• Share
  • Email
  • Embed
  • Like
  • Save
  • Private Content
Enzymes lectures
 

Enzymes lectures

on

  • 3,271 views

 

Statistics

Views

Total Views
3,271
Views on SlideShare
3,271
Embed Views
0

Actions

Likes
1
Downloads
151
Comments
0

0 Embeds 0

No embeds

Accessibility

Categories

Upload Details

Uploaded via as Adobe PDF

Usage Rights

© All Rights Reserved

Report content

Flagged as inappropriate Flag as inappropriate
Flag as inappropriate

Select your reason for flagging this presentation as inappropriate.

Cancel
  • Full Name Full Name Comment goes here.
    Are you sure you want to
    Your message goes here
    Processing…
Post Comment
Edit your comment

    Enzymes lectures Enzymes lectures Document Transcript

    • Enzymes Dr/ Faten LECTURE 1 DEFINITION, PROPERTIES AND CLASSIFICATIONEnzymes* Enzymes are proteins with highly specialized catalytic functions, produced by all living organisms.* Enzymes are responsible for many essential biochemical reactions in microorganisms, plants, animals, and human beings.* Enzymes are natural protein molecules that act as highly efficient catalysts in biochemical reactions, that is, they help a chemical reaction take place quickly and efficiently.* Enzymes are highly specific.* Enzymes not only work efficiently and rapidly, they are also biodegradable.* Enzymes are highly efficient in increasing the reaction rate of biochemical processes that otherwise proceed very slowly, or in some cases, not at all.* All enzymes are protein in nature enzymes are protein in nature, so have the same properties of proteins as denaturation, precipitation, electrophoresis ………….etc.* They are sensitive to changes in pH and temperature.* They function in minute amounts, remain unchanged chemically during the reaction.Chemical nature of enzymesAll enzymes are protein in nature and it may be:1. Simple protein enzyme: only protein chain(s) e.g. pepsin.2. Conjugated protein enzyme: (Holoenzyme)* Holoenzyme: it is an enzyme composed from protein part and non-protein part. a) Protein part (apoenzyme) b) Non-protein part : may beOrganic (coenzymes): loosely attached to apoenzyme Inorganic (activator): loosely attached to apoenzyme* Prosthetic group is a non-protein part firmly attached to the apoproteinBiochemistry Diploma 3OBAD
    • Enzymes Dr/ Faten Enzymes Inorganic catalyst Thermo- labile Thermo- stable Organic, biologically substances Inorganic, non biologically substances Protein in nature, Specific Non protein in nature, Non specific High catalytic efficiency Low catalytic effieciencyDifferentiate between holoenzymes, apoenzymes, co-factor, metal activated enzyme, prosthetic group,coenzyme, metalloenzyme and isoenzyme.A. Holoenzyme: it is an enzyme composed from proteinic part and non-proteinic part.B. Apoenzyme: it is the proteinic part of the holoenzyme.C. Co-factor: it is the non-proteinic part of the holoenzymeD. Metal activated enzyme: holoenzymes which have a loosely bound metals on its prosthetic inorganic group.E. Co-enzyme: specific thermo stable low mol.wt non-protein organic substance bound tightly in usual.F. Metalloenzyme: enzyme which has tightly bound metals as its prosthetic group.G. Isoenzymes: enzymes which have different structures and same function. - Cardiolipin is the prosthetic group of the enzyme cytochrome oxidase. - Cu++, Fe++ ions are the metalloenzyme of cytochrome oxidase.Turnover number:The “turnover number” is the number of substrate molecules converted into product by an enzyme moleculein a unit time when the enzyme is fully saturated.Biochemistry Diploma 3OBAD
    • Enzymes Dr/ FatenLocalization of the enzymeIntracellular enzymes: they act inside the cells that make them.Extracellular enzymes: they act outside the cells that secret them such as: - digestive enzymes in the GIT - coagulation enzymes in plasma.Zymogens* Digestive and coagulation enzymes are secreted in inactive forms, zymogens or proenzyme.* Zymogens are activated by trimming of a short peptide blocking the active site, or by covalentmodification of the zymogen.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: HCL1- pH – changes: Pepsinogen Pepsin trypsin2- Auto-activation: Trypsinogen Trypsin Entrokinase3- By other enzymes: Trypsinogen TrypsinTerminology of enzymes1-Some enzymes still retain their old names as digestion enzymes still use –in pepsin, trypsin.2-End in –ase: Identifies a reacting substance sucrase – reacts sucrose lipase - reacts lipid3-Describes function of enzyme: oxidase – catalyzes oxidation hydrolase – catalyzes hydrolysis4-Enzyme named by the name of both the substrate acted upon and the type of reaction catalyzed. eg.: Succinic Dehydrogenase that remove hydrogen from succinic acid.Biochemistry Diploma 3OBAD
    • Enzymes Dr/ FatenNomenclature of enzymesNomenclature systems are accepted for enzymes:1) 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 reactioncatalyzed, and the ending – ase. Lactate + dehydrogenation + ase = lactate dehydrogenase2) The systematic name:It is made up of the names of substrates of the chemical reaction catalyzed by the enzyme, the name of the typeof the catalyzed chemical reaction, and the ending –ase.L – Lactate NAD+ OxidoreductaseSubstrate I Substrate II type of chemical reactionThe substrateThe substrate of an enzyme are the reactants that are activated by the enzymeEnzymes are specific to their substratsThe specificity is determined by the active siteClassification of EnzymesAll the enzymes are classified into six groups. The name of the group indicates the type of the chemical reactioncatalyzed by the enzymes:1) Oxidoreductases: are involved in oxidation and reduction.2) Transferases: transfer functional groups (e.g., amino or phosphate groups) between donors and acceptors.3) Hydrolases: transfer water; that is, they catalyze the hydrolysis of a substrate.4) Lyases: add (or remove) the elements of water, ammonia, or carbon dioxide (CO2) to (or from) double bonds.5) Isomerases: catalyze changes within one molecule; they include racemases and mutases, as well as epimerases, cis-trans isomerases, intramolecular oxidoreductases, intramolecular transferases, and intramolecular lyases.6) Ligases (Synthetases): join two molecules together at the expense of a high-energy phosphate bond of adenosine triphosphate (ATP).Several subclasses of enzymes fall under each of these major classifications:* Each group is assigned a definite number. The groups are dividing into subgroups; the latter are further subdivided into subsubgroups.* The number of subgroups in a group varies, as well as the number of subsubgroups in a subgroup.Biochemistry Diploma 3OBAD
    • Enzymes Dr/ Faten* According to the numerical classification system, each enzyme receives a four- part number whose numerals are separated by a dot.Biochemistry Diploma 3OBAD
    • Enzymes Dr/ FatenB. TranseferaseDef: enzymes which catalyze the transfer of C- containing, N- containing and sulfur containing groups.Ex: ALT (GPT) Glutamic acid + pyruvic acid α- ketoglutaric acid + AlanineALT (GPT) enzyme can be found in liver normally, but if its found in the serum of the blood in high quantityThis indicates the presence of a disease in the liver. EC 2 Enzyme Transferred group Acyl- transeferase (e.g: synthesis of TG and Acetyl, Succinyl, Aminoacyl phospholipids) Phospho-transeferase (e.g: Hexokinase, Glucokinase) -H2PO3 (phosphoryl) Amino- transeferase(e.g: ALT, AST) -NH2 (amino) Sulfo- transeferase (e.g: GAG synthesis) -SO3H (sulfuryl) E.C. 3 Enzyme Substrate Bond Estrases (e.g: lipase, Neutral fats, phospholipids, ester phospholipase, acetylcholine acetylcholine esterase) Phosphodiesterases (e.g: cAMP Nucleotides (cAMP – 5Amp) phosphodiester phosphodiesterase) Phosphotases (e.g Glucose-6- Phosphatester (G-6-P) phosphomonoester phosphotases) Glycosidases (e.g: amylase, Polysaccharides, disacharides glycoside lipase) Proteases, peptidases (e.g: Proteines, peptides Trypsin)Biochemistry Diploma 3OBAD
    • Enzymes Dr/ Faten E.C. 4 Enzyme Removed groupDecarboxylase (e.g: amino acid decarboxylase) - CO2Aldolase (e.g: aldolase A/B) - aldehydeSynthase (e.g: GLY synthase) CO2 + NH3  GLYDehydratse (e.g: cysthationin synthase) - H2ODesaminase (e.g: Gln desaminase) -NH3E.C 5 Enzyme Isomerated group Changing position of the groupGlucose – 6- phosphate isomerase carbonyl C1  C2Phosphoglycerate phosphomutase phosphoryl C2  C3Epimerase (e.g: UDP-GLU, UDP- Hydroxyl D  LGAL)Biochemistry Diploma 3OBAD
    • Enzymes Dr/ Faten LECTURE 2 ENZYME STRUCTURE, SPECIFICITY AND ACTIONStructure of the Enzyme* Enzymes are proteins, and their function is determined by their complex structure. The reaction takes place in a small part of the enzyme called the active site, while the rest of the protein acts as "scaffolding".* The enzyme is tertiary or quaternary structure that has spatial configuration. This makes the enzyme specific for one reaction only, as other molecules wont fit into the active site –their shape is wrong.* It has pockets on its surface. Each pocket has its own function. The catalytic or active site. The allosteric site.1) The catalytic or active site:- It is the site at which the substrate binds to the enzyme. - It should be fit to the substrate (fitness is made by the tertiary structure of the enzyme molecule). - Any factor affecting this structure will alter the fitness and formation of enzyme-substrate complex.2) The Allosteric site: - It is a site for fitting of a small molecule whose binding alters the affinity of the catalytic site to the substrate. - This small molecule is called allosteric modifier for binding of the substrate: Stimulatory (making it more fit) Inhibitory (making the catalytic site unfit)Biochemistry Diploma 3OBAD
    • Enzymes Dr/ FatenEnzyme Action - An enzyme binds a substrate in a region called the active site - Only certain substrates can fit the active site forming enzyme substrate complex. - The active site contain specific groups of Amino acid help substrate bind. - Enzyme-substrate complex decomposes, giving rise to free enzyme and products of the reaction. E+S  ES  E+ PMode of enzyme action• The reactants should raise their energy levels to reach a transition state.• The transition state represents the energy barrier between the reactants and products.• This energy is known as energy of activation.• The enzyme makes the reaction needs lower activation energy.Biochemistry Diploma 3OBAD
    • Enzymes Dr/ FatenHow do enzymes work? - There are three parts to our thinking about enzyme catalysis.- They each describe different aspects of the same process, and you should know about each of them.1. Reaction Mechanism - In any chemical reaction, a substrate (S) is converted into a product (P). - In an enzyme-catalysed reaction, the substrate first binds to the active site of the enzyme to form an enzyme-substrate (ES) complex, then the substrate is converted into product whilst attached to the enzyme, and finally the product is released, thus allowing the enzyme to start all over again.2. Molecular Geometry: - The substrate molecule is complementary in shape to that of the active site. It was thought that the substrate exactly fitted into the active site of the enzyme molecule like a key fitting into a lock (the now discredited ‘lock and key’ theory). - It is now known that the substrate and the active site both change shape when the enzyme-substrate complex is formed, bending (and thus weakening) the target bonds.3. Energy Changes: - The way enzymes work can also be shown by looking at the energy changes during a chemical reaction. - In a reaction where the product has a lower energy than the substrate, the substrate naturally turns into product (i.e. the equilibrium lies in the direction of the product). - Before it can change into product, the substrate must overcome an "energy barrier" called the activation energy. - The larger the activation energy is, the slower the reaction will be. This is because only a few substrate molecules will have sufficient energy to overcome the activation energy barrier.Biochemistry Diploma 3OBAD
    • Enzymes Dr/ Faten Enzymes reduce the activation energy of a reaction so that the kinetic energy of most molecules exceeds the activation energy required and so they can react.Enzyme specificity The specificity of an enzyme is determined by: - The functional groups of the substrate (or product). - The functional groups of the enzyme and its cofactors. - The physical proximity of these various functional groups.Two theories have been proposed to explain the specificity of enzyme action;A) The lock and key theory: The active site of the enzyme is complementary inconformation to the substrate, so that enzyme and substrate "recognize" one another.Biochemistry Diploma 3OBAD
    • Enzymes Dr/ Faten E S complex  E + P  E + S B) Induced Fit Model - The active site of the enzyme is flexible, not rigid - When the active site identifies the substrate it brings a change in the active site shape so it can accommodate the substrate. - It has a shape complementary to that of the substrate only after the substrate is bound to the enzyme. - This model similar to the rubber gloves. E + S  ES complex  E + PBiochemistry Diploma 3OBAD
    • Enzymes Dr/ FatenThere are 5 types of specificity:1) Absolute specificity :e.g. urease enzyme acts on urea, uricase enzyme acts on uric acid and arginase enzyme acts on arginine.2) Relative Specificity:In this type, the enzyme acts on a group of closely related substrates i.e. which are similar in structure andposses the same type of bonds e.g.Lipase catalyzes the process of hydrolysis of ester linkage present in triglycerides containing different types offatty acids.3) Stereo-specificity or optical specificity: The enzyme works on one of two isomers e.g.: L – Amino acid oxidase acts cn L – amino acids only. & Enzymes of glycolysis act on D-sugars only.Exception: There is only one exception which is racemase enzyme catalyses reversible interconversion of D and L isomers.4) Dual specificity: - The enzyme acts on 2 different substrates e.g.: Xanthine oxidase can oxidize hypoxanthine and xanthine to uric acid. Isocitric acid dehydrogenase acting on isocitric acid causing dehydrogenation and decarboxylation.5) Group – specificity or structural specificity: -The enzyme shows specificity not only to the bond and its position but also towards the chemical groups or atoms surrounding this bond e.g. carboxypeptidase acts on the free carboxyl end of polypeptide chain . aminopeptidase acts on the free amino end of polypeptide chain .Autolysis - More commonly known as self-digestion, refers to the destruction of a cell through the action of its own enzymes. It may also refer to the digestion of an enzyme by another molecule of the same enzyme. - In the living body these enzymes are usually inactive since their optimum pH is slightly acidic, while the pH of the body fluids is slightly alkaline and unsuitable for their activity. - After death, lactic acid accumulate in the tissues and cathepsins become activated .Biochemistry Diploma 3OBAD
    • Enzymes Dr/ Faten LECTURE 3 FACTORS AFFECTING ENZYMESFactors Affecting Enzyme Action- The activity of the enzyme is evaluated by measuring the rate or the velocity of the reaction.- velocity of the reaction is measured by how many moles of the substrate are converted into products perunit of time (minute).The factors include the following:1- Substrate 2- Temperature 3- PH4- Time 5- Cofactor 6- Enzyme Inhibitors7- Hormones 8- Radiation & Light 9- Product concentration10- Enzyme activators 11- Antienzyme & antibodies 12- Concentrations of Co- Factors1- Temperature- Little activity at low temperature.- Rate increases with temperature.- Each enzyme has an optimum temperaturesat which the enzyme acts maximally.- Most active at optimum temperatures(usually 37°C in humans and 65°C for plantenzymes).- Activity lost with denaturation at hightemperatures.2- pH- Each enzyme has an optimum pH at whichthe enzyme acts maximally.- Maximum activity at optimum pH- Most lose activity in low or high pH3-Substrate ConcentrationAs substrate concentration increases,- the rate of reaction increases (at constantenzyme concentration).- the enzyme eventually becomes saturatedgiving maximum activity.Biochemistry Diploma 3OBAD
    • Enzymes Dr/ Faten4-Enzyme Concentration-Increasing enzyme concentration increases the rate of reaction.- Further increase in the enzyme can not increase the velocity ofreaction because the amount of substrate may not be sufficient topermit of maximum velocity.Michaelis Constant (Km)Km is equal to the substrate concentration [S] atwhich the reaction is half of its maximum(1/2Vmax).It expresses the affinity of the enzyme to itssubstrate.Low Km means high affinity of the enzyme to thesubstrate.High Km means low affinity of the enzyme to thesubstrate.Enzymes – Lineweaver-Burk Equation5-Hormonese.g. insulin hormone stimulates glucokinase enzyme while glucocorticoids inhibit it.Steroid hormones are known to increase the rate of synthesis of many enzyme.Biochemistry Diploma 3OBAD
    • Enzymes Dr/ Faten6-TimeAs time is passed the rate of the enzyme catalyzed reaction diminishes due to:*Decline of substrate concentration.*The accumulated product may cause feedback inhibition of the enzyme.7-Product concentrationAs you increase the product concentration youdecrease the rate of the reaction.The excess amount of product accumulates andoccupies the active site of the enzyme.8-Radiation and lightLight inhibit most enzyme activity although some enzymes e.g. amylase is activated by red or green light.Ultraviolet rays and ionized radiations cause denaturation of most enzymes.9-Enzyme activators - Certain inorganic ions e.g.: CL¯activate salivary and pancreatic amylase. Ca++ activate thrombokinase. Bile salts activate lipase enzyme.- Some enzymes are secreted in inactive form and are activated by: pH: pepsinogen is activated by HCL giving pepsin. Autoactivation: pepsinogen pepsin Kinase: which activate zymogen or proenzymes.10- Enzyme InhibitorsDefinition: substances which inhibit (stop) the enzyme activity. It causes a loss of catalytic activity Change the protein structure of an enzyme.It may be : Non-specific or specific.Biochemistry Diploma 3OBAD
    • Enzymes Dr/ Faten11-Antienzymes and antibodiesAntienzymes: ascaris living in the lumen of intestine secrete antipepsin and antitrypsin to prevent digestionof the worm by these enzymes.Antibodies: if enzyme is injected, the immune system of the body produces antibodies which will inactivatethese enzymes.12-Concentration of cofactorsThe rate of enzyme reaction is directly proportional to the concentration of the cofactors.Definition: An additional non-protein molecule that is needed by some enzymes to help the reactionTightly bound cofactors are called prosthetic groupsCofactors that are bound and released easily are called coenzymesMany vitamins are coenzymes.Coenzyme = thiamine pyrophosphate (TPP)  used in decarboxylation and transketolation contains pyrimidine and thiazole. disease due to deficiency: beri-beri, Wernicke’s disease. peripheral nerves, muscle cramps, numbnessCoenzyme :flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD) both act as prosthetic groups -- use = redox reactions -- its vitamin = riboflavin or B2Nicotinamide-adenine dinucleotide (NAD), nicotinamide-adenine dinucleotide phosphate(NADP)used in redox reactions with H transfer.Its vitamin =niacin or B3 =nicotinamide& nicotinic acidDisease due to deficiency pellagra, skin lesions,swollen tongue, nervous/mental disordersBiochemistry Diploma 3OBAD
    • Enzymes Dr/ FatenCoenzyme = pyrodoxal phosphateUsed in decarboxylations, transaminations and racemases.Its vitamin = pyridoxine, or vitamin B6Coenzyme = Coenzyme A (CoA)use = activates carbonyl groups and in acyl transfer(acetyl- CoA, synthesis of fats and steroids)its vitamin = pantothenic aciddisease due to deficiency GI problems, emotional instability, burning sensation in extemitiesCoenzyme = folate or tetrahydrofolate (the reduced form)Used in transfer of one carbon unit or formateIts vitamin = folic acid Disease due to deficiency megablastic anemia, birth defectsCoenzyme = biotina prosthetic group-- use = carboxylations-- its vitamin = biotinCoenzyme = cyanocobalaminUsed in methyl group transfer; folate metabolism, myelin synthesisIts vitamin is cyanocobalamin or vitamin B12 disease due to deficiency pernicious anemiaCoenzyme = lipoic acid(reduced SH or oxidized form -S-S-)prosthetic groupUsed in redox reactions Its vitamin = lipoic acid(humans probably produce enough so it is not always considered a vitamin)Biochemistry Diploma 3OBAD
    • Enzymes Dr/ Faten LECTURE 4 ENZYME INHIBITORSDefinition: substances which inhibit (stop) the enzyme activity. It causes a loss of catalytic activity Change the protein structure of an enzyme.It may be : Non-specific or specific.Non-specific inhibitors:These are inhibitors which exert their effect on all enzymes or onwide variety of enzymes; e.g.Agents which precipitate or denaturate proteins.Specific inhibitors:These are inhibitors which exert their effect on one enzyme oron a small group of related enzymes.May be competitive or noncompetitive*The molecule resembling the substrate.*Can bind to the active site of the enzyme and so it can form enzyme inhibitor complex EI.*Decreases the affinity of enzymes for substrate.*Excessive concentrations of substrate will break the EI complex and then S can bind to the enzyme*Reversible.*It depends on Substrate and Inhibitor.Biochemistry Diploma 3OBAD
    • Enzymes Dr/ FatenExamples of competitive inhibitors:Allopurinol competes with hypoxanthine for xanthine oxidase inhibiting the formation of uric acid, so it is usedin treatment of hyperuricemia (gout).Dicumarol or Warfarine compete with vitamin K, for epoxidereductase, so they are used to reduce prothrombin synthesis.Statins (e.g. atorvastatin) competes with HMGCoA for its reductase,so, it inhibits cholesterol synthesis.Methotrexate competes with dihydrofolic acid for dihydrofolatereductase, so, it inhibits DNA synthesis and used in treatment of cancers. Succinate dehydrogenase Succinate Fumarate + 2H++ 2e-Competitive Inhibition Product Substrate Competitive Inhibitor Succinate DehydrogenaseDoes not have a structure like substrateBinds to the enzyme but not active siteChanges the shape of enzyme and active siteSubstrate cannot fit altered active siteNo reaction occursEffect is not reversed by adding substrateBiochemistry Diploma 3OBAD
    • Enzymes Dr/ FatenEnzyme Inhibition (Mechanism)Competitive Inhibition: Non-Competitive Inhibition: * A competitive inhibitor molecule has a * A non-competitive inhibitor molecule is quitesimilar structure to the substrate molecule, different in structure from the substrate andand so it can fit into the active site of the does not fit into the active site.enzyme. * It binds to another part of the enzyme * It therefore competes with the substrate for molecule, changing the shape of the wholethe active site, so the reaction is slower. enzyme, including the active site, so that it can no longer bind substrate molecules. * Increasing the concentration of substraterestores the reaction rate and the inhibition is * Non-competitive inhibitors therefore simplyusually temporary and reversible. reduce the amount of active enzyme. - Allosteric inhibitors are low-molecular weight substances, they regulate the enzyme activity. - The inhibitor is not similar in structure to the substrate and it is bound to apoenzyme at sites far from the active site this site is called allosteric site.- It is not reversable by increasing the concentration of the substrate.- This interaction causes conformational changes in the catalytic site that makes it unfavorable for binding to Substrate.Biochemistry Diploma 3OBAD
    • Enzymes Dr/ Faten - When Allosteric inhibitor is consumed the activity of the enzyme is regained ,so Allosteric inhibition isreversible. e.g. glucose-6-phosphate is allosteric inhibitor for hexokinase enzyme. - Some time conformational change of apoenzyme produce by binding to the allosteric effector, makw theactive center more fit to bind with the substrate, thus the enzyme activity is increased. In this case theeffector is called allosteric activator e.g. AMP and ADP are allosteric activator to phosphofructokinase.End- product of a metabolic pathwayinhibits the initial enzyme in the pathway,this called feed-back inhibition. * Agents that block the coenzyme will stop enzyme action e.g. phenylhydrazine will block the aldehyde group of pyridoxal phosphate, which is the coenzyme of transaminase. * Agents that block the prosthetic group will stop enzyme action e.g.cyanide and carbon monoxide block the iron of heme of cytochrome oxidase enzyme.Biochemistry Diploma 3OBAD
    • Enzymes Dr/ Faten1. Covalent modification:(short term regulation) The enzyme may be phosphorylated in the OH group of serine, threonine, or tyrosine. This process iscatalyzed by kinase. ATP is the source of phosphate.The phosphoprotein produced is dephosphorylated by phosphatase.  phosphoprotein enzyme is the active form of the enzyme, e.g. glycogen phosphorylase.  the dephosphorylated form is the active form of the enzyme, e.g. glycogen synthase.2. Induction and repression of enzyme synthesisEnzymes synthesis (long term regulation ).• Synthesis is stimulated by "induction" or inhibited by "repression".• Control of enzyme synthesis is under hormonal control that affects geneexpression related to that particular enzyme.• Example:• Insulin induces synthesis of glucokinase and phosphofructokinase, but repressesglucose-6-phosphatase and fructose 1,6,bisphosphatase.• Steroid and thyroid hormones effects on enzyme generation.• Control of enzyme activity by induction or repression consumes hours ordays to be achieved.3. Allosteric modification (short term regulation):occurs within seconds or minutes LECTURE 5Biochemistry Diploma 3OBAD
    • Enzymes Dr/ Faten ISOENZYMESThese are different isomers of the same enzyme which differ by having a different electrophoretic mobilityand different tissue source.Each of these is called isoenzyme and all of them have the same catalytic activity e.g. lactate dehydrogenase(LDH) and creatin kinase (CK).Isozymes are different molecular forms of enzymes that may be isolated from the same or different tissues.Analysis of the distribution of isozymes of particular enzymes is sometimes a useful tool in clinicaldiagnosis.1) Creatine kinase (CK)occurs as a dimer consisting of two subunits can be present as two distinct molecular formsbrain type (B) and muscle type (M).Thus, three isozymes are possible CK-MM, CK-BB, and CK-MB.These isozymes can be readily distinguished and quantitated by electrophoresis.CK-MB is normally present in trace amounts only in the myocardium.Elevation of CK-MB levels to greater than 6% of the total CK is diagnostic of a myocardial infarction.CPK is made of three slightly different substances:CPK-1 (also called CPK-BB) is found mostly in the brain and lungs.CPK-2 (also called CPK-MB) is found mostly in the heart.CPK-3 (also called CPK-MM) is found mostly in skeletal muscle.2) Lactate dehydrogenase (LDH)is a tetramer of four subunits which can be present as two distinct molecular forms.Type H is found primarily in heart, and type M is found primarily in muscle or liver.Five isozmes of LDH composed of different combinations of these subunits are possible:M4, M3H, M2H2, MH3 and H4. In normal serum the H3M isozyme is present. In an individual who has suffered a myocardial infarction,the serum levels of the H-containing isozymes, particularly H4, are elevated.The increase in H4 such that ratio of H4/H3M is greater than 1 confirms the diagnosis that the patientsuffered a myocardial infarction.LDH exists in 5 isoenzymes , which differ slightly in subunits:LDH-1 HHHH is found primarily in heart muscle and red blood cells.LDH-2 HHHM is concentrated in white blood cells.LDH-3 HHMM is highest in the lung.LDH-4 HMMM is highest in the kidney, placenta, and pancreas.LDH-5 MMMM is highest in the liver and skeletal muscle.Biochemistry Diploma 3OBAD
    • Enzymes Dr/ FatenThe use of isozymes in the diagnosis of myocardial infarctionHigh levels of the MB isozyme of creatine (CK-MB) relative to other CK isozymes and the analysis oflactate dehydrogenase (LDH) isozymes indicating higher serum levels of the H-containing isozymes,particularly H4 with elevated ratio of H4/H3M greater than 1, are indicative with the clinical picture ofmyocardial infarction.Conditions other than myocardial infarction can cause similar changes in the isozyme patterns of either CKor LDH, but it is unlikely that both patterns would be affected in this manner.Therefore, data such as those described above can be reliably used to distinguish between the occurrence ofmyocardial infarction and other conditions that may caused the reported symptoms.Examples for medical important enzymes:Lipase: it is elevated in acute pancreatitis and pancreatic carcinoma.Amylase: it is elevated in parotitis, acute pancreatitis and pancreatic carcinoma.Trypsin: also increased in pancreatic diseases.Cholinesterase: is lowered in exposure to insecticides and is elevated in conditions of active heamopoiesis.Serum alkaline phosphatase : it is elevated in :1- Liver diseases as obstructive jaundice.2- Bone diseases: rickets, osteogenic sarcoma and Pagets disease.3- Hyperparathyroldism and malignancy.Serum Acid Phosphatase is elevated in cancer prostate.Lactic Acid Dehydrogenase (LDH) is elevated in cases of myocardial infarction.Creatine Phosphokinase (CK) is elevated in cardiac and skeletal muscle diseases.Transaminases :1- Glutamic pyruvic transaminase GPT increased in any disease causing damage or destruction to livercells e.g. infective hepatitis.2- Glutamic oxaloactic transaminase GOT is elevated in damage of heart e.g. coronary thrombosis andliver disease.Biochemistry Diploma 3OBAD
    • Enzymes Dr/ FatenNon-Functional plasma enzymesThese are enzymes which have no specific function in blood and having no substrate to act upon.They are present in low concentration in blood and originally they are present in different organs inside theircells.If the cells of theses organs are destroyed by any disease, these enzymes are liberated and appear in higherconcentrations in blood.So, the presence of these non-functional enzymes in higher concentrations than normal in blood is of greatclinical importance for diagnosis of such diseases. Assayed Enzyme Diagnostic UsesAcid phosphatase Prostate cancerAlanine aminotransferase (ALT) Viral hepatitis, liverAlkaline phosphatase Liver disease, bone disordersAmylase Acute pancreatitisCreatine kinase (CK) Muscle disorders, heart attackLactate dehydrogenase (LDH) Heart attackBiochemistry Diploma 3OBAD