Enzymes PDF

Enzymes  Catalysis Think of sugar: Our bodies use it to give energy within seconds (converting it to CO2 + H2O) But a bag of sugar can be on the shelve for years even though the process is very thermodynamically favorable The difference is catalysis ...

Enzymes  Catalysis Think of sugar: Our bodies use it to give energy within seconds (converting it to CO2 + H2O) But a bag of sugar can be on the shelve for years even though the process is very thermodynamically favorable The difference is catalysis  What Are Enzymes? The study of enzymatic processes is the oldest field of biochemistry, dating back to late 1700s. Enzymes are biological catalysts. Increase reaction rates without being used up Effective in small amounts, very efficient Do not affect reaction equilibrium High degree of specificity Extraordinary catalytic power Function in aqueous solutions under very mild conditions of temperature and pH Very few non-biological molecules have all these properties Most enzymes are globular proteins. However, some RNA (ribozymes and ribosomal RNA) also catalyze reactions.  What are Enzymes? Some enzymes need other chemical groups Cofactors – either one or more inorganic ions Coenzymes – complex organic or metalloorganic molecules Some enzymes require both Prosthetic group – a coenzyme or cofactor that is very tightly (or covalently) bound to an enzyme Holoenzyme – a complete active enzyme with its bound cofactor or coenzyme Apoprotein – the protein part of a holoenzyme  What are Enzymes? Enzyme classification Common names (DNA polymerase, urease; pepsin; lysozyme, etc.) 6 classes each with subclasses  What are Enzymes? Enzyme example(s) Group Reaction catalyzed Typical reaction with trivial name To catalyze oxidation/reduction reactions; EC 1 AH + B → A + BH (reduced) transfer of H and O atoms or electrons from Dehydrogenase, oxidase Oxidoreductases A + O → AO (oxidized) one substance to another Transfer of a functional group from one EC 2 substance to another. The group may be AB + C → A + BC Transaminase, kinase Transferases methyl-, acyl-, amino- or phosphate group EC 3 Formation of two products from a substrate Lipase, amylase, AB + H2O → AOH + BH Hydrolases by hydrolysis peptidase Non-hydrolytic addition or removal of groups EC 4 RCOCOOH → RCOH + CO2 from substrates. C-C, C-N, C-O or C-S bonds Decarboxylase Lyases or [X-A-B-Y] → [A=B + X-Y] may be cleaved Intramolecule rearrangement, i.e. EC 5 isomerization changes within a single AB → BA Isomerase, mutase Isomerases molecule Join together two molecules by synthesis of EC 6 new C-O, C-S, C-N or C-C bonds with X + Y+ ATP → XY + ADP + Pi Synthetase Ligases simultaneous breakdown of ATP  E.C. 1: Oxidoreductases Redox reactions: Lactate + NAD+ + H+ LDH→ Pyruvate + NADH Subclasses: – Oxidases – Oxygenases – Oxidative deaminases – Hydroxylases – Peroxidases  E.C. 2: Transferases Functional group transfer reactions: Acetyl CoA + Choline --choline acyltransferase→ Acetylcholine + CoASH Examples: – Aminotransferases – Kinases – Phosphotransferases – Glycosyltransferases  E.C. 3: Hydrolases Hydrolysis reactions: Sucrose + H2O --sucrase→ Glucose + Fructose All digestive enzymes belong here – Peptidases – Proteases – Lipases – Glycosidases – Esterases – Phosphodiesterases – Phosphatases  E.C. 4: Lyases Bond breaking reactions: Fructose 1,6-bisphosphate --aldolase→ Dihydroxyacetone phosphate + Glyceraldehyde 3- phosphate  E.C. 5: Isomerases Interconversion of isomers reactions: Dihydroxyacetone phosphate triose phosphate isomerase→ Glyceraldehyde 3-phosphate Subclasses: – Racemases – Epimerases – Cis-trans isomerases – Aldose-ketose isomerases – Mutases  E.C. 6: Ligases Also called synthetases Binding two reactants reactions using energy from high energy molecules such as ATP: Glutamate + NH3 + ATP --glutamine synthetase→ Glutamine + ADP + Pi Examples: – Synthetases – Carboxylases  Example: ATP + D-glucose → ADP + D-glucose 6-phosphate Formal name: ATP:glucose phosphotransferase Common name: hexokinase E.C. number (enzyme commission): 2.7.1.1 2 → class name (transferases) 7 → subclass (phosphotransferases) 1 → phosphotransferase with –OH as acceptor 1 → D-glucose is the phosphoryl group acceptor  Enzymatic Specificity Absolute specificity Catalysis by certain enzymes on one substrate in one reaction only (Urea –urease→ ammonia) Stereochemical specificity Catalysis by certain enzymes on one stereoisomer only (LDH can only catalyze reactions with L-lactate and not D-lactate) Linkage specificity Certain enzyme require a certain bond to work on with little regard to the nature of neighboring groups (e.g. esterases and lipases) Group specificity Require the presence of particular linkages and additional neighboring groups (chymotrypsin hydrolyzes peptide bond near aromatic amino acids)  Factors Affecting Enzymatic Activity 1. Enzyme Concentration 3. Temperature 2. Substrate Concentration 4. pH  Mechanism of Enzyme Action Active site Substrate Binding of a substrate to an enzyme at the active site (chymotrypsin)  Enzymatic Catalysis A simple enzyme reaction: E + S → ES → EP → E + P – The function of a catalyst is to increase the rate of the reaction. – Catalysts do not affect the equilibrium – Reaction coordinate diagram  Enzymatic Catalysis Slow reactions face significant activation barriers (ΔG‡) that must be surmounted during the reaction –Enzymes increase reaction rates (k) by decreasing ΔG‡ Reaction coordinate diagram. The free energy of the system is plotted against the progress of the reaction S → P. Activation energy –ve (eq. favors P) Position & direction of Starting points for forward or eq. is not affected by reverse reactions ANY catalyst  Enzymes affect reaction rates not equilibria A favorable equilibrium does not mean the S → P conversion would be detectable The rate of the reaction depends on the energy barrier (the activation energy): - a higher activation energy → slower reaction Enzymes Decrease ΔG‡ GB  Rate Enhancement by Enzymes Some Rate Enhancements Produced by TABLE 6-5 Enzymes Cyclophilin 105 Carbonic anhydrase 107 Triose phosphate isomerase 109 Carboxypeptidase A 1011 Phosphoglucomutase 1012 Succinyl-CoA transferase 1013 Urease 1014 Orotidine monophosphate decarboxylase 1017  How to Lower G Enzymes bind transition states best Induced fit The idea was proposed by Linus Pauling in 1946 – Enzyme active sites are complimentary to the transition state of the reaction – Enzymes bind transition states better than substrates – Stronger/additional interactions with the transition state as compared to the ground state lower the activation barrier  Weak interactions are optimized in TS “Lock and key” model – substrate fits the enzyme like a key in a lock This hypothesis can be misleading in terms of enzymatic reactions An enzyme completely complementary to its substrate is a very poor enzyme!  Enzyme Activation Activators are compounds that increase enzyme’s activity Activators are positive modifiers of enzyme activity Usually metal ions Alcohol dehydrogenase -- Zn2+ Hexokinase -- Mg2+ Xanthine oxidase -- Fe3+, Mo4+ Removal of these metal ions results in partial or total loss of enzymatic activity Restoration of lost metal ions regains the lost activity  Enzyme Inhibition Inhibitors are compounds that decrease enzyme’s activity 1. Irreversible inhibitors (inactivators) bind covalently to the enzyme One inhibitor molecule can permanently shut off one enzyme molecule They are often powerful toxins but also may be used as drugs (e.g. aspirin inactivates cyclooxygenase) 2. Reversible inhibitors bind to and can dissociate from the enzyme Effect of inhibitor may be reversed They are often structural analogs of substrates or products They are often used as drugs to slow down a specific enzyme Reversible inhibitor can bind: to the free enzyme and prevent the binding of the substrate to the enzyme-substrate complex and prevent the reaction  Competitive Inhibition Competes with substrate for binding – Binds active site – Does not affect catalysis – many competitive inhibitors are similar in structure to the substrate, and combine with the enzyme to form an EI complex No change in Vmax; apparent increase in Km Competitive Inhibition  Uncompetitive Inhibition Only binds to ES complex Does not affect substrate binding Inhibits catalytic function Decrease in Vmax; apparent decrease in Km No change in Km/Vmax Uncompetitive Inhibition  Mixed Inhibition Binds enzyme with or without substrate — Binds to regulatory site — Inhibits both substrate binding and catalysis Decrease in Vmax; apparent change in Km Noncompetitive inhibitors are mixed inhibitors such that there is no change in Km Mixed Inhibition  Regulatory Enzymes Each cellular metabolism pathway has one or more regulatory enzymes (enzymes that have a greater effect on the rate of the overall sequence) They show increased or decreased activities in response to certain signals (function as switches) Generally, the first enzyme in a pathway is a regulatory enzyme (not always true!)  Regulatory Enzymes Classes of regulatory enzymes: ❖ allosteric enzymes (affected by reversible noncovalent binding of allosteric modulators) ❖ nonallosteric/covalent enzymes (affected by reversible covalent modification) ❖ regulatory protein binding enzymes (stimulated or inhibited by the binding of separate regulatory proteins) ❖ proteolytically activated enzymes (activated by the removal of some segments of their polypeptide sequence by proteolytic cleavage)  Allosteric Enzymes Allosteric enzymes function through reversible, noncovalent binding of regulatory compounds (allosteric modulators, aka allosteric effectors) Allosteric enzymes are generally larger and more complex than nonallosteric enzymes with more subunits Modulators can be stimulatory or inhibitory Sometimes, the regulatory site and the catalytic site are in different subunits Conformational change from an inactive T state to an active R state and vice versa  (C) catalytic (R) regulatory subunits Not to be confused with uncompetitive or mixed inhibitors… Those inhibitors are kinetically distinct and they do not necessarily induce conformational changes Diagnostic Importance of Blood Enzymes Damaged cells secrete their enzymes into the blood Normal levels of certain enzymes are present When diseases occur more damaged cells cause increase in certain enzymes in the blood Blood enzyme levels help in the diagnosis of disease ❖ Creatine phosphokinase (CPK) is an important indication of heart attack ❖ Levels increase within 4-8 hours of infarction and can therefore help in the early detection  Isoenzymes Isoenzymes catalyze the same reaction with different efficiency Structurally similar proteins with similar enzymatic activity May be located in different tissues and/or organelles within a cell ❖ LDH isozymes ❖ First to be identified to exist in various forms: LDH1, LDH2, LDH3, LDH4, LDH5  LDH Isoenzymes Tetramer of two polypeptide chains Increased LDH1 → myocardial infarction Increases within 12-24 hours and remains high for ~ 1 week  CPK Isoenzymes Dimer of two polypeptide chains Increased CPK3 → muscle injury  ALP Isoenzymes Alkaline phosphatase has 5 isoenzymes In a healthy adult only two are present The same protein but different content of the carbohydrate sialic acid 1. ALP-1 (Liver) increased in obstructive jaundice and biliary cirrhosis 2. ALP-2 (bone) increased in rickets nd other bone diseases