PROTEINS AND ENZYMES =ROTEINS AND ENZYMES I. PROTEINS Introduction * Proteins are in different forms and innumerable amount * They occupy a prominent position in a cell and were summarized by Muldar asa substance existing in a cell whose absence would mean no life * Another philosopher summarized that there is life if there isa protein and where there isa protein there is life Definition of a Prot * Polymers of amino-acids linked together by peptide bonds to form peptides which link to form polypeptides (proteins) * The number of amino acid., chemical in nature and sequential order of amino acids determines the structure and function of a protein * Amino acids are linked by peptide bonds to form polypeptide bonds * The a-COO" of one amino acid, is joined to a — NH group of another amino acta bya peptide bond (amide bond) Bo foe [ese ieee eee 4n0— ca =O of HyN— ¢~ €-O . iu “y" we Noe id coer piel ee . Ternunal HgN es ca Fermmal i Amide. Bond 1a) b) * The equilibrium ofthis reaction les far on the side of hydrolysis rather than synthesis hence biosynthesis of the peptide bond requires an input of fee erg whereas hydrolysis in thermodynamically downhill, * Many amino acids. are joined by peptide bonds to form a polypeptide chain which isan unbalanced structure © The amino acid. unit in a polypeptide is called a residue * A polypeptide chain has direetion because its building blocks have different ends namely the a ~amino and « ~ carboxyl groups * By convention the amino end is taken tothe beginning ofa polypeptide chain * The sequence of amino acid. in a polypeptide chain is writen starting with the amino terminal residue * There is a free NH group at the N-terminal and a fice COOH group atthe C-terminal end * The properties of a peptide depend on the nature of R groups, NH and COOH group and the electrical charge of the whole peptide * The structure and function of a peptide depends on The number and kind of amino acid, ‘The sequential order of amino acid. (also called the primary structure) Eunctions/Importance of Proteins Proteins play a crucial role in virtually all biological processes, The remarkable scope of their functions are exemplified in a) b) d) Fnsymatic catalysis — nearly all chemical reactions in biological system are catalyzed by spevific macromolecules called enzymes. They enhance reaction rates by at least a million fold. Several thousand enzymes have been characterized and many have been crystallized. All known enzymes are proteins Generation and transmission of nerve impulses. The response of nerve cells to specitic stimuli 'smedliated by receptor proteins .g. rhodopsin isthe photo receptor protein in retinal rod cells Control of growth and differentiation e.g, DNA and RNA control the sequential expression of senetic information essential for the orderly growth and differentiation of cells Regulatory ~ polypeptide hormones e.g, insulin and glucagon secreted by the Panereas are transported to other organs and regulate sugar (glucose) metabolism 2©) Mechanical and structural: mechanical involves contractile proteins found in muscles whereas Structural involves fibrous proteins e.g, elastin, fibroin and keratin 1) Transport e.g. hemoglobin and myoglobin transport O2 and COs, Others are BI lipoprotein and serum albumin 8) Defensive e.g, immune-proteins involved in specific immunity, fibrinogen and thrombin involved in blood clotting hh) Nutrient and. Storage e.g, Gliadin, Ovalbumin, Casein, Ferritin }) Contractile and Motile eg. actin, myosin, tubulin, dynein J) Others ~ antifreeze, proteins in arctic animals 1) Covalent: formed by 2 atoms where each donates electrons eg disulphide, peptide bonds 2) Coordinate covalent bonding: similar to covalent only that the electrons shared are NOT donated by both atoms but only ONE atom. It is weaker than covalent readily made and broken 5) Jonie forces: traction of 2 groups of opposite charges e.g. NH and COOH 4) Hydrogen bonding: shaving of H atoms between 2 electronegative atoms which unbounded ‘lectrons, They contribute tothe thermodynamic stability ofa folded proteins confirmation 9) Van-de-Waals attractive forces ~ operate between all atoms, ions and molecules and are due ‘0 axed dipole in one molecule including an oscillating dipole. No electrons are shared 6) Hydrophobic interactions caused by non-polar R groups ~ This is a force rather than a bond 1) Electrostatic bonds — can be repulsive ot attractive depending on whether the interacting charges are the same or opposite, The strength of the electrostatic force is directly dependent on the charge of the ion Structural Level of Protein: Structural Level of Proteins a) Configuration The arrangement of substituent groups of molecules in space to form stereoisomers, which cannot be interconverted unless one or more covalent bonds are broken b) Conformation Arrangement of certain groups in such a way that they assume different forms in space without breaking covalent bondsProteins are well defined 3-dimensional structures The peptide unit is rigid and planar The hydrogen of the substituted amino group is nearly always trans to the O» of the carbonyl group ‘Tete is no freedom of rotation about the bond between the carbonyl carbon atom and the Neatom of the peptide unit because this link has partial double bond character 4 ior —o N ta c= ae H H ‘The Tink between the a — carbon atom and a cavbonyl carbon atom is a single bond, There 'salso a single bond between the a carbon and the N2 atom hence there is a large degree of rotational freedom about these bonds on ether side ofthe rigid peptide bond Quatemary Tertiary 1 Domains t Super Secondary I Secondary f PrimaryPrimary Structure The sequence of amino acid and location of disulphide bonds if any in a polypeptide Ttis the complete description of the covalent connections ofa protein Tt gives the protein its physical properties and causes a polypeptide chain to fold into a unique structure giving it a characteristic function and role The primary structure gives rise to other levels Secondary Structure The steric relationship of amino acid, that are close to one another in a linear sequence Some of these steric relationships are of a regular kind giving rise to a periodic structure i) a-helix Itis rod-like structure ‘The tightly coiled polypeptide main chain forms the inner part of the rod The side chains extend outwards in a helical array The helix is stabilized by hydrogen bonds between the NH and COO groups of the main chain The COO” group of each amino acid. is hydrogen bonded to the NH group of the amino acid. that is situated 4 residues ahead in the linear sequence i) B-pleated sheet The polypeptide chain is fully extended rather than tightly coiled The axial distance between adjacent amino acid, is 3.5A° rather than 1.5A® in the achelix Stabilized by hydrogen bonds between NH and COO groups in different polypeptide strands Adjacent strands can run in the same direction (parallel — B-sheet) or run in opposite directions (antiparallel — B-shect) e.g, silk fibroin is antiparallel N G cC—__n c Nor cC——_n Anti-parallel Paralleliii) Tertiary ‘This isthe steric relationship of an amino acid, that are far apart in a linear sequence to the ‘otal 3-D structure ofthe protein including geometric relationship between distant segments of the 1° structure and the relationship of the side chain group with respect to each other in 3 dimensional space iv) Quaternary Structure Refers to the way the polypeptide chains of @ protein that contains more than one Polypeptide chain are packed together. Each chain is called a sub-unit or domain (compact globular unit) The helix has rotation of 100° which gives ~4 amino acids, resides per tum ‘The helix can be right or left handed, however helices found in ‘most proteins are the former EG lec! lof Faced Right HYanele Fach or repeat is~0.54nm in length, As the helix turns, the a —helix creates a hypothetical cylinder There are other helices such as, 1 and 31 although the mis regarded as hypothetical while the 310s very rare and only observed in a few parts ofa backbone ‘The a ~ helix forms more readily than others since it makes maximum use of internal H bonding ‘The a —helix can be formed either by D or L amino acid, however, helices are not formed by a mixture of both L and D amino acid, Right handed a — helices are found mainly in structural proteins e 8. o-keratin and silk (fibroinyExamples of Protein Conformations 1. Simple Proteins 4) Fibrous Proteins * Composed of individual polypeptides chains which are arranged in the form of coils * The proteins are though and insoluble in Hs thus making their ideal for structural functions * Examples are collagen, a-keratin, silk, * The coiling polypeptide chains are held by disulphide bonds in the case of a-keratin thus keratin has a lot of eysteine, * Collagen coils are held by hydrogen bonds. Collagen is rich in glycine, 8) Globular Proteins * Unlike fibrous proteins they are soluble in water and salt solutions Usually have tertiary or quaternary structures, These are biologically active proteins e.g, enzymes, hormones, toxins and antibodies + Examples; )) Albumins ~ They are the majority of globulins, They are soluble in H20 and coagulate when heated ii) Globulins ~ They are insoluble in the H2O and coagulate when heated. Soluble in salt sol ns iil) Histones — basic proteins that bind DNA iv) Protamine — basic proteins that complex with nucleic acids 2. Conjugated Proteins ~ _ These are proteins complexed to other molecules or moieties e.g, 8) Nucleoproteins ~DNA or ma b) Glycoproteins ©) Lipoproteins 4) Phospho-proteinsDenaturation of Proteins The majority of proteins when subjected to extreme temps and gH values and pressure they undergo chemical physical and biological changes. This is referred to as denaturation 1. Changes which take place + Decrease in solubility of proteins * Alteration in internal structure of the proteins ~ the arrangement of the peptide chains, Denaturation does not usually affect the primary structure of the protein * Changes in the symmetry of the protein * There can be increased reactivity ofthe proteins due to ionizable-R groups * The proteins are easily hydrolyzed by proteolytic enzymes * The proteins lose their biological activity 2, Factors leading to denaturation * Changes in the pH value of the solution * High temperatures © UNV radiation * Ultrasonic vibration — sonication * Vigorous shaking and stirring of proteins * High concentration of neutral polar. Compounds e.g. urea, uri ine which breakdown the He bonds and allow new ones to be formed thus changing the conformation of the protein * Treatment with organic solvents e.g, ethanol * Denaturation is a reversible reaction if the S-S bonds are not broken * The denaturation process does not create a new biological function. Even after enaturation, the protein doesn’t acquire a new biological function * The amino acid sequence of a PP chain determines the biological role of proteins hecause the amino acid. sequence never changes and determines the native conformation of the protein and since the conformation is never changed the proteins role never changes Amino Acid ~—» ative conformation —____ biological roleMethods of Protein Separation Proteins behave like solutes in aqueous and salt solutions and can be electrolytes or macromolecules whose separation depends on 1) Electrical charge 2) Molecular size 3) Solubility 4) Adsorption 5) Biological affinity for other molecules I, ENZYMOLOGY 1, INTRODUCTION 1.1 Historical background In the nineteenth century it was considered that processes such as fermentation could only take place through the action of living organisms 1m 1835, diastase (Amylase) was partially isolated later pepsin was extracted from gastric Juice, pepsin, diastase and other active preparations were given the name ferments The name enzyme was first proposed by Kuhn in 1878 and this name replaced the term ferment {n 1897 the Buchner’s showed that sugar fermentation could take place when a yeast cell extract was added even though no living cells were present 4m 1926, Sumner erystallize urease from Jack — bean extracts and in the following years many other enzymes were purified and crystallized 1.2 What are Enzymes ‘They are biological catalysts They increase the rate of chemical reactions taking place within living cells without themselves suffering any overall change The reactants of enzyme-catalyzed reactions are called substrates each enzyme acts on a articular substrate (s) to produce products Allenzymes are proteins However, enzymes required a non-protein component called a cofactor for catalytic activity* The cofactor maybe an organic molecule this is called a coenzyme or maybe a metal ion * When a cofactor is bound so tightly that itis difficult to remove without damaging the enzyme itis called a prosthetic group Classification of Enzymes * Neatly all enzyme names end with the prefix “ASE” except proteolytic enzymes whose names end with “in” * The names of enzymes usually indicate the substrate involved e.g. Lactase hydrolyses Lactose, Fumerase hydrolyses Fumerate * Other names may indicate the nature of the reaction without specifying the substrate eg, ‘transcarboxylase while other names indicate neither the substrate nor the reaction Enzyme Commission’s (E.C.) System of Classification In this classification, the systematic name includes the name of the substrate () in full and a word ending in “ASE” indicating the nature of the process catalyzed 1. Systematic Name a) Oxido-reduetases Catalyze the transfer of H, O2 or electrons from one substrate to another, Trivial names of oxidoreductases include oxidases dehydrogenases b) Transferases ‘Transfer atoms or groups between 2 molecules (excluding reactions in other classes eg Ax+B #———_+ Bx4 A Others - oxidoreductase and hydrolases examples i) Methyltransferases ii) Hydroxymethyl (CH2OH) Transferases iii) Carbonyl transferases iv) Phosphotransferases (trivial name ending in “Kinase” e.g, D-Hexose-6-Phosphotransferase also called Hexokinase) ©) Hydrolases 10Catalyze hydrolytic reactions AX+H2O +———+ XoH + HA Hydrolyze a) Ester bonds e.g, Carboxylic Ester Hydrolases b) Thiol bonds e.g. Thiol Hydrolases ©) Phosphoric Monoester Hydrolases ) Peptide bonds ©) Glycosidic linkages ) Lysases Catalyze the non-hydrolytic removal of groups from substrates often leaving double bonds e.g. remove Carboxyl, Aldehyde and Keto-acid groups, ©) Isomerases Catalyze isomerization reactions e.g. i) Racemization or Epimerization it) Cis-Trans Isomerization Will act on Q,G, Hydroxyacids, carbohydrates eg. L-Alanine «+» D-Alanine Alanine Racemase (Epimerase / Isomerase) 1) Ligases Catalyze the synthesis of new bonds coupled to the breakdown of ATP ot other nucleoside triphosphates X+Y+ATP «+ X_-y+App+p, ‘Trivial names are Synthases or Synthetases aIL Class Specificity 8) Absolute specificity Enzyme exhibits absolute specificity when it only catalyzes one reaction and act on only one substrate b) Group Enzyme acts on a class of substrates that have common functional groups e.g. phosphatase can act ona variety of substrates which must have a phosphate group ©) Linkage Some enzymes are specific for a particular type of bond e.g, Esterase can only hydrolyze ester bonds 4) Stereo-specificity Enzymes have the eapacity to discriminate D and L isomers and can only catalyze reactions of one isomer and not the other Cofaetors * Some enzymes are only active in the presence of non-protein compounds referred to as cofactors + These compounds can be; i) Metal ions ii) Inorganic compounds * A Structure or complex consisting of the enzyme and cofactor is referred to as a Holo- enzyme * A Holo-enzyme without its cofactor is referred to as an Apo-enzyme * Enzymes requiring metal ions are referred to as Metallo-enzymes 2Functions of Cofactors * Actas binding bridges building the substrate to the enzyme through the formation of a coordination complex + May act as primary catalytic centers * Act of agents that stabilize the confirmation of the enzyme in its catalytically active form Factors affecting the rate of a chemical r 1 Temperature * The activity of proteins depends on temperature. Proteins function optimally at their optimum temp * Generally, an increase in temp by 10% doubles the enzyme activity until it reaches its optimum temp * Enzyme activity is dependent on sH of the medium. The enzyme with function optimally ‘within its optimum pH range e.g. Pepsin is 1-2 while Trypsin is 8-9. 2. Concentration of Enzyme * The rate of chemical reaction is directly proportional to the concentration of the enzyme. Ata fixed substrate concentration. The rate of reaction is used as a measure of enzyme concentration, 3. Concentration of Substrate * At fixed enzyme concentration, the initial rate of reaction increases with an increase in substrate concentration until the enzyme is saturated with the substrate 4. Presence of inhibitors * Inhibitors are compounds that regulate or stop the rate of reaction but are not enzymes. 5. Presence of Allosteric Effectors * Allosteric enzymes apart from their active sites have other sites known as Allosteric sites * Allosteric effectors are compounds that are either products of the enzymes reaction or Products of other metabolic pathway that modulate the activity of enzymes, 2BCatalytic Effect of Enzymes on Chemical Reaction i) {in any population of molecules individual molecules have individual potential energy PE) Variation in P-E. can be described using a special curve. Some molecules have a higher eneray than others. Others have a low energy while the majority have an average amount OfP.E. Suppose we have the reaction. A ———+ Pp A Certain function of molecules of A are being converted to P order to be converted to P, the faction of A should possess an amount of energy to enter into the transition state at which there is a high probability that chemical bonds can be broken and created easily. Therefore, energy of activation is required t ee Lrecg Bi Brguss GMeachon The above reaction is reversible and reaches a dynamic equilibrium, ‘The rate of reaction is a to the amount of molecules in the Transition State, The rate of reaction can be accelerated by; Reising the temperature ~ this increases the chances of the molecules reaching the transition state Addition of a catalyst (enzyme). The enzyme will 8) accelerate the forward to reverse reaction ice. it doesn’t alter the equilibrium b) the lower the energy of activation by forming a transition state [ES] that has a lower energy of activation than if the substrate was alone 4Active Site Concept of Enzymes ‘The substrate is bound to a specific region of the enzyme called an active site Most enzymes are highly selective in their binding substrates The catalytic specificity of enzymes depends on the specificity of the binding process The active site is the region that binds the substrates and contributes the residues that directly participate in the making and breaking of bonds Features of the Active Sites a) b) °) dQ °) 8) h) The active site takes up a relatively small part of the total volume of an enzyme The active site is a three-dimensional entity. tis not a point, line or plane but an intricate 3-dimensional form made up of groups that come from different parts of the linear amino-acid sequence Substrates are bound to enzymes by relatively weak forces Active sites are clefis or crevices. The substrate molecules are bound to a cleft or crevice from which water is usually excluded unless it is a reactant. The cleft also contains several polar residues that ate essential for binding and catalysis. The non-polar character of the cleft enhances the binding of the substrate. The cleft creates a microenvironment in which certain polar residues acquire special properties essential for their catalytic role ‘The specificity of binding depends on the precisely defined arrangement of atoms in an active site ‘An enzyme usually has one active site per sub-unit 1m addition to an active ‘site? we may have an allosteric modifier site (regulatory site) Charged groups in the active site may include ‘) amino acid. side chains ii) Prosthetic groups iii) Metal ions e.g. Mg?*, Zn ‘The active site may consist of short stretches of the achelix and high % of the 6° pleated sheet structure 15‘Models proposed to account for enzyme specificity Lock and Key Hypothesis * Also called the Fischer hypothesis * Proposed by Fischer in 1890 * He suggested that enzyme specificity implied the presence of complementary structural features between the enzyme and substrate * The substrate fits into its complementary active site on the enzyme just as a key fits into a lock © All the structures remain fixed throughout the binding process Koshland hypothesis * Also called the induced fit hypothesis * Suggested by Koshland in 1958 * Suggests thatthe structure of a substrate maybe complementary to that ofthe active site in the enzyme-substrate complex but notin the free enzyme * A conformational change takes place inthe enzyme during the binding of substrate which fesults in the required matching of structures * Proposes that the active site is floppy and the substrate is rigid allowing the enzyme to wrap ‘self around the substrate hence bringing together the comesponding catalytic sites and reacting groups * Haldane in 1930 pointed out that ifthe binding energy was used to distort the substrate in Such a way as to facilitate the subsequent reaction, then less enerey would be required for the reaction to take place. This concept was further developed by Pauling in 1948. * Thus if the structure of the active site is rigid, the substrate must be distorted slightly in order to bind to the enzyme 16This distortion might result in the stretching and this weakening of a bond which is subsequent to be cleaved thus assisting the forward reaction The transition state stabilization form but the proposal assumes that the substrate is bound in understored form but the enzyme-substrate complex possess various unfavorable interactions Which tend to distort the substrate in such a way as to favor the following sequence: Enzyme ~ Substrate — Complex — Transition State ~ Products It is thought some enzymes preferentially bind a strained form of the substrate corresponding to the transition state. 7