PHTH1011 003 Proteins and Enzymes.pdf

Protein and Enzymes Dr. L. Lindo Sequencing Polypeptides Determine which aa is present at the beginning of the sequence: A) Use a CHEMICAL REACTION B) A Proteolytic Enzyme to get the entire sequence Chemical Reaction 1.Use 1-fluoro-2-4-dinitrobenzene (DNB) or known as Sanger Reagent Nobel prize for...

Protein and Enzymes Dr. L. Lindo Sequencing Polypeptides Determine which aa is present at the beginning of the sequence: A) Use a CHEMICAL REACTION B) A Proteolytic Enzyme to get the entire sequence Chemical Reaction 1.Use 1-fluoro-2-4-dinitrobenzene (DNB) or known as Sanger Reagent Nobel prize for aa sequence for insulin Done under slightly alkaline condition React Sanger Reagent with a simple polypeptide chain SANGER REAGENT Eg. NH2-Ala *-Gly—*Phe……..Lys-COOH Under Basic conditions so OH- displaces the F-(NO2)2Phenyl-NH-Ala-Gly-Phe….Lys-COOH But if in acid-breaks the bonds at *: Phenyl-NH-Ala-COOH (yellow ) + +NH3-Gly-COOH + +NH -Phe-COOH 3 Use Organic Solvent to extract the derivative of Ala Do TLC get Rf value and compare and determine the aa by comparing with Standard Sanger Reagent cont. Problem: Will obtain all the other aa’s in solution as well Destroys the rest of the polypeptide chain Edman Degradation (ED) Edman Degradation –phenylisothiocyanate Ph-N=C=S Basic conditions: it labels the first amino acid-PTH-alanine Phenyl-N=C=S + NH2-Ala+Gly+Phe+……Lys-COOH forms a COMPLEX of Phenyl-N-C-S-Ala + Gly-Phe ….Lys-COOH (the remainder of the aa remains intact) The reagent can be used repeatedly therefore will be done several times to determine the sequence of the polypeptide. This is for 50 aa’s -ED not efficient so use gel electrophoresis First, cleave the polypeptide with an enzymeproteolytic cleavage so have smaller fragments – Then trypsin Then chymotrypsin then use polyacrylamide gel electrophoresis Dansyl Chloride 3. Dansyl chloride or 5-(DimethylAmino)Naphthalene-1- sulfonyl chloride a reagent that reacts with primary amino groups in both aliphatic and aromatic amines to produce stable blue- or blue-green– fluorescent sulfonamide adducts. It can also be made to react with secondary amines. reacts like Sanger Reagent-more useful for a brominated sample and aa in small quantity eg. cyanogenbromide- (CNBr) This will react with ONLY the N-terminus of AA OTHERS Cyanogen bromide (CNBr) cleaves at methionine (Met) res BNPS-skatole - (2-(2-Nitrophenylsulfenyl) cleaves at tryptophan (Trp) residues formic acid cleaves at aspartic acid-proline (Asp-Pro) peptide bonds hydroxylamine cleaves at asparagine-glycine (Asn-Gly) peptide bonds and 2-nitro-5-thiocyanobenzoic acid (NTCB) cleaves at cysteine (Cys) Proteolytic Enzymes Trypsin-cleaves at the peptide bond on the carboxyl side of Arg, Lys,-basic positively charged aa’s Chymotrypsin-cleaves on the carboxyl side of aromatic aa’s eg Phe, Tyr, and Tryp papaya (papain) destructive- wide specificity-does not act on acidic residues – Used in Meat tenderizer-the enzyme breaks down collagen in meat hence becomes tender. Thermolysin Bacillus thermoproteolyticus thermolysin is a thermostable zinc endopeptidase contains zinc ion (for catalysis) and four calcium ions necessary for its thermal stability.... Proposed mechanism for thermolysin - catalysed cleavage of peptides Amino linked bonds of aliphatic aa’s- ie. Non-polar R groups Eg Val, Ile, Leu- resist heat Eg with trypsin and chymotrypsin H2N-Ala-Gly-Ser-Arg-Val-Phe-Leu-Gly-COOH with TRYPSIN: cleaves at COOH of Arg (pos charged) to give H2N-Ala-Gly-Ser-Arg-COOH + -H2N-Val-Phe-Leu-Gly-COOH With chymotrypsin:cleaves at aromatic aa on the carboxyl side to give H2N-Ala-Gly-Ser-Arg-Val-Phe-COOH + H2N-Leu-Gly-COOH Eg CNBr Methionine specific for C terminus-carboxy peptidase H2N-Ala-Gly-Phe-Met-Ala-COOH to give H2N-Ala-Gly-Phe-Met-COOH + H2N-Ala-COOH Hydrazinolysis-NH2-NH2 Hydrazinolysis the most frequently employed chemical method for the identification of Cterminal amino acids and occasionally, of their neighboring residue. The quantitative aspects of hydrazinolysis are more problematical than the qualitative identification of the C-terminal amino acid. Proteins Are large molecules with many different shapes and structures. Are the primary structural component of all tissues in humans and all other animals. Build, maintain and repair the tissues in the body. Are highly specialized and have specific purposes in the body. 15 PROTEINS All proteins are composed of amino acids linked together. There are 20 different types of amino acids that constitute the monomer units of proteins. Importance of Proteins Structural components Signal or communication molecules Enzymes Transport molecules Hormones 17 Proteins The bond that holds two amino acids together is called a peptide bond. Short chains of amino acids, generally containing fewer than 50 amino acids are referred to as peptides. Longer chains of amino acids are referred to as polypeptides. Each protein has its own combination of different amino acids. 18 PROTEIN STRUCTURE 19 Protein shapes The reasons for the shapes (bonding arrangements): Peptide bonds (primary) “Hydrogen bonding” (secondary & tertiary) Ionic attraction/bonds (tertiary) Hydrophobic & hydrophilic interactions (tertiary) Covalent sulfur bonds (tertiary) Quaternary is dependent on the tertiary structure of the individual polypeptides and so is influenced by these bonds. 20 Question: Class of Protein? 21 * h bonds-hydrogen bonds 22 Protein denaturation Proteins are denatured by: Heat- eg. Eggs fried Mechanical agitation-shaking Detergents – tide, breeze Organic compounds-eg. ethanol pH changes- sour milk Inorganic salts – salt 25 Enzymes (as Biological Catalysts) Enzymes are proteins that increase the rate of reaction by lowering the energy of activation They catalyze nearly all the chemical reactions taking place in the cells of the body. Not altered or consumed during reaction. Reusable ACTIVE SITE The area on the enzyme where the substrate or substrates attach to is called the active site. Enzymes are usually very large proteins and the active site is just a small region of the enzyme molecule. SUBSTRATE In enzymatic reactions, the substance at the beginning of the process, on which an enzyme begins its action is called substrate. When a substrate binds to an enzyme it forms an enzyme-substrate complex Enzyme molecules contain a special pocket or cleft called the active sites. APOENZYME and HOLOENZYME The enzyme without its non-protein moiety is termed as apoenzyme and it is inactive. Holoenzyme is an active enzyme with its non-protein component. Cofactors a non-protein chemical compound that is boun(either tightly or loosely) to an enzyme and is required for catalysis. Types of Cofactors: Coenzymes. Prosthetic groups. Types of Cofactors Coenzyme: The non-protein component, loosely bound to apoenzyme by non-covalent bond. Examples : vitamins or compound derived from vitamins. Prosthetic group The non-protein component, tightly bound to the apoenzyme by covalent bonds is called a Prosthetic group. Enzyme Specificity Enzymes have varying degrees of specificity for substrates Enzymes may recognize and catalyze: - a single substrate - a group of similar substrates - a particular type of bond Mechanism of Action of Enzymes Enzymes increase reaction rates by decreasing the Activation energy: Enzyme-Substrate Interactions: ‒Formation of Enzyme substrate complex by: ‒Lock-and-Key Model ‒Induced Fit Model Lock-and-Key Model In the lock-and-key model of enzyme action: - the active site has a rigid shape - only substrates with the matching shape can fit - the substrate is a key that fits the lock of the active site This is an older model, however, and does not work for all enzymes Induced Fit Model In the induced-fit model of enzyme action: - the active site is flexible, not rigid - the shapes of the enzyme, active site, and substrate adjust to maximumize the fit, which improves catalysis - there is a greater range of substrate specificity This model is more consistent with a wider range of enzymes Enzyme Catalyzed Reactions When a substrate (S) fits properly in an active site, an enzyme-substrate (ES) complex is formed: E + S  ES Within the active site of the ES complex, the reaction occurs to convert substrate to product (P): ES → E + P The products are then released, allowing another substrate molecule to bind the enzyme - this cycle can be repeated millions (or even more) times per minute The overall reaction for the conversion of substrate to product can be written as follows: E + S  ES → E + P What Affects Enzyme Activity? Three factors: 1. Environmental Conditions 2. Cofactors and Coenzymes 3. Enzyme Inhibitors 1. Environmental Conditions 1. Extreme Temperatures are the most dangerous high temps may denature (unfold) the enzyme 2. pH (most like pH 6 - 8 near neutral) 3. substrate concentration. Environmental factors Optimum temperature: The temp at which enzymatic reaction occur fastest. Environmental factors pH also affects the rate of enzyme-substrate complexes Most enzymes have an optimum pH of around 7 (neutral) However, some prefer acidic or basic conditions Substrate Concentration and Reaction Rate The rate of reaction increases as substrate concentration increases (at constant enzyme concentration) Maximum activity occurs when the enzyme is saturated (when all enzymes are binding substrate) 2. Cofactors and Coenzymes Inorganic substances (zinc, iron) and vitamins (respectively) are sometimes needed for proper enzymatic activity. Example: Iron must be present in the quaternary structure - hemoglobin in order for it to pick up oxygen. COMPETITIVE INHIBITORS Enzyme inhibitors- Some chemicals inhibit the action of an enzyme. A competitive inhibitor is a molecule that resembles the substrate enough that it can bind to the active site in place of the substrate. This will slow down the reaction rate as a certain percentage of the enzyme will combine with the inhibitor. Competitive Inhibitors block enzyme activity by mimicking the substrate 48 NON-COMPETITIVE INHIBITORS A noncompetitive inhibitor is one that does not bind to the receptor site but to some other place on the molecule causing a conformational change in the enzyme (protein). This causes the active site to change shape so that substrate cannot bind. This also slows down the reaction rate. Effect of Noncompetitive Inhibitors and Noncompetitve inhibitors Enzymatic Reaction Rates block enzyme function too, but attach a different point than the active site 50 Naming Enzymes The name of an enzyme in many cases end in –ase For example, sucrase catalyzes the hydrolysis of sucrose The name describes the function of the enzyme For example, oxidases catalyze oxidation reactions Sometimes common names are used, particularly for the digestion enzymes such as pepsin and trypsin Some names describe both the substrate and the function For example, alcohol dehydrogenase oxides ethanol Enzymes Are Classified into six functional Classes (EC number Classification) by the International Union of Biochemists (I.U.B.) on the basis of the Types of Reactions that they catalyze: Oxidoreductases Transferases Hydrolases Lyases Isomerases Ligases Oxidoreductases Biochemical Activity: Catalyse Oxidation/Reduction Reactions Act on many chemical groupings to add or remove hydrogen atoms. Examples: Lactate dehydrogenase Glucose Oxidase Peroxidase Catalase Phenylalanine hydroxylase Transferases Biochemical Activity: Transfer a functional groups (e.g. methyl or phosphate) between donor and acceptor molecules. Examples: Transaminases (ALT & AST). Phosphotransferases (Kinases). Transmethylases. Transpeptidases. Transacylases. Hydrolases Biochemical Activity: Catalyse the hydrolysis of various bonds Add water across a bond. Examples: Protein hydrolyzing enzymes (Peptidases). Carbohydrases (Amylase, Maltase, Lactase). Lipid hydrolyzing enzymes (Lipase). Deaminases. Phosphatases. Lyases Biochemical Activity: Cleave various bonds by means other than hydrolysis and oxidation. Add Water, Ammonia or Carbon dioxide across double bonds, or remove these elements to produce double bonds. Examples: Fumarase. Carbonic anhydrase. Isomerases Biochemical Activity: Catalyse isomerization changes within a single molecule. Carry out many kinds of isomerization: L to D isomerizations. Mutase reactions (Shifts of chemical groups). Examples: Isomerase. Mutase. Ligases Biochemical Activity: Join two molecules with covalent bonds Catalyse reactions in which two chemical groups are joined (or ligated) with the use of energy from ATP. Examples: Acetyl-CoA Carboxylase. Glutamine synthetase