Enzymes Learning Material PDF

V. ENZYMES ENZYMES are the biological catalysts produced by living cells. Each cell in the human body contains thousands of different enzymes, because almost every reaction in a cell requires its own specific enzyme. Enzymes cause cellular reactions to oc...

V. ENZYMES ENZYMES are the biological catalysts produced by living cells. Each cell in the human body contains thousands of different enzymes, because almost every reaction in a cell requires its own specific enzyme. Enzymes cause cellular reactions to occur millions of times faster than corresponding uncatalyzed reactions. An enzyme speeds a reaction by lowering the activation energy, changing the reaction pathway. This provides a lower energy route for conversion of substrate to product. As catalysts, enzymes are not consumed during the reaction but merely help the reaction occur more rapidly. If there are no enzymes, most reactions occurring in the body would be so slow that they would not be able to support life. Composition of enzymes: - Most enzymes are globular proteins; some are simple proteins, others are conjugated proteins - Until the 1980’s it was thought that all enzymes were proteins, a few enzymes are now known that are made of ribonucleic acids and catalyze cellular reactions involving nucleic acids. 1) simple proteins - consist entirely of amino acid units; it is the 3o structure of the simple proteins (enzymes) that makes it biologically active; eg, pepsin, trypsin, ribonuclease, etc. 2) conjugated proteins a) apoenzyme  protein part; inactive in itself b) coenzyme (cofactor)  nonprotein organic moiety; the activator; loosely bound to protein; called prosthetic group if coenzyme/cofactor is permanently bound a metallic ion (Fe2+ ; Mg+ ; Co2+ ; Zn2+, Mn2+, etc) HOLOENZYME  the resulting complete, active enzyme when the apoenzyme has been activated by the coenzyme Nomenclature of enzymes: Enzymes are most commonly named by using a system that attempts to provide information about the function (rather than the structure) of the enzyme. Type of reaction catalyzed and substrate identity are focal points for the nomenclature. A substrate is the reactant in an enzyme-catalyzed reaction - the substance upon which the enzyme “acts”. Important aspects in naming enzymes are as follows: 1. The suffix –ase identifies a substance as an enzyme; e.g., urease, sucrase, lipase are all enzyme designations. The suffix –in is still found in many of the digestive enzymes; e.g., trypsin, pepsin 2. The type of reaction catalyzed by an enzyme is often noted with a prefix; e.g., oxidase catalyzes oxidation reaction, hydrolase catalyzes hydrolysis reaction. 1 3. The identity of the substrate is often noted in addition to the type of reaction; e.g., glucose oxidase, pyruvate carboxylase, succinate dehydrogenase. 4. Infrequently, the substrate but not the reaction type is given; urease, lactase. Classification of enzymes: To systematize the nomenclature for enzymes the International Union of Biochemists has grouped enzymes into six major classes 1. Oxidoreductases - catalyze oxidation-reduction involving substrate molecules a. Oxidases – oxidation of a substrate b. Reductases – reduction of a substrate c. Dehydrogenase – introduction of a double bond (oxidation) by formal removal of H2 from substrate, the hydrogen being accepted by a coenzyme 2. Transferases - catalyze transfer of functional groups between two substrates a. Kinases – transfer of a phosphate group between substrates b. Transaminase – transfer of an amino group between substrates 3. Hydrolases - catalyze the addition of a water molecule to a bond causing the bond to break a. Proteases – hydrolysis of peptide linkages in proteins b. Carbohydrases – hydrolysis of glycosidic bonds in carbohydrates: sucrase, lactase, cellulase c. Lipases – hydrolysis of ester linkages in lipids 2  d. Nucleases – hydrolysis of sugar phosphate ester bonds in nucleic acids e. Phosphatases – hydrolysis of phosphate ester bonds 4. Lyases - catalyze the addition of a group to a double bond or the removal of a group to create a double bond in a manner that does not involve hydrolysis a. Dehydratases – removal of water from substrate b. Decarboxylases – removal of carbon dioxide from substrate c. Deaminases – removal of ammonia from substrate 5. Isomerases - catalyze the conversion of a substrate into another compound that is isomeric with it a. Racemases – conversion of D to L isomer or vice versa b. Mutases – transfer of a functional group within a molecule 3  c. Epimerases – conversion of one sugar epimer into another 6. Ligases - catalyze the bonding together of two substrate molecule with participation of ATP a. Synthetases – formation of new bond between two substrates with participation of ATP b. Carboxylases – formation of new bond between substrate and carbon dioxide with participation of ATP The Models of Enzyme Action Explanations of how enzymes function as catalysts in biochemical systems are based on the concepts of an enzyme active site and enzyme-substrate complex formation. 4  Enzyme active site It is the relatively small part of an enzyme that is actually involved in catalysis; a small portion of an enzyme surface where the substrate(s) becomes bound by noncovalent forces, e.g., hydrogen bonding, electrostatic attractions, van der Waals attractions. It is a three-dimensional entity formed by groups that come from different parts of the protein chain(s); these groups are brought together by the folding and bending (2o & 3o structure) of the protein. The active site is usually a “crevice-like” location in the enzyme. Enzyme-substrate complex Catalysts offer an alternative pathway with lower activation energy through which a reaction can occur. In enzyme-controlled reactions, this alternative pathway involves the formation of an enzyme-substrate complex as an intermediate species in the reaction. An enzyme-substrate complex is the intermediate reaction species that is formed when a substrate binds to the active site of an enzyme. Within the enzyme-substrate complex, proximity effects and orientation effects create more favorable reaction conditions than if substrates were free. The result is faster formation of products. The forces that draw the substrate into the active site are many of the same forces that maintain tertiary structure in the folding of the polypeptide chains. Electrostatic interactions, hydrogen bonds, and hydrophobic interactions all help attract and bind substrate molecules. For example, a protonated (positively charged) amino group in a substrate could be attracted and held at the active site by a negatively charged aspartate or glutamate residue. Alternatively, cofactors such as positively charged metal ions often help bind substrate molecules. Lock-and-Key model Just as the notches on a key are designed to fit a specific lock, active sites on a protein are designed to fit a specific substrate. In this model, the active site in the enzyme has a fixed, rigid geometrical conformation. Only substrates with a complementary geometry can be accommodated at such site, much as a lock accepts only certain keys. This model fails to take into account proteins’ conformational changes to accommodate a substrate molecule Induced-Fit model The induced-fit model of enzyme action assumes that the enzyme active site is a more flexible pocket whose conformation changes to accommodate the substrate molecule. It allows for small changes in the shape or geometry of the active site of an enzyme in order to accommodate a substrate. This is a more thorough explanation for the active-site properties of an enzyme because it includes the specificity of the lock-and-key model coupled with the flexibility of the enzyme protein 5 Specificity of Enzymes 1) Absolute specificity - catalyze a particular reaction for one particular substrate only and will have no catalytic effect on substrates which are closely related - eg, urease  acts only on urea H2N – CO – NH2 not for methylurea H2N – CO – NHCH3 nor biuret H2N – CO – NH – CO – NH2 2) Stereochemical specificity - catalyze a specific stereochemical representation - eg. acid dehydrogenase catalyzes the oxidation of L-lactic acid (in muscle tissues) but not D-lactic acid (in microorganisms) 3) Group specificity - less selective and will act upon structurally similar molecules - eg, carboxypeptidase catalyzes the hydrolysis of C-terminal groups regardless of what amino acid 4) Linkage specificity - the least specific; attack a particular kind of chemical bond, irrespective of the structural features in the vicinity of the linkage - eg, lipase catalyzes the hydrolysis of any kind of ester Factors Affecting Enzyme Activity Enzyme activity is a measure of the rate at which an enzyme converts substrate to products. Four factors affect enzyme activity: substrate concentration, enzyme concentration, temperature, & pH. 1) Concentration of substrate - enzyme activity increases proportionally with [S] until a limiting rate is reached (when all enzyme surface has been used for reaction). - enzyme activity increases up to a certain substrate concentration and thereafter remains constant – this activity pattern is called a saturation curve. - as substrate concentration increases, the point is eventually reached where enzyme capabilities are used to their maximum extent, the rate remains constant from this point on. Each substrate must occupy an enzyme active site for a finite amount of time, and the products must leave the site before the cycle can be repeated. When each enzyme molecule is working at full capacity, the incoming substrate molecules must “wait their turn” for an empty active site. At this point, the enzyme is said to be under saturating conditions. 6 - the rate at which an enzyme accepts and releases substrate molecules at substrate saturation is given by its turnover number, which is equal to the number of substrate molecules transformed per second by one molecule of enzyme under optimum conditions of temperature, pH, and saturation; some enzymes have a much faster mode of operation than others; e.g., carbonic anhydrase (600,000), glutamate dehydrogenase (500), phosphoglucomutase (21), chymotrypsin (2) 2) Concentration of the enzyme - If the amount of substrate present is kept constant and the enzyme concentration is increased, the reaction rate increases because more substrate molecules can be accommodated in a given amount of time; enzyme activity increases proportionally with [E] 3) Temperature - Temperature is a measure of the kinetic energy (energy of motion) of molecules; higher temperatures mean molecules are moving faster and colliding more frequently. - As the temperature of an enzymatically catalyzed reaction increases, so does the rate of the reaction. When the temperature increases beyond a certain point, however, the increased energy begins to cause disruptions in the tertiary structure of the enzyme; denaturation is occurring. Tertiary structure change at the active site impedes catalytic action, and the enzyme activity quickly decreases as the temperature climbs past this point. The temperature that produces maximum activity for an enzyme is known as the optimum temperature for that enzyme; for human enzymes, the optimum temperature is often 37oC, normal body temperature. 4) pH - The pH of an enzyme’s environment can affect its activity because the charge on acidic and basic amino acids located at the active site depends on pH; small changes in pH (less than one unit) can result in enzyme denaturation and subsequent loss of catalytic activity. - Most enzymes exhibit maximum activity over a very narrow pH range; only within this narrow pH range do the enzyme’s amino acids exist in properly charged forms - Optimum pH is the pH at which an enzyme has maximum activity; biochemical buffers help maintain the optimum pH for an enzyme - Each enzyme has a characteristic optimum pH, which usually falls within the physiological pH range of 7.0- 7.5 , except digestive enzymes pepsin (functions best at pH 2.0) and trypsin (functions best at pH 8.0) - A variation from normal pH can also affect substrates, causing either protonation or deprotonation of groups on the substrate. The interaction between the altered substrate and the enzyme active site may be less efficient than normal – or even impossible. 7 Regulation of Enzyme Activity Enzyme activity is often regulated by the cell to conserve energy. If the cell runs out of chemical energy, it will die; therefore many mechanisms exist to conserve cellular energy. 1. Produce the enzyme only when the substrate is present – the simplest mechanism 2. Allosteric enzymes – effector molecules change the activity of an enzyme by binding at a second site a) active site or catalytic site - where the substrate binds lock-and-key b) allosteric site (meaning “another site”) - where the effector/inhibitor binds; distorts active site some effectors speed up enzyme action (positive allosterism) some effectors slow enzyme action (negative allosterism) 3. Feedback inhibition (negative feedback control) - the enzyme catalyzing the first reaction in the pathway is inhibited by the final product which the pathway produces; an effective way of controlling the rate of synthesis of a molecule according to the cell’s needs. 4. Production of proenzymes (zymogen) – enzyme in an inactive form; converted by proteolysis to the active form when it has reached the site of its activity; e.g., pepsinogen H+ pepsin 5. Protein modification – another mechanism that the cell can use to turn an enzyme on or off is protein modification where the most common is addition (phosphorylation) or removal (dephosphorylation) of a phosphate group. For example, phosphorylation can either activate (orange) a protein or inactivate it (green). Kinase is an enzyme that phosphorylates proteins. Phosphatase is an enzyme that dephosphorylates proteins, effectively undoing the action of kinase. Inhibition of Enzyme Activity 1. Irreversible inhibitors - substances that cause inhibition that cannot be reversed; bind tightly to the enzyme and thereby prevent formation of the E-S complex; covalently or noncavalently bound to the target enzyme and dissociates very slowly from the enzyme; e.g., diisopropylphosphofluoridate (DIPF) and iodoacetamide. 8  2. Reversible inhibitors - substances that bind to an enzyme to inhibit it, but can be released Competitive inhibition - any compound which bears a close structural resemblance to a particular substrate and which competes with that substrate for the same active sites on the enzyme is said to be a competitive enzyme inhibitor - in competitive inhibition, a substrate and an inhibitor compete for the active sites on the enzyme. They are so similar in structure that the enzyme binds to the inhibitor by mistake. As long as the competitive inhibitor occupies the active site, no reaction of the substrate can take place. However, competitive inhibition may be reversed by adding more substrate that competes with the inhibitor for the active site - competitive enzyme inhibitors are also called structural analogs Noncompetitive inhibition - this type of inhibition involves inhibitors that can bind at sites different from the active sites of the enzyme; these inhibitors do not interfere with the binding of the substrate to the enzyme directly but interfere with the reaction of the enzyme-substrate complex. - occur when a foreign substance (inhibitor) attaches to only a portion of the enzyme tying up one or two active sites , or if a bulky foreign substance blocks the entrance of the substrate to the enzyme Chemotherapy - refers to the use of chemicals to destroy infectious microorganisms without damaging the cells of the host A. Antimetabolites (act through competitive inhibition) - eg. Inhibition by a Sulfa drug : Sulfa drugs are similar to PABA, a compound that bacteria must have to synthesize folic acid. Sulfa drugs react with the enzymes that usually complex with PABA, preventing them from forming folic acid. Humans get their folic acid preformed from foods, not synthesized by our system 9 B. Antibiotics - bacteria have one structural feature not found in animal cells – a cell wall. Penicillin acts by complexing with the enzymes responsible for cell wall synthesis, effectively killing the bacteria. Since animal cells do not have cell walls, there are no such enzymes to be affected and penicillin has no effect on animal cells. - the bacterial cell wall precursor is a polymer comprising a repeating disaccharide unit with attached polypeptide side chains that end with a d-alanyl-d-alanine unit. The transpeptidase enzyme cleaves the terminal d-alanine and the amino group of the glycine then reacts with the penultimate d-alanine on a neighbouring chain to produce the mature cross-linked matrix of the cell wal. The structural similarity between the penicillins and d-alanyl-d-alanine allows the antibiotics to act as inhibitory substrates for the transpeptidase enzyme. Michaelis – Menten Kinetics of Enzyme Action k1 k3 For a monosubstrate reaction: E + S ↔ ES ↔ E + P k2 k4 if k3 is very small, initial velocity, υ = k3 ES when substrate concentration is very high vmax = k3 ET (ET = total E ; free + bound E) Rate of formation of ES = dES/dt = k1 (E – ES) (S) Rate of breakdown of ES = - dES/dt = k2 (ES) + k3 (ES) At steady state, with [ES] remaining constant, where there is a dynamic steady state with substrates continually supplied and products continually removed removed as in the usual situation in the living cell k1 (E – ES) (S) = k2 (ES) + k3 (ES) (E - ES) (S) / (ES) = k2 + k3 / k1 = Km (Michaelis constant) to solve for ES: (E - ES) (S) / (ES) = Km (E)(S) / (ES) - (ES)(S) / (ES) = Km (E) (S) / (ES) = Km + (S) (ES) = (E) (S) / Km + (S) if υ = k3 ES and vmax = k3 E υ = k3 (E) (S) / Km + (S) = vmax / (E). (E) (S) / Km + (S) υ = (vmax) (S) / Km + (S) → Michaelis Equation (Fig. 6.4 - p126 McKee) where υ = observed initial velocity of the reaction (S) = substrate concentration Km = Michaelis constant vmax = maximum velocity of enzyme reaction The equation states that, when the [S] is very high, Km is negligible compared to the [S] and the equation reduces to υ = vmax , that is , the observed velocity is maximal at high [S]. Conversely, when [S] is very low, then Km + (S) becomes appreciable compared to (S) and the observed velocity, υ, is a fraction of the vmax ; υ = (S) / Km + (S) · vmax when υ = ½ vmax vmax / 2 = vmax (S) / Km + (S) ; dividing both sides by vmax ½ = (S) / Km + (S) Km + (S) = 2 (S) Km = (S) Km is equal to substrate concentration if υ = ½ vmax - Km is the dissociation constant for ES; the greater the value of Km, the less tightly S is bound to E; - there is a characteristic Km value for each enzyme (lying bet. 10-1 - 10-6 ) expressed in moles of substrate per liter - Vmax is the turnover number 10 Lineweaver – Burke Plots - an alternative linear transformation of the M-M equation - estimation of the value of Km is inconvenient from Michaelis Equation plot and several more convenient forms of the equation have been developed. The reciprocal of the equation, a linear form called the Lineweaver – Burke plot is used. 1/υ = Km + (S) / vmax (S) = Km / vmax (S) + (S) / vmax (S) = Km / vmax x 1 / (S) + 1 / vmax (eqn for st. line) As shown in the figure, if one plots 1/υ vs. 1/(S), the slope of the line is Km/vmax & the intercept on the ordinate is 1/vmax..Since vmax can be obtained from the intercept it is possible to calculate Km.The intercept on the abscissa is equal to – 1/ Km 11 Coenzymes - the water-soluble vitamins, which include all B-vitamins and Vitamin C, act as coenzymes or coenzyme precursors Vitamin Coenzyme Function Deficiency Symptoms 1) Thiamine (B1) Thiamine Decarboxylation of α-keto beriberi (fatigue, anorexia, pyrophosphate acids; cetain rexns of keto nerve degeneration, paralysis (TPP) sugars heart failure) 2) Riboflavin (B2) Flavin mononucleotide Several kinds of redox dermatitis, glossitis (tongue Flavin adenine reactions inflammation),cataracts dinucleotide (FMN, FAD) 3) Niacin/Nicotinic Nicotinamide adenine Numerous redox reactions pellagra(scaly skin, muscle acid (B3) dinucleotide fatigue, diarrhea, mouth Nicotinamide adenine sores, mental disorders dinucleotide phosphate (NAD, NADP) 4) Pantothenic acid Coenzyme A Many reactions of fatty no deficiency known, may (B5) acids particularly those cause fatigue, anemia involving the transfer of acetyl groups 5) Pyridoxine (B6) Pyridoxal phosphate Several kinds of reactions dermatitis, fatigue, anemia (PP) involving aa’s;eg, decarbo- irritability, convulsions xylation, transamination in infants 6) Biotin Biotin CO2 fixation reactions dermatitis, fatigue, anemia nausea, mental depression 7) Folic acid Tetrahydrofolic acid Various reactions involving abnormal rbc & wbc, gi (THF) single C-compounds disturbances 8) Cyanocobalamine “Cobamide” Carbon chain isomerization; pernicious anemia, coenzymes methyl group transfers (eg, malformed rbc, neurological in rbc biosynthesis) disorders 9) Ascorbic acid (Vitamin C) - needed for collagen formation, protein metabolism, iron absorption, healing of wounds; scurvy (bleeding gums, slow healing wounds, muscle pain, anemia) Role of Vit. C in collagen formation Collagen also contains hydroxylysine and hydroxylproline. Hydroxylation of lysine and proline in collagen formation are catalyzed by enzymes and require ascorbic acid (Vit. C). In Vit. C deficiency, hydroxylation is impaired, and the triple helix of the collagen is not assembled properly. Persons deprived of Vit. C develops scurvy, a disease whose symptoms include skin lesions, fragile blood vessels, loose teeth, and bleeding gums (Voet, p 157) 12 Cofactors ================================================================================= Metal Ion Enzymes ----------------------------------------------------------------------------------------------------------------------------------------- Ca 2+ Thromboplastin Cu2+ Tyrosinase, cytochrome oxidase Fe2+ ; Fe3+ Cytochrome oxidase, catalase, dehydrogenase Mg2+ Pyruvate kinase Mn2+ Arginase, pyruvate carboxylase, phosphatase, succinic dehydrogenase, glycosyl transferases, cholinesterase K+ Pyruvate kinase Zn2+ Carbonic anhydrase, carboxypeptidase, lactic dehydrogenase, alcohol dehydrogenase ================================================================================= CLINICAL APPLICATIONS OF ENZYMES Isoenzymes in Medical Diagnosis Different cells in the body produce enzymes for the same type of reactions. Although the proteins are similar, they are not identical. Enzymes that catalyze the same reactions but vary slightly in structure are called isoenzymes. For example, there are five isoenzymes for lactate dehydrogenase (LDH), an enzyme that converts lactic acid to pyruvic acid. Each LDH consists of a quaternary structure containing four subunits. In heart muscle, the most prevalent subunit is designated as H. In skeletal muscle, the major subunit is designated as M. Isoenzyme LDH1 LDH2 LDH3 LDH4 LDH5 Subunits H4 H3M H2M2 HM3 M4 Abundant in Heart, kidneys Heart, kidneys, Kidneys, brain Spleen Liver, skeletal brain, rbc muscle In healthy tissues, these enzymes are contained within cellular membranes. However, if the cells of a particular organ are damaged, the contents including the enzymes spill into the blood. By identifying the isoenzyme that becomes elevated in the blood serum, it is possible to determine which type of tissue has been damaged. For example, liver diseases can be detected by a rise in the serum LDH5 level. When a myocardial infarction (MI), or heart attack, damages the cells in the heart muscle, there is an increase in the serum LDH1 level. 13 Serum Enzymes used in diagnosis of tissue damage Organ Condition Diagnostic Enzymes Heart Myocardial infarction Lactate dehydrogenase (LDH1) ; Creatine kinase (CK2) ; Glutamic oxaloacetic transaminase (GOT) Liver Cirrhosis, carcinoma, Glutamic pyruvic transaminase (GPT) ; Lactate dehydrogenase Hepatitis (LDH5) ; Alkaline phosphatase (ALP) ; GOT Bone Rickets, carcinoma Alkaline phosphatase (ALP) Pancreas Pancreatic diseases Amylase ; Cholinesterase ; Lipase (LPS) Prostate Carcinoma Acid phosphatase (ACP) FUNCTIONS OF ESSENTIAL ELEMENTS ELEMENT MAJOR FUNCTIONS Carbon Structural – in carbohydrates, lipids, proteins, and nucleic acids Hydrogen Structural - in carbohydrates, lipids, proteins, and nucleic acids Oxygen Structural – in carbohydrates, lipids, proteins, and nucleic acids Nitrogen Structural – in proteins, nucleic acids, chlorophyll, certain coenzymes Phosphorus Structural – in nucleic acids, phospholipids, ATP (energy transfer compound) Calcium Structural – in middle lamella of cell walls Physiological – role in membrane permeability; enzyme activation Magnesium Structural – in chlorophyll Physiological – enzyme activator in carbohydrate metabolism Sulfur Structural – in certain amino acids and vitamins Potassium Physiological – osmosis and ionic balance; opening and closing of stomata; enzyme activator (for 40+ enzymes) Chlorine Physiological – ionic balance; involved in light reactions (oxygen evolution) of photosynthesis. Iron Physiological – part of enzymes involved in photosynthesis, respiration, and nitrogen fixation. Manganese Physiological – part of enzymes involved in respiration and nitrogen metabolism; required in oxygen evolution in photosynthesis Copper Physiological – part of enzymes involved in photosynthesis Zinc Physiological – part of enzymes involved in respiration and nitrogen metabolism Molybdenum Physiological – part of enzymes involved in nitrogen metabolism Boron Physiological – exact role unclear; involved in membrane transport and calcium utilization 14 15