthesis of biomolecules. 2. Transport Across Membranes- Cell membranes regulate the passage of ions & molecules from one compartment to another. 3. Cell Movement- Organized movement is one of the most obvious characteristics of living cells. The intricate & coordinated activities required to sustain life require the movement of cell components. 4. Waste Removal – Animal cells convert food molecules into CO2, H2O & NH3. If these not disposed properly can be toxic. LINKAGES IN BIOCHEMICAL COMPOUNDS Functional groups- specific parts of molecules 4. NUCLEIC ACID- information molecules of the cell. involved in biochemical reactions. The primary function of nucleic acid is to store and transfer information from the genes to proteins. Linkages between biochemical compounds Functional groups in a single biomolecule MANY IMPORTANT BIOMOLECULES ARE POLYMERS BIOPOLYMERS- macromolecules created by joining many smaller organic molecules (monomers). e.g. Condensation reactions join monomers (H2O is removed in the process). PORPHYRIN RING- Mg2+ RESIDUE- each monomer in a chain ESTER GROUP- -OCH3 EXERCISE: KETONE GROUP- C=O HYDROXYL GROUP- OH METHYL GROUP- CH3 MODULE 1.2: WATER AS MEDIUM OF CELL & HYDROGEN BONDING EXAMPLES BUFFERS AQUEOUS SOLUTION & PROPERTIES OF WATER 1. Organisms are mostly water (humans 70%) 2. Influences the shape of and function of biomolecules 3. Medium of most biochemical reactions 4. Important for transport of nutrients & waste 5. Water itself may be a participant in chemical reactions UNIQUE PROPERTIES OF WATER ✓ High cohesion ✓ High adhesion ✓ High surface tension ✓ High specific heat ✓ High heat of vaporization ✓ Liquid at room temperature WEAK INTERACTIONS IN AQUEOUS SOLUTION: 4 ✓ Excellent solvent 1. IONIC INTERACTIONS ✓ Hydrophobic exclusion 2. VAN DER WAALS INTERACTION Any two atoms in close proximity Two uncharged atoms are brought very close together, the surrounding electron clouds influence each other. HYDROGEN BONDING BETWEEN WATER & OTHER MOLECULES 3. HYDROGEN BONDS 4. HYDROPHOBIC INTERACTIONS Amphipathic Weak interactions are individually weak relative to covalent bond yet the cumulative effect many such interaction in protein or nucleic acid can be very NONPOLAR & AMPHIPATHIC BIOMOLECULES significant. E.g. substrate-enzyme POLAR BIOMOLECULES Why talk about acids & bases? Many biological molecules have functional groups that undergo acid-base reactions; therefore, the properties of these molecules are affected by acidity of the solutions in which they are surrounded. NON-POLAR & AMPHIPATHIC BIOMOLECULES Nonpolar  Fig. Titration curve of weak acid/conjugate base pairs. BUFFERS A solution that is capable of resisting substantial changes in pH upon addition of acidic or basic substances. The composition is a mixture of a weak acid (or base) and its conjugate base (acid), respectively. Characteristics of good buffers for biochemical studies are: ✓ pKa = 6 & 8 ✓ Highly soluble in water ✓ Minimum salt effects ✓ Minimal effects of Ki ✓ Does not form metal ion complexes Autoionization of water ✓ Non-absorbing in UV & Vis regions “Many of the solvent properties of water is explained ✓ Chemically stable by uncharged water molecule, the small degree of ✓ Available in pure form ionization of water to hydrogen ions (H+) and BUFFER RANGE hydroxide ions (-OH) must also be taken account.” pH range where the buffer is most effective in ✓ Described in an equilibrium constant resisting pH change ✓ Weak acid & base contribute to H+ by ionizing (acids) or consume H+ by being protonated pH – pKa ± 1 (base) ✓ Expressed by equilibrium constants BUFFER CAPACITY “Reversible ionization of water molecule is crucial to Number of equivalents (n) of either H+ or – OH the role of water in cellular functions.” required to change the pH of a given volume of buffer by one pH unit. “The total hydrogen ion concentration from all sources is experimentally measurable – pH of FACTORS AFFECTING BUFFER CAPACITY solution.” Concentration of the two buffer components Ph ! For pure water the concentration of hydroxyl & hydronium ions must be equal: The ion-product for water, K VARIETY OF WEAK ACID/CONJUGATE BASE PAIRS pH Buffering EXERCISE: Fig. Titration curve of acetic acid/acetate pH Buffering MODULE 2.1: AMINO ACID MOST AMINO ACIDS EXIST AS MIRROR-IMAGE FORMS 2 WAYS TO VIEW BIOMOLECULES The α-amino acids are chiral, and may exist either as Proteins have the ability to form three dimensional L-isomer or D-isomer. Proteins are made up of L- (3D) structures due to the different functional amino acids only. groups attached to the amino acid or the side chain. 1. Fischer projection- visualizing the C and H atoms (constituent atoms) is more important than seeing the shape of the molecule. horizontal line is projected towards the viewer vertical lines are assumed to point away from the viewer CHARGES OF AMINO ACIDS ✓ Free amino acids in solution at neutral pH exist ✓ predominantly as dipolar ions (ZWITTERIONS). ✓ In the dipolar form: -NH3+ & -COO- ✓ The ionization state of an amino acid varies with pH. ✓ In acidic solution (pH < 7), -NH3+ and –COOH 2. Stereochemical structure- are present. ✓ In basic solution (pH>7), -NH2 and –COO- are Wedges- used to depict the direction of a bond into or out of the plane of pages. present ✓ Under physiological conditions, amino acids Dash- indicates that the bond is going away from the exist inn dipolar form. viewer. Solid wedge- denotes a projection toward the viewer. The other two bonds are meant to be straight lines. Amino acid is considered as α-amino acid if it consists of a central carbon atom, α-carbon, linked to an amino group, carboxylic acid group, a hydrogen atom group and side chain of (R group). Each amino acid has a different R group or functional group. CLASSIFICATION OF AMINO ACIDS POLAR AMINO ACIDS WITH ELECTRONEGATIVE ATOMS -have varying size, shape, charge, hydrogen- bonding capacity, hydrophobic character and chemical reactivity conferred by their functional groups. The functional groups of amino acids include alcohol, thiols, thioethers, carboxylic acids, carboxamides, and a variety of basic groups. HYDROPHOBIC AMINO ACIDS mainly have hydrocarbons as R group (glycine, alanine) larger aliphatic side chains are branched amino acids (valine, leucine and isoleucine, methionine) have simple aromatic side chains (phenylalanine, tryptophan) Histidine can bind or release protons near possess a tendency to cluster together physiological pH. due to hydrophobic effect. NEGATIVELY CHARGED AMINO ACIDS HAVE ACIDIC SIDE CHAINS EXERCISE: What is the hidden message, translate the following amino acid sequence into a one-letter code? Glu-Leu-Val-Ile-Ser-Ile-Ser-Leu-Ile-Val-Ile-Asn-Gly- Ile-Asn-Leu-Ala-Ser-Val-Glu-Gly-Ala-Ser ELVISISLIVINGINLASVEGAS THE IONIZABLE SIDE CHAINS ENHANCE REACTIVITY & EXERCISE: BONDING 1. Show the ionization state of lysine. Identify the net Tyrosine, cysteine, arginine, lysine, histidine, aspartic charges. and glutamic acids have readily ionizable chains. 2. Which amino acid has no chiral carbon? Why? They can form ionic bonds, as well as donate or (Make your answer brief) accept protons to induce acid-base catalysis, a vital process in enzymes. 3. Draw the structure of a short peptide with an amino acid sequence of gylcyl-seryl-cysteine (Fischer and Stereochemical structure) ESSENTIAL AMINO ACIDS Amino acids that cannot be generated in the body must be supplied by the diet and are termed essential amino acids. The amino acids that do not fall under this classification are considered as nonessential amino acids. MODULE 2.2 PROTEIN LEVEL OF STRUCTURES About 50 to 2000 amino acid residues mostly consist the natural polypeptide chains or a protein. An PRIMARY STRUCTURE- definite amino acid sequence OLIGOPEPTIDES are consisted of small number of of a polypeptide formed by linking the α-carboxyl amino acid residues. group of one amino acid to the α -amino group of another amino acid covalently. Another feature of a polypeptide chain is a cross- linked, disulfide bond, formed by oxidation of a pair PEPTIDE BOND OR AMIDE BOND- linkage joining the of cysteine residues. amino acids. Loss of water molecule accompanies the formation of dipeptide bond. Cystine is the resulting two linked cysteine which could happen in same polypeptide chain or with another chain. POLYPEPTIDE CHAIN- a series of amino acids joined by peptide bonds. RESIDUE- amino acid unit in a polypeptide chain. It is always that one end of the polypeptide is an α- amino group and an α-carboxyl group, where the amino end is the beginning of a polypeptide. Thus, it is written starting with amino-terminal residue. CHARACTERISTICS OF THE PEPTIDE BOND ARE IMPORTANT: 1. The peptide bond is essentially planar. Six atoms lie in the same plane (α-carbon atom and CO group of the first amino acid and the NH group of the α—carbon atom of the second amino acid). POLYPEPTIDE CHAIN- consist of a main chain or the backbone and the side chain (R). The functional group of the amino acid differentiate one amino acid from the other which stabilizes the structure. 2. The peptide bond has considerable double- bond character – resonance structure. This prevents the rotation about this bond and thus constrains the conformation of the peptide backbone.  The Alpha Helix This is a coiled structure stabilized by intrachain hydrogen bonds. 3. The peptide bond is uncharged. Thus, polypeptides linked by peptide bonds to form tightly packed globular proteins inhibited charge repulsion. Since peptide bond tends to be planar, two configurations are possible. 1. Trans configuration – two α-carbon atoms are on opposite sides of the peptide bond. Almost all peptide bonds are in trans Examples of alpha helix configuration. 1. Alpha-keratin 2. Cis configuration – these groups are on the same side of the peptide bond. Poses steric 2. Ferritin- 75% of residues in ferritin, an iron-storage hindrance. protein, are helices. SECONDARY STRUCTURE- repetitive conformation of amino acids that are spatially close to one another. This is due to H-bonding between carbonyl group of one peptide bond and amino group of another THE BETA PLEATED SHEET peptide. There are two types of secondary structures, α-helix and β-pleated sheets. A single polypeptide strand with repeating units of a Linus Pauling and Robert Corey (1951) amino acids with small compact R groups. It is composed of two or more polypeptide chains, beta Proposed the α-helix and β-pleated sheet – ability of strands, and can be parallel or anti-parallel in certain polypeptides to fold into two periodic direction and the H-bonding exists among peptide structures. Other structures e.g. loops and turns bonds. were identified.  Fibrous proteins form long fibers that serve a structural and protective role. It is consisted of THE BETA PLEATED SHEET polypeptide chains arranged side by side in long In two beta strands that lie next to each other, the filaments. It tend to be mechanically strong and last amino acid of one strand and the first amino acid insoluble in water. of the adjacent strand are not necessarily neighbors A special type of fibrous proteins, collagen, a primary in the amino acid sequence. component of wool and hair, consists of two right- handed α-helices intertwined to form a typed of left- handed superhelix, coiled coil. Collagen- a primary component of skin, bone, tendon, cartilage and teeth. THE BETA PLEATED SHEET Fatty acid-binding proteins, which are important for lipid metabolism are built almost entirely from beta sheets. Coiled coil- special type of fibrous proteins that is consisted of two right-handed α-helices intertwined to form a typed of left-handed superhelix. TERTIARY STRUCTURE- three-dimensional structure resulting in R-group interactions e.g. H-bond, disulfide bond, hydrophobic interaction and electrostatic interaction. It is the spatial arrangement of amino acid residues that are far apart in the sequence and to the pattern of disulfide Globular proteins- have compact three-dimensional bonds. It is also called whole molecule folding. The structure and are water soluble proteins. Globular water-soluble proteins fold into compact structures. proteins are more intricate three-dimensional There are two types of proteins based on the three- structure which function of the chemical dimensional structure, fibrous and globular proteins. transactions in the cell. The tertiary structure is stabilized by R-group An example is myoglobin, a single polypeptide chain interactions and are therefore dictated by the amino of 153 amino acid residues, is an oxygen binding acid sequence. protein of the heart and skeletal muscle. The role of myoglobin the facilitation of the diffusion of oxygen from blood to the mitochondria where the primary site of oxygen utilization in the cell.  CLASSIFICATION OF PROTEIN BASED ON COMPOSITION 1. Simple proteins - consist of polypeptide chain QUATERNARY STRUCTURE- refers to the arrangement of subunits and the nature of their interactions which usually are the weak interactions i.e. H-bonds, ionic bonds and van der Waals interactions. DIMER- simplest quaternary structure which is consisted of two identical subunits. A quaternary structure is not limited to dimers but could be dozen subunits in a polypeptide chain. e.g. is the human hemoglobin (Hb), function as protein carrier of oxygen in blood. Hemoglobin is consisted of two subunits of one type (α) and two subunits of another type (β), thus one hemoglobin molecule exists as a protein carrier of oxygen in blood. α2β2 tetramer. 2. Coagulated proteins- composed of simple EXERCISE: proteins combined with some non-protein substances MODULE 2.3: ENZYMES COFACTORS ENZYMES- biopolymers that catalyze the chemical The enzymes and the inorganic ion required reactions that make life possible. Most reactions in biological systems do not take place at perceptible rates in the absence of enzymes. Even a reaction as simple as adding water to carbon dioxide is catalyzed by an enzyme—namely, carbonic anhydrase (Tymoczko, Berg and Stryer, 2013). Coenzymes are the organic cofactors All enzymes are proteins except the ribosomal RNAs (rRNAs) which include ribozymes, the self- clearing or self-splicing RNA molecules (Murray et al., 2012). COENZYMES Enzymes are highly specific in both the reactions that they catalyze and in their choice reactants 1. Nicotinamide adenine dinucleotide (NAD+) and (substrates). nicotinamide dinucleotide phosphate (NADP+) e.g. is proteolysis, the hydrolysis of peptide binds by An electron acceptor in the oxidation of fuel proteolytic enzymes. It is affected by the degree of molecules. The nicotinamide ring of these two substrate of specificity. coenzymes accept a hydrogen ion and two electrons (~ H- ion) when substrate is oxidized. Enzymes require inorganic ions or complex organic molecules to catalytically function called co-factor. These are non-protein components which are loosely held to the protein part of enzymes or permanently bound. Prosthetic group- is the cofactor which is tightly bound. The specific role of an enzyme may depend on the cofactor and the enzyme. An enzyme without its co-factor is the apoenzyme. 2. Flavin adenine dinucleotide (FAD) and Flavin On the other hand, the complete catalytic active mononucleotide (FMN) enzyme is the holoenzyme. Cofactors could be a coenzyme or a metal. The isoalloxazine ring is the active site of FAD and FMN. They can accept two electrons, but __________________________________________ take up a proton as well as hydride ion.  ENZYME CLASSIFICATION -large volume of identified and characterized enzymes are present, but enzymes are categorized only into six classes. This classification helped in recognizing the function of enzymes more simply. The system of naming and classification were adopted from international agreement. 3. Ubiquinone (Coenzyme Q, CoQ) This is a mobile electron carrier in the ETS. 4. Coenzyme A (CoA) The letter A stands for acetylation. The reactive site is the terminal sulfhydryl group on CoA. Acyl CoA is when acyl group linked to CoA but if acetyl group linked it is an acetyl CoA. 5. CYTOCHROMES This are groups of heme containing proteins which primary role is as an electron carrier in respiratory and photosynthetic ETS. The Fe ion may vary.  MODELS OF ENZYME ACTION The enzyme-substrate interaction is specific, but of different levels of specificity for substrates. Several models were proposed but the two simplest models are: ENZYMATIC CATALYSIS Crucial stage in catalysis is the formation of enzyme- substrate complex which is the first step. One of the functions of enzyme is bringing the substrates together in a favorable orientations, thus formation of transition state. Enzyme decreases the activation energy, accelerates reactions by decreasing free Gibbs energy. The substrates are bound to a specific region of the enzyme called active site. This interaction promotes the transition of state. The active site has the following features: 1. 3D cleft or crevice 2. Takes up a small part of the total volume of an enzyme 3. Unique micronutrients 4. Substrates are bound to enzymes by multiple weak attractions 5. The specificity of binding depends on the precisely defined arrangement of atoms in an active site. MODULE 3: BIOENERGETICS Biochemical thermodynamics, aptly referred to as Enthalpy, H – heat content of the species present bioenergetics- deals with transformations of energy in the system related to biochemical reactions (Murray et al., Entropy, S – represents the degree of disorder or 2013). randomness in a system Cells constantly require energy to do work and perform tasks such as maintaining organized FREE ENERGY structures and building up cellular components, Two energy quantities (G and H) as well as entropy among others. (S) can be related to each other by combining the first two laws, given by the equation. ∆G= ∆H -T∆S where ΔG = change in Gibbs free energy, ΔH = change in enthalpy, and ΔS = change in entropy. The sign of the change in Gibbs free energy indicates the state of the free energy of the system, whether it is lost or gained by the biological system. LAWS OF THERMODYNAMICS To achieve homeostasis, biological systems are regulated at constant temperatures (isothermic condition). Doing so requires and affects the transformation of energy from chemical energy into other forms via biochemical reactions. If the reactants are present in 1.0 mol/L at a pH of The energy transformations associated with 7.0, the standard free-energy change, ΔG° can be biological processes are always bound by the Laws of calculated from the equilibrium constant Keq Thermodynamics. ∆G°=-RTlnK’eq First Law of Thermodynamics, where it is established Where that “for any physical or chemical change, the total energy in the universe remains constant, but energy ΔG° = standard change in free energy, itself can be transformed from one form to another.” R = gas constant (8.314 J/mol-K), and Second Law of Thermodynamics specifies that to Keq = equilibrium constant. enable a process to proceed spontaneously, the total entropy of the system must increase. The actual free energy may vary from the standard Three variables (other than temperature) that we free energy change due to several factors, such as the use to quantify the energy possessed by biological [reactant] as well as the presence of ions and even systems. proteins. Gibbs free energy, G – amount of energy available COUPLED REACTIONS to by used by cells to perform work in a reaction at Since endergonic reactions need to consume free constant T and P energy to proceed, these reactions are coupled to exergonic reactions to form a coupled exergonic- endergonic system with an overall exergonic net change. The free energy diagram below shows an endergonic conversion of C → D coupled to the exergonic reaction A → B. The amount of energy associated with the hydrolysis of a phosphate intermediate can be compared to the ΔGo of ATP at 37 °C. Low-energy phosphates have free energy values lower than that of ATP, whereas the high-energy phosphates possess higher free energy values than ATP. In biological systems, some exergonic-endergonic systems are coupled through an obligatory intermediate, I. A+C→I→B+D Dehydrogenation reactions are coupled to hydrogenation reactions through an intermediate carrier, as shown below. HIGH-ENERGY COMPOUNDS Biological processes such as biosynthesis, muscular contraction, nervous excitation, and active transport, among others, use high-energy compounds to couple endergonic and exergonic BIOLOGICAL REDOX REACTIONS reactions. The principal high-energy compound in the living cell is adenosine triphosphate (ATP). Oxidation-reduction reactions, also known as redox reactions, involve the transfer of electrons by one chemical species (through oxidation) to another species (through reduction). This flow of electrons directly correlates to the amount of energy cells receive to perform work. For heterotrophic organisms, the electron donors are the food sources they consume. Cells possess molecular energy transducers that convert the Below is a list of some substrates with their energy associated to the flow of electrons into work corresponding coenzymes as they undergo redox (Lehninger, Nelson, and Cox, 2014). The oxidation of reactions. glucose, C6H12O6, a sugar, provides energy to drive the synthesis of ATP. The overall reaction comes from a pair of oxidation and reduction half reactions. Many biological systems employ dehydrogenation where one or two hydrogen atoms are transformed into an acceptor. The enzymes responsible for catalyzing this type of reaction are referred to as dehydrogenases. Electrons can be transported from one species to another in any one of these mechanisms: 1. Direct transfer 2. As hydrogen atoms – for example, AH2 reduces B via transfer of H atoms AH2+B⇌A+BH2 3. As a hydride ion – occurs for NAD-linked dehydrogenases 4. Direct combination with oxygen Many enzymes (>200) catalyze reactions by using NAD+ and/or NADP+ (ionized forms of Nicotinamide Adenine Dinucleotide and Nicotinamide Adenine Dinucleotide Phosphate, respectively) as an acceptor of hydride from the reduced chemical species NADH and/or NADPH. Many enzymes (>200) catalyze reactions by using NAD+ and/or NADP+ (ionized forms of Nicotinamide Adenine Dinucleotide and Nicotinamide Adenine Dinucleotide Phosphate, respectively) as an acceptor of hydride from the reduced chemical species NADH and/or NADPH. The general reactions involving these species are as follows: MODULE 4.1: SUGARS BIOLOGICAL FUNCTION Primary source of energy Storage of energy Cell structure Cell recognition Cell communication/signal Genetic information component Chirality BASIC COMPOSITION Monosaccharides contain 1 or more chiral carbon Glycans basic composition: (CH2O)n atom Monosaccharides – simple sugars with multiple OH Optically active isomeric forms groups. e.g. Triose, tetrose, pentose or hexose Disaccharides – 2 monosaccharides covalently linked Oligosaccharides – few monosaccharides covalently linked. Polysaccharides – polymers consisting of chains of monosaccharide or disaccharide units MONOSACCHARIDES STEREOISOMERS ALDOSES EPIMERS Sugars that differ in the configuration around one carbon atom. KETOSES D vs. L Designation For sugar with more than one chiral center, D or L refers to the assymetric C farthest from the ald or keto group. Most naturally occurring sugars are D-isomers FORMATION OF HEMIACETAL & HEMIKETAL GLUCOSE IN RING FORM DISACCHARIDE HAYWORTH PROJECTION FISCHER PROJECTION POLYSACCHARIDES  CHITIN STARCH CONFORMATION AT GLYCOSIDIC BOND CELLULOSE