Enzymes Lecture Notes PDF

‫‪ENZYMOLOGY‬‬ ‫جامعه وارث االنبياء‬ ‫كليه الطب‬ ‫قسم الكيمياء الحيوية‬ ‫د‪.‬رياض حنيوه‬ ‫‪LEC 4 & 5 & 6‬‬ ‫والطبية‬ Enzymology is the study of enzymes, their kinetics, structure, and function, as well as their relation to each other. Characteristics of Enzymes i. Almost all enzymes are protein...

‫‪ENZYMOLOGY‬‬ ‫جامعه وارث االنبياء‬ ‫كليه الطب‬ ‫قسم الكيمياء الحيوية‬ ‫د‪.‬رياض حنيوه‬ ‫‪LEC 4 & 5 & 6‬‬ ‫والطبية‬ Enzymology is the study of enzymes, their kinetics, structure, and function, as well as their relation to each other. Characteristics of Enzymes i. Almost all enzymes are proteins. Enzymes follow the physical and chemical reactions of proteins. ii. They are heat labile. iii. They are water-soluble. iv. They can be precipitated by protein precipitating reagents (ammonium sulfate or trichloroacetic acid). . v. They contain 16% weight as nitrogen i.E. 1- accelarate the rate of chemical reaction 2- not change by entering the reaction 3- does not effect the end product of reaction Chemical reactions    Life is possible due to the co-ordination of numerous metabolic reactions inside the cells. Proteins can be hydrolyzed with hydrochloric acid by boiling for a very long time; but inside the body, with the help of enzymes, proteolysis takes place within a short time at body temperature. Enzyme catalysis is very rapid; usually 1 molecule of an enzyme can act upon about 1000 molecules of the substrate per minute Chemical reactions need an initial input of energy = THE ACTIVATION ENERGY During this part of the reaction the molecules are said to be in a transition state. Reaction pathway Making reactions go faster      Increasing the temperature make molecules move faster Biological systems are very sensitive to temperature changes. Enzymes can increase the rate of reactions without increasing the temperature. They do this by lowering the activation energy. They create a new reaction pathway “a short cut” An enzyme controlled pathway  Enzyme controlled reactions proceed 108 to 1011 times faster than corresponding non-enzymic reactions. IUBMB System of Classification International Union of Biochemistry and MolecularBiology (IUBMB) in 1964, suggested the IUBMB system of nomenclature of enzymes the name starts with EC (enzyme class) followed by 4 digits. First digit represents the class Second digit stands for the subclass Third digit is the sub-subclass or subgroup Fourth digit gives the number of the particular enzyme in the list. Alcohol dehydrogenases (ADH) (EC 1.1.1.1) R-CH2-OH + NAD EC 1 EC 1.1 EC 1.1.1 EC 1.1.1.1 R-CHO+ NADH + H class of enz. ( oxidoreductase ) group enz. Act CH-OH the coenzyme NAD ethanol is substrate 1st digit 2nddigit 3rd digit 4thdigit Class no. ( 1- 6 ) Functional group Coenzyme Substate Enzyme structure    Enzymes are proteins They have a globular shape A complex 3-D structure Human pancreatic amylase CO-ENZYMES 1- Enzymes may be simple proteins, or complex enzymes, containing a non-protein part, called the prosthetic group. The prosthetic group is called the co-enzyme. It is heat stable. 2-The protein part of the enzyme is then named the apo-enzyme. It is heat labile. iii. These two portions combined together is called the holo-enzyme. 3-Co-enzymes may be divided into two groups a. Those taking part in reactions catalyzed by oxidoreductases by donating or accepting hydrogen atoms or electrons. b. Those co-enzymes taking part in reactions transferring groups other than hydrogen. apoenz. (protein part ofenz. ) Simple protein Enz conjucated protein (holoenz.) cofactor ( non protein part of enz. ) First Group of Co-enzymes examples are NADP–NADPH; FAD FADH2 and FMN–FMNH2. Nicotinamide Adenine Dinucleotide (NAD+) i. This is a co-enzyme synthesized from Nicotinamide, a member of vitamin B complex. ii. The reversible reaction of lactate to pyruvate is catalyzed by the enzyme lactate dehydrogenase, but the actual transfer of hydrogen is taking place on the coenzyme, NAD+ iii. In this case, two hydrogen atoms are removed from lactate, out of which one hydrogen and two electrons are accepted by the NAD+ to form NADH, and the remaining H+ is released into the surrounding medium. Second Group of Co-enzymes These co-enzymes take part in reactions transferring groups other than hydrogen. A particular group or radical is transferred from the substrate to another substrate. Most of them belong to vitamin B complex group. Examples of 2nd type of co-enzymes Co-enzyme Group transferred 1- Thiamine pyrophosphate................ ........(TPP) Hydroxy ethyl 2- Pyridoxal phosphate (PLP)............... .....Amino group 3- Biotin................................................... ..Carbon dioxide 4- Coenzyme-A (Co-A).............................. Acyl groups 5- Tetra hydrofolate (FH4) ..........................One carbon groups 6- Adenosine triphosphate (ATP) ................Phosphate Adenosine Triphosphate (ATP) i. ATP is considered to be the energy currency in the body. ii. In the ATP molecule, the second and third phosphate bonds are ‘high energy' bonds iii. During the oxidation of food stuffs, energy is released, a part of which is stored as chemical energy in the form of ATP. iv. The endergonic reactions are carried out with the help of energy released from hydrolysis of ATP. Metallo-enzymes Metalloenzymes are enzyme proteins containing metal ions (metal cofactors), which are directly bound to the protein or to enzymebound nonprotein components (prosthetic groups). About one-third of all enzymes known so far are metalloenzymes. Also ribozymes, i.e., RNA molecules with enzyme function may contain structurally and/or functionally important metal ions (mostly divalent metal ions such as Mg2+) and may be therefore termed as metalloenzymes Metal Enzyme containing the metal Zinc Carbonic anhydrase, carboxy peptidase, alcohol dehydrogenase Magnesium Hexokinase, phospho fructo kinase, enolase, glucose-6phosphatase Manganese Phospho gluco mutase, hexokinase, enolase, glycosyl transferases Copper Tyrosinase, cytochrome oxidase, lysyl oxidase, superoxide dismutase Iron Cytochrome oxidase, catalase, peroxidase, xanthine oxidase Calcium Lecithinase, lipase Molybdenum Xanthine oxidase The active site   One part of an enzyme, the active site, is particularly important The shape and the chemical environment inside the active site permits a chemical reaction to proceed more easily Allosteric site : site or region in the enz. Surface away from active site which increase or decrease the enz. Activity for substrate The substrate    The substrate of an enzyme are the reactants that are activated by the enzyme Enzymes are specific to their substrates The specificity is determined by the active site Enzyme Classification class 1. Oxidoreductases. To this class belong all enzymes catalysing oxidoreduction reactions. The substrate that is oxidized is regarded as hydrogen donor. The systematic name is based on donor:acceptor oxidoreductase. The common name will be dehydrogenase, wherever this is possible; as an alternative, reductase can be used. Oxidase is only used in cases where O2 is the acceptor. S( oxidised) + Y(reduced) S (reduced) + Y( oxidised) Class 2. Transferases. Transferases are enzymes transferring a group. The common names are normally formed according to acceptor grouptransferase or donor grouptransferase. In many cases, the donor is a cofactor (coenzyme) charged with the group to be transferred. aspartateTransaminases , alanine Transaminases ( AST , ALT ) . SX + Y S + XY class 3. Hydrolases. Hydrolases are hydrolytic enzymes, which catalyze the hydrolysis reaction by adding water to cleave the bond and hydrolyze it ,for ex. Peptidase . Class 4. Lyases. Cleavage without add water, carbon dioxide or ammonia across double bonds or eliminate these to create double bonds., like decarboxylase, aldolase, dehydratase Class 5. Isomerases. catalyze the formation of an isomer of a compound. Example phosphoglucose isomerase Glucose-6P fructose-6P Class 6. Ligases. Ligases are enzymes catalysing the joining together of two molecules coupled with the hydrolysis of a diphosphate bond in ATP,for ex. glutamine synthetase How enzyme act The Lock and Key Hypothesis        Fit between the substrate and the active site of the enzyme is exact Like a key fits into a lock very precisely The key is analogous to the enzyme and the substrate analogous to the lock. Temporary structure called the enzyme-substrate complex formed Products have a different shape from the substrate Once formed, they are released from the active site Leaving it free to become attached to another substrate The Lock and Key Hypothesis S E E E Enzymesubstrate complex Enzyme may be used again P P Reaction coordinate The Induced Fit Hypothesis      Some proteins can change their shape (conformation) When a substrate combines with an enzyme, it induces a change in the enzyme’s conformation The active site is then moulded into a precise conformation Making the chemical environment suitable for the reaction The bonds of the substrate are stretched to make the reaction easier (lowers activation energy) The Induced Fit Hypothesis Hexokinase (a) without (b) with glucose substrate  This explains the enzymes that can react with a range of substrates of similar types Factors affecting Enzymes        substrate concentration Enzyme concentration Product concentration pH temperature Inhibitors activators Substrate concentration: Non-enzymic reactions Reaction velocity Substrate concentration  The increase in velocity is proportional to the substrate concentration Substrate concentration: Enzymic reactions Vmax Reaction velocity Substrate concentration   Faster reaction but it reaches a saturation point when all the enzyme molecules are occupied. If you alter the concentration of the enzyme then Vmax will change too. Features of Km 1. 2. 3. 4. 5. Km value is substrate concentration (expressed in moles/L) at half-maximal velocity. It denotes that 50% of enzyme molecules are bound with substrate molecules at that particular substrate concentration Km is independent of enzyme concentration. If enzyme concentration is doubled, the Vmax will be double . But the Km will remain exactly same. Km is the Signature of the Enzyme. Km value is thus a constant for an enzyme. Km denotes the affinity of enzyme for substrate. The lesser the numerical value of Km, the affinity of the enzyme for the substrate is more. The lesser the numerical value of Km, the affinity of the enzyme for the substrate is more!! For example, Km of glucokinase is 10 mmol/L and that of hexokinase is 0.05 mmol/L. Therefore,50% molecules of hexokinase are saturated even at a lower concentration of glucose. In other words, hexokinase has more affinity for glucose than glucokinase Enzyme Concentration 1. 2. 3. Rate of a reaction or velocity (V) is directly proportional to the enzyme concentration, when sufficient substrate is present. Velocity of reaction is increased proportionately with the concentration of enzyme. Hence, this property is made use of in determining the level of particular enzyme in plasma, serum or tissues. Known volume of serum is incubated with substrate for a fixed time, then reaction is stopped and product is quantitated (end point method). Since the product formed will be proportional to the enzyme concentration, the latter could be assayed effect of product In a reversible reaction, S P when equilibrium is reached, the reaction rate is slowed down. So, when product concentration is increased, the reaction is slowed, stopped or even reversed. In inborn errors of metabolism, one enzyme of a metabolic pathway is blocked. For example, A E1 B E2 C || E3 D If E3 enzyme is absent, C will accumulate, which in turn, will inhibit E2. Consequently, in course of time, the whole pathway is blocked. The effect of pH     Extreme pH levels will produce denaturation The structure of the enzyme is changed The active site is distorted and the substrate molecules will no longer fit in it At pH values slightly different from the enzyme’s optimum value, small changes in the charges of the enzyme and it’s substrate molecules will occur The effect of pH Optimum pH values Enzyme activity Trypsin Pepsin 1 3 5 7 pH 9 11 The effect of temperature      Q10 (the temperature coefficient) = the increase in reaction rate with a 10°C rise in temperature. For chemical reactions the Q10 = 2 to 3 (the rate of the reaction doubles or triples with every 10°C rise in temperature) Enzyme-controlled reactions follow this rule as they are chemical reactions BUT at high temperatures proteins denature The optimum temperature for an enzyme controlled reaction will be a balance between the Q10 and denaturation. The effect of temperature Denaturation Q10 Enzyme activity 0 10 20 30 Temperature / °C 40 50 The effect of temperature     For most enzymes the optimum temperature is about 37°C Many are a lot lower, cold water fish will die at 30°C because their enzymes denature A few bacteria have enzymes that can withstand very high temperatures up to 100°C Most enzymes however are fully denatured at 70°C Inhibitors     Inhibitors are chemicals that reduce the rate of enzymic reactions. The are usually specific and they work at low concentrations. They block the enzyme but they do not usually destroy it. Many drugs and poisons are inhibitors of enzymes in the nervous system. enzyme inhibitor Irreversible reversible Irreversible inhibitors: Combine with the functional groups of the amino acids in the active site, irreversibly. Examples: nerve gases and pesticides, containing organophosphorus, combine with serine residues in the enzyme acetylcholine esterase.  The effect of enzyme inhibition Reversible inhibitors: Combine with other or with the functional groups of the amino acids in the active site, reversibly. There are 3 categories.  The effect of enzyme inhibition 1. Competitive: These compete with the substrate molecules for the active site. The inhibitor’s action is proportional to its concentration. Resembles the substrate’s structure closely. E+I Reversible reaction EI Enzyme inhibitor complex Competitive inhibitor Suppose 100 molecules of substrate and 100 molecules of inhibitor are competing for 100 molecules of the enzyme. So, half of enzyme molecules are trapped by the inhibitor and only half the molecules are available for catalysis to form the product Competitive inhibition is usually reversible? excess substrate abolishes the inhibition. In the previous example of 100 moles of E and 100 moles of I, if 900 moles of S are added, only 1/10th of enzyme molecules are attached to inhibitor and 90% are working with substrate. Thus 50% inhibition in the first example is now decreased to 10% inhibition it is clear that in the case of competitive inhibition, the Km is increased in presence of competitive inhibitor. Thus competitive inhibitor apparently increases the Km. In other words, the affinity of the enzyme towards substrate is apparently decreased in presence of the inhibitor. But Vmax is not changed. The effect of enzyme inhibition 2. Non-competitive: These are not influenced by the concentration of the substrate. It inhibits by binding irreversibly to the enzyme but not at the active site. Examples  Cyanide combines with the Iron in the enzymes cytochrome oxidase.  Heavy metals, Ag or Hg, combine with –SH groups. These can be removed by using a chelating agent such as EDTA. Since these inhibitors have no structural resemblance to the substrate, an increase in the substrate concentration generally does not relieve this inhibition. The inhibitor combines with the enzymes by forming a covalent bond and. The velocity (Vmax) is reduced. But Km value is not changed, because the remaining enzyme molecules have the same affinity for the substrate. Increasing the substrate concentration will abolish the competitive inhibition, but will not abolish noncompetitive inhibition. Uncompetitive Inhibition Here inhibitor does not have any affinity for free enzyme. Inhibitor binds to enzyme–substrate complex; but not to the free enzyme. In such cases both Vmax and Km are decreased. Inhibition of placental alkaline phosphatase (Regan iso-enzyme) by phenylalanine is an example of uncompetitive inhibition Uncompetitive Inhibition Competitive&non Competitive inhibitor Competitive inhibition 1-Acting on 2-Structure of Substrate Active site analog Non-competitive inhibition May or may not Unrelated molecule inhibitor 3-Inhibition is 4- Excess substrate 5- Km 6- Vmax 7- Significance Reversible Generally irreversible Inhibition relieved No effect Increased No change No change Decreased Drug action Toxicological End of lectuer of enzymology