Enzymes
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Enzymes, terminology, factors affected activity, classification, kinetic, enzymes uses

Enzymes, terminology, factors affected activity, classification, kinetic, enzymes uses

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    Enzymes Enzymes Presentation Transcript

    • 1
    • Enzymes Dr Mohamed Mostafa Omran Faculty of Sciences, Helwan University 2
    • Introduction to Biochemistry
    • Biochemistry • Chemistry of living organisms. • Each second/ 8,000 trillion reactions in our bodies
    • Biomolecules • • • • • • Carbohydrates Lipids Proteins Nucleic acid Vitamins Enzymes
    • Enzymes Made of protein Present in all living cells Enzymes Biological catalysts Converts substrates into products Affected by cellular conditions: any condition that affects protein structure temperature, pH, salinity Increase the rate of Remain unchanged chemical reactions by chemical reaction proceeding from10^3–10^8 times faster than uncatalyzed reactions.
    • Properties of enzymes • Reaction specific – each enzyme works with a specific substrate • chemical fit between active site & substrate – H bonds & ionic bonds • Not consumed in reaction – single enzyme molecule can catalyze thousands or more reactions per second • enzymes unaffected by the reaction • Affected by cellular conditions – any condition that affects protein structure • temperature, pH, salinity
    • Specificity of the Enzyme • For enzyme and substrate to react, surfaces of each must be complementary • Enzyme specificity: the ability of an enzyme to bind only one, or a very few, substrates thereby catalyzing only a single reaction • Compare these 2 reactions: • Urease is VERY Specific or has a HIGH DEGREE of Specificity
    • Specificity of Enzymes One of the properties of enzymes that makes them so important as diagnostic and research tools is the specificity they exhibit relative to the reactions they catalyze. In general, there are four distinct types of specificity: 1. Absolute specificity - the enzyme will catalyze only one reaction. 2. Group specificity - the enzyme will act only on molecules that have specific functional groups, such as amino, phosphate and methyl groups. 3. Linkage specificity - the enzyme will act on a particular type of chemical bond regardless of the rest of the molecular structure. 4. Stereochemical specificity - the enzyme will act on a particular steric or optical isomer. Though enzymes exhibit great degrees of specificity, cofactors may serve many apoenzymes.
    • Enzyme Specificity
    • Function of Enzymes • Enzymes speed up the rate of chemical reactions in the body; both breaking down (e.g.: starch into maltose) and building up reactions. (e.g: amino acids into proteins). • Enzymes lower the activation energy required to start a chemical reaction
    • Characteristics of Enzymes • Enzymes are highly specific in action. • Enzymes remain chemically unchanged at the end of the reaction. • Enzymes are required in minute amounts.
    • is a non-protein part firmly attached to the apoprotein 13
    • Cofactors are bound to the enzyme for it to maintain the correct configuration of the active site 14
    • How Coenzymes work • A coenzyme is required by some enzymes – An organic molecule bound to the enzyme by weak interactions / Hydrogen bonds – Most coenzymes carry electrons or small groups – Many have modified vitamins in their structure
    • Water-Soluble Vitamins and Their Coenzymes
    • 17
    • Turnover number (Enzyme Unit) The “turnover number” is the number of substrate molecules converted into product by an enzyme molecule in a unit time under optimal condition of measurement. 18
    • Zymogens (proenzymes) • Digestive and coagulation enzymes are secreted in inactive forms, zymogens or proenzymes. • Zymogens are activated by trimming of a short peptide blocking the active site, or by covalent modification of the zymogen. 19
    • Mechanism of activation * The mechanism of activation may involve unmasking of a polypeptide chain that may be blocking or masking the active centers of apoenzyme. * Proteolytic enzymes are secreted inactive to prevent digestion of protein of the cell that synthesized them. Activation takes place by: HCL 1- pH – changes: Pepsinogen Pepsin trypsin 2- Auto-activation: Trypsinogen Trypsin Entrokinase 3- By other enzymes: Trypsinogen Trypsin
    • 21
    • ase Protein Substrate Name + -ase
    • Carbohydrate ase
    • Terminology of enzymes 1-Some enzymes still retain their old names as digestion enzymes still use –in pepsin, trypsin. 2-End in –ase: Identifies a reacting substance lipase - reacts lipid 3-Describes function of enzyme: oxidase – catalyzes oxidation hydrolase – catalyzes hydrolysis 24
    • Terminology of enzymes Nomenclature systems are 4. The trivial name: which give no indication of the function of the enzyme, are commonly used. In some cases, the trivial name is composed of the name of the substrate involved, the type of the reaction catalyzed, and the ending – ase. Lactate + dehydrogenation + ase = lactate dehydrogenase 5. The systematic name: It is made up of the names of substrates acted upon , coenzyme involved in the reaction , the type of reaction catalyzed+ ase eg Lactate NAD+ Oxidoreductase (the old name was lactate dehydrogenase). 25
    • Classification of enzymes: six classes according to reaction type (Each class comprises other subclasses) Enzyme class General scheme of reaction 1. Oxidoreductases Ared + Box  Aox + Bred 2. Transferases A-B + C  A + C-B 3. Hydrolases A-B + H2O  A-H + B-OH 4. Lyases A-B  A + B 5. Isomerases A-B-C  A-C-B 6. Ligases (synthetases) A + B + ATP  A-B + ADP + Pi (reverse reaction: synthases) 26
    • 1 Oxidoreductases catalyze the oxidation or reduction of substrate subclasses: 1. dehydrogenases catalyze transfers of two hydrogen atoms 2. oxygenases catalyze the incorporation of one/two O atoms into the substrate (monooxygenases, dioxygenases) 3. oxidases catalyze transfers of electrons between substrates (e.g. cytochrome c oxidase, ferroxidase) 4. peroxidases catalyze the breakdown of peroxides Example: lactate + NAD+  pyruvate + NADH + H+ lactate dehydrogenase 27
    • 2 Transferases catalyze the transfer of a group from one to another substrate subclasses: • aminotransferases, methyltransferases, • phosphomutases – the transfer of the group PO32– within molecule • kinases phosphorylate substrate by the transfer of phosphoryl group PO32– from ATP (e.g. hexokinases, proteinkinases) Example: glucose + ATP  glucose 6-P + ADP glucokinase 28
    • 3 Hydrolases catalyze the hydrolytic splitting of esters, glycosides, amides, peptides etc. subclasses: • esterases (lipases, phospholipases, ribonucleases, phosphatases) • glycosidases (e.g. sucrase, maltase, lactase, amylase) • proteinases and peptidases (pepsin, trypsin, cathepsins, dipeptidases, carboxypeptidases, aminopeptidases) • amidases (glutaminase, asparaginase) • ATPases (split anhydride bonds of ATP) Example: glucose 6-P + H2O  glucose + Pi glucose 6-phosphatase 29
    • 4 Lyases catalyze non-hydrolytic splitting or forming bonds C–C, C–O, C–N, C–S through removing or adding, respectively, a small molecule (H2O, CO2, NH3) Some frequent recommended names: • ammonia lyases (e.g. histidine ammonia lyase: histidine  urocanate + NH3) • decarboxylases (amino acid  amine + CO2) • aldolases (catalyze aldol cleavage and formation) • (de)hydratases (carbonate dehydratase: CO2 + H2O  H2CO3) Example: fumarate + H2O  L-malate fumarate hydratase Fumarate is hydrated to malate in a freely reversible reaction catalyzed by fumarase (also called fumarate hydratase) 30
    • 31
    • 6 Ligases catalyze formation of high-energy bonds C–C, C–O, C–N in the reactions coupled with hydrolysis of ATP Frequent recommended names: carboxylases synthetases (e.g. glutamine synthetase: glutamate + ATP + NH3  glutamine + ADP + Pi) Example: pyruvate + CO2 + ATP + H2O  oxaloacetate + ADP + Pi pyruvate carboxylase 32
    • ! Three enzymes have something to do with phosphate Enzyme (Class) Reaction scheme / Reaction type Kinase substrate-OH + ATP  substrate-O-P + ADP (Transferase) phosphorylation = transfer of phosphoryl PO32– from ATP to substrate Phosphatase (Hydrolase) substrate-O-P + H2O  substrate-OH + Pi the hydrolysis of phosphoester bond (glycogen)n + Pi  (glycogen)n-1 + glucose 1-P Phosphorylase inosine + Pi  hypoxanthine + ribose 1-P (Transferase) phosphorolysis = the splitting of glycoside bond by phosphate = transfer of glucosyl to inorganic phosphate 33
    • ! Distinguish: Three types of lysis (decomposition of substrate) the decomposition of substrate by water, frequent in intestine: Hydrolysis sucrose + H2O  glucose + fructose (starch)n + H2O  maltose + (starch)n-2 Phosphorolysis the cleavage of O/N-glycoside bond by phosphate: (glycogen)n + Pi  (glycogen)n-1 + glucose 1-P the cleavage of C-C bond by sulfur atom of coenzyme A Thiolysis in β-oxidation of FA or ketone bodies catabolism RCH2COCH2CO-SCoA + CoA-SH  RCH2CO-SCoA + CH3CO-SCoA 34
    • 0 Time Salt concentration 35
    • reaction rate Temperature 37° temperature
    • • Factors affecting enzyme Temperature function – Optimum T° • greatest number of molecular collisions • human enzymes = 35°- 40°C – body temp = 37°C – Heat: increase beyond optimum T° • increased energy level of molecules disrupts bonds in enzyme & between enzyme & substrate – H, ionic = weak bonds • denaturation = lose 3D shape (3° structure) – Cold: decrease T° • molecules move slower • decrease collisions between enzyme & substrate
    • Enzymes and temperature • Different enzymes function in different organisms in different environments reaction rate human enzyme hot spring bacteria enzyme 37°C temperature 70°C (158°F)
    • pH trypsin reaction rate pepsin pepsin trypsin 0 1 2 3 4 5 6 pH 7 8 9 10 11 12 13 14
    • Factors affecting enzyme function • pH – changes in pH • adds or remove H+ • disrupts bonds, disrupts 3D shape – disrupts attractions between charged amino acids – affect 2° & 3° structure – denatures protein – optimal pH? • most human enzymes = pH 6-8 – depends on localized conditions – pepsin (stomach) = pH 2-3 – trypsin (small intestines) = pH 8 0 1 2 3 4 5 6 7 8 9 10 11
    • reaction rate Salinity salt concentration
    • Factors affecting enzyme function • Salt concentration – changes in salinity • adds or removes cations (+) & anions (–) • disrupts bonds, disrupts 3D shape – disrupts attractions between charged amino acids – affect 2° & 3° structure – denatures protein – enzymes intolerant of extreme salinity • Dead Sea is called dead for a reason!
    • 43
    • 44
    • Substrate Concentration As substrate concentration increases, the rate of reaction increases (at constant enzyme concentration). • the enzyme eventually becomes saturated giving maximum activity. 45
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    • 49
    • Important conclusion about Km 1. 2. 3. 4. Substrate are usually in physiological fluids in amounts nearly equal to Km values. Km is a constant characteristic of an enzyme and its substrate. Km reflect the affinity of the enzyme for the substrate. Low Km reflect high affinity of the enzyme for substrate High Km reflect low affinity of the enzyme for substrate • Glucose • • Hexokinase is more active than glucokinase because the amount of glucose (substrate) needed to produce ½ V max in case of hexokinaes is less than in case of glucokinase i.e Km of hexokinase is less than glucokinase. • Hexokinase or glucokinase Glucose 6 phosphatase 50
    • 51
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    • 53
    • Uncompetitive
    • 55
    • 56
    • Classes of Inhibition Two real, one hypothetical • Competitive inhibition - inhibitor (I) binds only to E, not to ES • Noncompetitive inhibition - inhibitor (I) binds either to E and/or to ES • Uncompetitive inhibition - inhibitor (I) binds only to ES, not to E. This is a hypothetical case that has never been documented for a real enzyme, but which makes a useful contrast to competitive inhibition
    • 58
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    • Mechanisms for Regulating Enzyme Activity 1. Allosteric Enzymes • Allosteric means "other site" or "other structure". • Enzymes whose activity can be changed by molecules (effector molecules) other than substrate. • The interaction of an inhibitor at an allosteric site changes the structure of the enzyme so that the active site is also changed.
    • 2 Processes Involving the Allosteric Enzyme 1. Negative Allosterism: effector binding sites alters the shape of the active site of the enzyme making it to an inactive configuration.
    • 2. Positive Allosterism - effector binding sites that alters the shape of inactive site of enzyme to an active configuration. Therefore, binding of the effector molecule regulates enzyme activity by determining whether it will be active or not.
    • Vo vs [S] for Allosteric Enzymes
    • 2. Feedback Inhibition • An enzyme regulation process in which formation of a product inhibits an earlier reaction in the sequence. It controls the allosteric enzymes.
    • 3. Proenzymes (Zymogen) • The inactive form of enzyme which can be activated by removing a small part on their polypeptide chain. • Mostly are the digestive enzymes and blood clotting enzymes. • Why is it that digestive enzymes are in inactive state before it becomes active? • This is necessary to prevent digestion of pancreatic and gastric tissues.
    • Zymogen • Pepsinogen • Trypsinogen • Prothrombin Active Form of Enzyme • Pepsin • Trypsin • Thrombin
    • 4. Phosphorylation: • Some enzymes may be regulated by addition or removal of phosphate groups from enzymes • phosphate is transferred from an activated donor (usually ATP) to an amino acid on the regulatory enzyme, is the most common example of this type of regulation.
    • Phosphorylation • Phosphorylation reactions are catalyzed by protein kinase and removed by protein phosphatase. • The phosphorylated enzyme may be more or less active the unphosphorylated enzymes. • Phosphorylation of glycogen phosphorylase enzyme result in increase enzyme activity. • Phosphorylation of glycogen synthase enzyme result in less enzyme activity. 71
    • 5. Isoenzymes: are enzymes catalyze the same chemical reaction (identical functions), isolated from different tissues, have same number but different amino acid sequence. Differences may be: – A.acid sequence – Some covalent modification – 3-D structure – The existence of isozymes permits the fine-tuning of metabolism to meet the particular needs of a given tissue or developmental stage. – Isoenzymes e.g – Creatine kinase (CK) – Creatine phosphokinase CPK – alkaline phosphatase (ALP)
    • Percentage of Enzymes Usage in Industries:
    • Detergentes • Contain: - lipase: greasy stains - protease: eggs, blood • Advantage: they work at lower temperatures, so less water heating is needed, and clothes don´t shrink.
    • Food industry • Fruit juices: are extracted using pectinase. It breaks down pectin and is much easier to squeeze juice from the fruit. It also makes the juice clear rather than cloudy. • Biscuits: - isomerase: converts glucose to fructose, which is sweeter so less needs to be used in slimmers biscuits. - protease: softens glutens, making the roller of biscuits easier
    • Three utilizations of enzymes in medicine 1. enzymes as indicators of pathological condition 2. enzymes as analytic reagents in clinical chemistry 3. enzymes as drugs 76
    • Enzymes as tumor markers Enzyme Disease Serum acid phosphatase Cancer prostate Serum Alkaline phosphatase *Metastasis in liver, jaundice due to carcinoma head of pancreas, osteoblastic metastasis in bones *Metastasis, or metastatic disease, is the spread of a cancer from one organ or part to another non-adjacent organ or par Serum LDH Advanced malignancies and Leukemias Β- Glucuronidase Cancer of urinary bladder Leucine Amino Peptidase (LAP) Liver cell carcinoma Neuron specific Enolase Malignancies of nervous tissue and brain 77
    • Summary-Enzymes as diagnostic markers NAME OF THE ENZYME Conditions in which level of activity in serum is elevated Aspartate Amino transferase (AST) Serum glutamate-oxaloacetate transaminase (SGOT) Myocardial infarction, Liver disease especially with liver cell damage Alanine Amino transferase (ALT) Serum glutamate-pyruvate transaminase (SGPT) Liver disease especially with liver cell damage Alkaline Phosphatase (ALP) Liver disease- biliary obstruction Osteoblastic bone disease-rickets Acid Phosphatase (ACP) Prostatic carcinoma  glutamyl Transferase ( GT) Liver disorder like liver cirrhosis and alcoholism Creatine kinase (CK) Myocardial infarction and skeletal muscle disease(muscular dystrophy Lactate Dehydrogenase (LDH) Myocardial infarction, other diseases like liver diseases, some blood diseases  Amylase Acute pancreatitis 78
    • Enzymes as diagnostic reagents Enzyme Used for testing Urease Urea Uricase Uric acid Glucose oxidase Glucose Cholesterol oxidase Cholesterol Lipase Triglycerides Alkaline phosphatase ELISA Horse radish Peroxidase ELISA Restriction endonuclease Recombinant DNA technology Reverse transcriptase Polymerase chain reaction 79
    • Enzymes as therapeutic agents Enzyme Therapeutic Application Trypsin, lipase and amylase Pancreatic insufficiency Asparaginase/Glutaminase Acute lymphoblastic leukemias Hyaluronidase Enhanced local anesthesia and for easy diffusion of fluids Papain Anti inflammatory Serratopeptidase Pain killer and Anti inflammatory Chymotrypsin Pain killer and Anti inflammatory Alpha- 1 Antitrypsin Deficiency and Emphysema 80