MLS 064 Biochemistry: Proteins and Enzymes PDF

MLS 064 BIOCHEMISTRY FOR MLS: PROTEINS DEFINITION Proteins are the most abundant biological macromolecules, occurring in all cells. It is also the most versatile organic molecule of the living systems and occur in great variety; thousands of different kinds, ranging in size from r...

MLS 064 BIOCHEMISTRY FOR MLS: PROTEINS DEFINITION Proteins are the most abundant biological macromolecules, occurring in all cells. It is also the most versatile organic molecule of the living systems and occur in great variety; thousands of different kinds, ranging in size from relatively small peptides to large polymers. Individual amino acids (residues) are joined by peptide bonds to form the linear polypeptide chain. This linear polypeptide chain is folded into specific structural conformations. A protein can have up to four levels of structural conformations. COMPOSITION Carbon, hydrogen, oxygen, nitrogen, (sulfur) The nitrogen content of serum proteins is approx. 16% Mostly synthesized by the LIVER and secreted by the HEPATOCYTE into the circulation Water soluble Amphoteric: can be positively/negatively charged  CHARACTERISTICS a. Solubility in water The relationship of proteins with water is complex. The secondary structure of proteins depends largely on the interaction of peptide bonds with water through hydrogen bonds. Hydrogen bonds are also formed between protein (alpha and beta structures) and water. The protein-rich static ball is more soluble than the helical structures. At the tertiary structure, water causes the orientation of the chains and hydrophilic radicals to the outside of the molecule, while the hydrophobic chains and radicals tend to react with each other within the molecule (hydrophobic effect). b. Denaturation and Renaturation Agents such as heat and urea that cause unfolding of polypeptide chains without causing hydrolysis of peptide bonds can denature proteins. The denaturing agents destroy secondary and tertiary structures, without affecting the primary structure. If a denatured protein returns to its native state after the denaturing agent is removed, the process is called renaturation. c. Posttranslational modifications It occurs after the protein has been synthesized on the ribosome. Phosphorylation, glycosylation, ADP ribosylation, methylation, hydroxylation, and acetylation affect the charge and the interactions between amino acid residues, altering the three-dimensional configuration and, thus, the function of the protein. STRUCTURE OF PROTEINS STRUCTURE OF PROTEINS STRUCTURE OF PROTEINS A. Primary structure Primary structure of a protein gives the details of the amino acid sequence of a protein. By convention, the sequence of information is written from left to right, starting with the N-terminal amino acid and ends with the C-terminal. The primary structure of a protein will determine all other levels of structural organization of a protein. The primary structure is stabilized by peptide bonds, which are covalent in nature. At isoelectric pH (pI), the net charge is zero, and the protein does not migrate to anode or cathode. STRUCTURE OF PROTEINS B. Secondary structure The secondary structure includes various types of local conformations in which the atoms of the side chains are not involved. Secondary structures are formed by a regular repeating pattern of hydrogen bond formation between backbone atoms. Thus, secondary structures are stabilized mainly by hydrogen bonds. Two of the most important secondary structure in protein are: α- helix and the β-plates. STRUCTURE OF PROTEINS B. Secondary structure a. α- helix The alpha-helix is the most common secondary structure. An alpha-helix is generated when each carbonyl of a peptide bond forms a hydrogen bond with the –NH of a peptide bond four amino acid residues further along the chain. They are regular structures that repeat every 5.4 Armstrong Each helical turn in an alpha-helix contains 3.6 amino acids. The helical twist of the alpha-helix in all protein is right-handed. STRUCTURE OF PROTEINS B. Secondary structure b. β-conformations (β-plates) Beta-sheets are formed by hydrogen bonds between two extended polypeptide chains of between two regions of a single chain that folds back on itself. The chains of beta-sheets may run in the same direction (parallel) or in opposite directions (antiparallel). STRUCTURE OF PROTEINS c. Tertiary structure The tertiary structure of a protein refers to its overall three-dimensional conformation/overall shape of the protein molecule. It is produced by interactions between amino acid residues that may be located at a considerable distance from each other in the primary sequence of the polypeptide chain. Several types of bonds can contribute to a protein’s tertiary structure. The strongest but least common bonds, S-S covalent bonds called disulfide bridges, form between the sulfhydryl groups of two monomers of the amino acid cysteine. Many weak bonds—hydrogen bonds, ionic bonds, and hydrophobic interactions—also help determine the folding pattern. Some parts of a polypeptide are attracted to water (hydrophilic), and other parts are repelled by it (hydrophobic). Often, helper molecules known as chaperones aid the folding process STRUCTURE OF PROTEINS d. Quaternary structure The quaternary structure refers to the spatial arrangement of subunits in a protein that consists of more than one polypeptide chain. The subunits are joined together by the same types of noncovalent interactions that join various segments of a single chain to form its tertiary structure, as well as disulfide bonds. For a protein to have a quaternary structure, it should fulfil two conditions: a. It should have more than one polypeptide subunit and b. There should not have permanent (covalent) interaction between the subunits (like disulfide bond). Bonds stabilizing quaternary structure include hydrogen bonds, hydrophilic interactions, hydrophobic interaction and van der Waals interactions. CLASSIFICATION OF PROTEINS A. SIMPLE B. CONJUGATED C. DERIVED CLASSIFICATION OF PROTEINS a. Simple proteins: They are composed of only amino acid residue. On hydrolysis these proteins yield only constituent amino acids. It is further divided into: Fibrous protein: Elongated, asymmetrical polypeptide chains e.g., Keratin, Elastin, Collagen, troponin, fibrinogen Globular protein: Symmetrical, compactly folded polypeptide chains e.g., Albumin, Globulin, Glutelin, Histones CLASSIFICATION OF PROTEINS b. Conjugated proteins: They are combined with non-protein moiety. Eg. Nucleoprotein, Phosphoprotein, Lipoprotein, Metalloprotein etc. **It consists of protein (apoproteins) and a non protein group (prosthetic group: are commonly metal, lipid, and CHO in nature) -these proteins impart certain characteristics to the proteins 1. Metalloproteins: ferritin, ceruloplasmin, hemoglobin and flavoproteins 2. Lipoproteins: VLDL, LDL, HDL, chylomicrons 3. Glycoproteins: haptoglobin, a1-antitrypsin (with 10-40% CHO) 4. Mucoproteins/Proteoglycans: mucin (higher CHO content than CHON) 5. Nucleoproteins: chromatin (combined with NA) CLASSIFICATION OF PROTEINS c. Derived proteins: They are derivatives or degraded products of simple and conjugated proteins. They may be: Primary derived protein: Protean, Metaproteins, Coagulated proteins Secondary derived proteins: Proteases or albunoses, peptones, peptides. LIST OF SOME IMPORTANT FUNCTIONS OF PROTEINS FUNCTIONS OF PROTEINS LIST OF SOME IMPORTANT FUNCTIONS OF PROTEINS: 1. Regulate colloidal oncotic pressure 2. Act as carrier for transport of different substances 3. Coagulation cascade 4. Complement fixation 5. Serves as biocatalysts or enzymes 6. Maintenance of acid-base balance, proteins act as buffers. 7. Immunologic functions 8. Tissue repair 9. Generate energy through catalysis and electron transfer 10. Assemble molecules 11. Serves as ion channels and pumps 12. Serves as receptors, hormones and cytokines for intercellular regulation 13. Constitute signaling networks for intracellular regulation LABORATORY IDENTIFICATION OF AMINO ACIDS & PROTEINS  LABORATORY IDENTIFICATION OF AMINO ACIDS & PROTEINS A. Ninhydrin test Principle of test: Ninhydrin is specific for amino acids and proteins – to differentiate between carbohydrates and amino acids & proteins. Ninhydrin reacts with α-amino acids (-NH2) in proteins giving a purple complex, except proline and hydroxyproline gives yellow color (no -NH2). Ninhydrin is most commonly used as a forensic chemical to detect “fingerprints”, as amines left over from proteins sloughed off in fingerprints react with ninhydrin giving a characteristic purple color. B. Biuret’s test Principle of test: Biuret test is specific for proteins – to differentiate between proteins and amino acids. The biuret reagent (copper sulfate in a strong base) reacts with peptide bonds in proteins to form a blue to violet complex known as the “biuret complex.” At least two peptide bonds are required for the formation of this complex.  LABORATORY IDENTIFICATION OF AMINO ACIDS & PROTEINS C. Sakaguchi’s test Principles of test: Sakaguchi test is specific for arginine. Sakaguchi’s test is positive for the amino acid containing the guanidine group in arginine. Guanidine group present in the amino acid reacts with α-Naphthol and alkaline hypobromite to give a red-colored complex. D. Nitroprusside test Principle of test: It is specific for proteins containing sulfur, -SH (in cysteine & cystine), which give a red-purple color called “Morner test.”  LABORATORY IDENTIFICATION OF AMINO ACIDS & PROTEINS E. Millon’s test Principle of test: It is specific for tyrosine. Millon’s reagent (Hg/HNO3) gives positive results with proteins containing the phenolic amino acid “tyrosine.” F. Aldehyde test principle of test: It is specific for tryptophan. Sulfuric acid in presence of mercuric sulfate oxidized the indole nucleus of tryptophan. The product formed reacts with aldehydes to form violet colored complex. G. Xanthoproteic test Principle of test: This test is specific to the amino acids containing the benzene ring (aromatic amino acids). Phenylalanine, tyrosine and tryptophan reacts with concentrated HNO3 at high temperature to form nitro-compounds, which are yellow in color, and turn to orange color in an alkaline medium. MLS 064 BIOCHEMISTRY FOR MLS: ENZYMES  DEFINITION Enzymes are considered the heart of biochemistry Enzymes are proteins that help speed up metabolism, or the chemical reactions in our bodies Enzymes serve as biological catalysts for reactions in all living organisms. Remember: All enzymes are proteins but not all proteins are enzymes (Additional: Structurally, the vast majority of enzymes are proteins but in but since the 1980s the catalytic ability of certain nucleic acids, called ribozymes (or catalytic RNAs), has been demonstrated) CHARACTERISTICS They increase the rate of a reaction (10^6 to 10^12 times faster) They lower the activation energy Enzymes are very specific; each enzyme catalyzes a certain reaction or type of reaction only. Sensitive to pH and temperature Highly efficient Not consumed and are not permanently altered as they participate in a reaction.  NOMENCLATURE The International Union of Biochemistry and Molecular Biology assigns each enzyme a name and a number to identify them. Enzymes are named by adding the suffix -ase to the - name of the substrate that they modify (urease and tyrosinase) - the type of reaction they catalyze (dehydrogenase, decarboxylase). Some have arbitrary names (pepsin and trypsin) Each enzyme has a code number called Enzyme Commission Number or ‘EC’ Number Ex: Hexokinase = 2.7.1.1 Each number represents: 2 - Class 7 - Subclass 1 - 1st subclass 1 - Individual or 1st Enzyme  CLASSIFICATION OF ENZYMES 1. Oxidoreductase - enzymes involved in the transfer of electrons; they catalyze reactions involving removal of electrons from an electron donor and transfer them to an appropriate electron. 2. Transferase – involved in transferring functional groups between donors and acceptors. Transfer of chemical groups other than hydrogen from one substrate to another 3. Hydrolase – cause hydrolysis or splitting of a bond by the addition of water  CLASSIFICATION OF ENZYMES 4. Lyases - Catalyze the removal of groups from substrates without hydrolysis. The product contains double bond 5. Isomerases – Catalyzes the interconversion of optical geometric or positional isomers 6. Ligases - Catalyze the joining of two substrate molecules at the expense of an ATP “high energy phosphate bond”. 7. Translocase - Catalyzes the movement of ions or molecules across membranes  HOW ENZYMES WORK? Enzymes work by binding to reactant molecules(substrate) and holding them in such a way that the chemical bond-breaking and bond-forming processes take place more readily. HOW ENZYMES WORK? The mechanism of action of enzymes depends on the ability of enzymes to accelerate the reaction rate by decreasing the activation energy. During the course of the reaction, the enzyme (E) binds to the substrate/s (S) and forms a transient enzyme–substrate complex (ES). At the end of the reaction, the product/s are formed, the enzyme remains unchanged, can bind another substrate and can be reused many times. Active site or catalytic site is the specific place in the enzyme where the substrate binds.  FACTORS AFFECTING ENZYMATIC REACTION Temperature - chemical reactions get accelerated with a rise in temperature - Optimal: 37C A higher temperature generally makes for higher rates of reaction, enzyme-catalyzed or otherwise. However, either increasing or decreasing the temperature outside of a tolerable range can affect chemical bonds in the active site, making them less well-suited to bind substrates. Very high temperatures may cause an enzyme to denature, losing its shape and activity  FACTORS AFFECTING ENZYMATIC REACTION pH - Extreme pH level may denature enzyme or influence its ionic state resulting in structural change - Optimal: neutral 7 (5-8 range) for common enzyme - Optimal: pH 2 (1.5-3.5 range) for pepsin Active site amino acid residues often have acidic or basic properties that are important for catalysis. Changes in pH can affect these residues and make it hard for substrates to bind  FACTORS AFFECTING ENZYMATIC REACTION Enzyme concentration – the higher the enzyme concentration, the faster the reaction, because more enzyme is present to bind with the substrate Substrate Concentration – With the amount of enzyme exceeding the amount of substrate, the reaction rate steadily increases as more substrate is added Time - The longer an enzyme is incubated with its substrate, the greater the amount of product that will be formed Inhibitors and Activators ENZYMATIC REACTIONS 1. Zero- order reaction – the reaction rate depends only on the enzyme concentration 2. First – order reaction – the reaction rate is directly proportional to substrate concentration  ENZYME MODELS/THEORIES The Lock and Key Model - proposed by Emil Fischer in 1894 - According to the model, the shape of the substrate and the active site of the enzyme are thought to fit together like a key into its lock - The two shapes are considered as rigid and fixed, and perfectly complement each other when brought together in the right alignment. LOCK AND KEY THEORY  ENZYME MODELS/THEORIES The Induced Fit Model - proposed by Daniel E. Koshland, Jr. in 1958 - the induced fit model shows that enzymes are rather flexible structures in which the active site continually reshapes by its interactions with the substrate until the time the substrate is completely bound to it. INDUCED FIT THEORY  ENZYME INHIBITORS Enzyme inhibitors are molecules that interact with enzymes (temporary or permanent) in some way and reduce the rate of an enzyme-catalyzed reaction or prevent enzymes to work in a normal manner Enzyme inhibition can be either reversible or irreversible. Reversible inhibitor – binds to an enzyme but then enzyme activity is restored when the inhibitor is released a. Competitive inhibitor – has a shape and structure similar to substrate so it competes with the substrate for binding to active site b. Uncompetitive inhibitor binds only to the enzyme– substrate complex. c. Non-Competitive inhibitors – binds to the enzyme but does not bind at the active site Irreversible inhibitors - bind tightly to the enzyme and inactivate it. REVERSIBLE INHIBITORS  COFACTOR Not all enzymes consist exclusively of protein,many include additional chemical components or cofactors which serve as tools. It is a non-protein chemical that is bound to an enzyme and is required for catalysis Cofactors can consist of one or more inorganic ions (such as Fe3+, Mg2+, Mn2+, or Zn2+) or more complex organic molecules, known as coenzymes. Some enzymes require both types of cofactors. Coenzymes are organic compounds that bind to the active site of enzymes or near it. Many coenzymes are derived from vitamin precursors Some enzymes are active without coenzymes. However many, require a coenzyme to be active. An enzyme that is inactive in the absence of its coenzyme is called an apoenzyme. In the presence of its coenzyme to produce the active form of the enzyme, it is called a holoenzyme:  ENZYME KINETICS How enzyme functions Enzymes lower the activation energy, of a particular reaction. They can do this because they have a high affinity for a transition state. The activation energy is the minimum energy needed for a reaction to occur. Enzymes assist in the reaction so that less energy is needed. This means the reaction can occur more easily. This speeds up the rate of the reaction as it allows the product to be formed faster.  ENZYME KINETICS REACTION KINETICS When an enzyme is added to a substrate, the reaction that follows occurs in three stages with distinct kinetics: Phase Concentration of ES Rate of product formation Pre-steady state Rapid burst of ES complexes form Initially slow, waiting for ES to form, then speeds up Steady state(equilibrium) ES concentration remains Constant rate of formation, constant as it is being formed as faster than pre-steady state quickly as it breaks down Post-steady state Substrate depletes so fewer ES Slow as there are fewer ES complexes form complexes; slows down as substrate runs  ENZYME KINETICS Michaelis-Menten Kinetics Michaelis-Menten kinetics is a model of enzyme kinetics which explains how the rate of an enzyme-catalysed reaction depends on the concentration of the enzyme and its substrate. Let’s consider a reaction in which a substrate (S) binds reversibly to an enzyme (E) to form an enzyme-substrate complex (ES), which then reacts irreversibly to form a product (P) and release the enzyme again. S + E ⇌ ES → P + E Two important terms within Michaelis-Menten kinetics are: Vmax – the maximum rate of the reaction, when all the enzyme’s active sites are saturated with substrate. Km (also known as the Michaelis constant) – the substrate concentration at which the reaction rate is 50% of the Vmax. Km is a measure of the affinity an enzyme has for its substrate, as the lower the value of Km, the more efficient the enzyme is at carrying out its function at a lower substrate concentration.  ENZYME KINETICS The Michaelis-Menten equation for the reaction above is: This equation describes how the initial rate of reaction (V) is affected by the initial substrate concentration ([S]). It assumes that the reaction is in the steady state, where the ES concentration remains constant.  ENZYME KINETICS When a graph of substrate concentration against the rate of the reaction is plotted, we can see how the rate of reaction initially increases rapidly in a linear fashion as substrate concentration increases (1st order kinetics). The rate then plateaus, and increasing the substrate concentration has no effect on the reaction velocity, as all enzyme active sites are already saturated with the substrate (0 order kinetics). This plot of rate of reaction against substrate concentration has the shape of a rectangular hyperbola.  ENZYME KINETICS Lineweaver–Burk plot a more useful representation of Michaelis–Menten kinetics, plots the inverse of the reaction rate (1/r) against the inverse of the substrate concentration (1/[S]). The equation used to generate this plot is given by:  ENZYME KINETICS Lineweaver–Burk plot - This produces a straight line, allowing for the easier interpretation of various quantities and values from the graph. For example, the y-intercept of the graph is equivalent to the Vmax. - also useful when determining the type of enzyme inhibition present by, comparing its effect on Km and Vmax. “There are no shortcuts to any place worth going.” - Beverly Stills