Enzymes (1).ppt PDF

BCH 201/216 ENZYMES Enzymes as Catalyst, Active Sites, Substrate Specificity  Enzymes as Catalyst Enzymes are protein (organic) catalysts that increase the velocity of a chemical reaction Note: Some RNAs can act like enzymes, usually catalyzing the cleavage and...

BCH 201/216 ENZYMES Enzymes as Catalyst, Active Sites, Substrate Specificity  Enzymes as Catalyst Enzymes are protein (organic) catalysts that increase the velocity of a chemical reaction Note: Some RNAs can act like enzymes, usually catalyzing the cleavage and synthesis of phosphodiester bonds. RNAs with catalytic activity are called ribozymes.  Enzymes as Biological Catalysts/Properties of Enzymes Enzymes are proteins that increase the rate of reaction by lowering the energy of activation Enzymes are highly specific, interacting with one or a few substrates and catalyzing only one type of chemical reaction Enzyme-catalyzed reactions are highly efficient, proceeding from 10ᴧ3 – 10ᴧ8 times faster than uncatalyzed reactions. Not altered or consumed during reaction. Reusable Enzymes work by reducing the Activation Energy of a Reaction 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 Every chemical reaction is characterized by an equilibrium constant, Keq, which is a reflection of the difference in energy between reactants, aA, and products, bB An enzyme-controlled pathway  Diagram of Energy Difference Between Reactants and Products The uncatalyzed reaction has a large activation energy, Ea, seen at left In the catalyzed reaction, the activation energy has been lowered significantly increasing the rate of the reaction  The Concept of the Active Site The part of the enzyme combining with the substrate is the active site The shape and the chemical environment inside the active site permits a chemical reaction to proceed with a greater ease. Active sites characteristics include: – Pockets or clefts in the surface of the enzyme R groups at active site are called catalytic groups – Shape of active site is complimentary to the shape of the substrate – The enzyme attracts and holds the substrate using weak noncovalent interactions – Conformation of the active site determines the specificity of the enzyme Enzymes are specific to their substrates and this specificity is determined by the active site.  Lock and Key (Emil-Fischer’s) Enzyme Model In the lock-and-key model, the enzyme is assumed to be the lock and the substrate the key – The enzyme and substrate are made to fit exactly like a key fits into a lock. – This explains enzyme specificity – Also explains the loss of activity when enzymes denature. – However, it fails to take into account protein- conformational changes to accommodate a substrate molecule.  Induced Fit (Daniel Koshland’s) Enzyme Model The induced-fit model of enzyme action assumes that the enzyme active site is more a flexible pocket. It however undergoes conformational changes to accommodate the substrate. These changes then provide the chemical environment suitable for the reaction. The bonds of the substrate are stretched to make the reaction easier (lowers activation energy) This explains how enzymes can react with a range of substrates of similar types as well as allosterism.  Enzyme Catalyzed Reactions When a substrate (S) fits properly in an active site, an enzyme-substrate (ES) complex is formed: E + S  ES Within the active site of the ES complex, the reaction occurs to convert substrate to product (P): ES E + P The products are then released, allowing another substrate molecule to bind the enzyme - this cycle can be repeated millions (or even more) times per minute The overall reaction for the conversion of substrate to product can be written as follows: E + S  ES E + P  Enzyme Specificity Enzymes have varying degrees of specificity for substrates 1.Absolute Specificity: catalyzes one type of reaction for a single substrate e.g urease catalyzes only hydrolysis of urea. 2.Group Specificity: catalyzes reaction involving any molecules with the same functional group e.g hexokinase adds phosphate group to hexoses 3.Linkage Specificity: enzyme catalyzes the formation or break up of only certain category or type of bond e.g chymotrypsin catalyzes hydrolysis of peptide bonds 4.Stereochemical Specificity: enzyme recognizes only one of two enantiomers. Enzyme Nomenclature and Classification  Nomenclature / enzyme classification IUBMB (International Union of Biochemistry and Molecular Biologists) has recommended a system of nomenclature for enzymes in which each enzyme is assigned with two names:  Trivial name (common name, recommended name).  Systematic name ( official name ).  Trivial, Recommended or Common name The name of an enzyme identifies the reacting substance - usually ends in -ase e.g sucrase catalyzes the hydrolysis of sucrose The name may also describe the function of the enzyme e.g oxidases catalyze oxidation reactions Sometimes common names are used, particularly for the digestive enzymes such as pepsin and trypsin Some names describe both the substrate and the function e.g alcohol dehydrogenase oxidizes ethanol  IUBMB System of Enzyme Classification Enzymes are classified according to the type of reaction they catalyze: Class Reactions catalyzed  EC 1 Oxidoreductases Oxidation-reduction  EC 2 Transferases Transfer groups of atoms  EC 3 Hydrolases Hydrolysis  EC 4 Lyases Adds atoms/removes atoms to/from a double bond  EC 5 Isomerases Rearranges atoms  EC 6 Ligases Uses ATP to combine molecules  Systematic/Official/IUBMB Name Each enzyme is characterized by a code no. called Enzyme Code no or EC number and contains four figures (digits) separated by a dot e.g EC m. n. o. p First digit (m) represents the class; Second digit (n) stands for subclass; Third digit (o) stands for the sub-sub class or subgroup; Fourth digit (p) gives the serial number of the particular enzyme in the list.  Systematic/Official/IUBMB Name Using the IUBMB naming system, the official, systematic name for Hexokinase is: ATP:D-hexose 6-phosphotransferase E.C. 2.7.1.1. – This name identifies hexokinase as a member of class 2 (transferases), – subclass 7 (transfer of a phosphoryl group), – sub-subclass 1 (alcohol is the phosphoryl acceptor), – and "hexose-6" indicates that the alcohol phosphorylated is on carbon six of a hexose. Despite the clarity of the IUB system, the names are lengthy and relatively cumbersome, biochemists continue to use the common name e.g hexokinase. Oxidoreductases, Transferases and Hydrolases Lyases, Isomerases and Ligases Factors Affecting Enzyme Activity/Enzyme Regulation  Factors Affecting Enzyme Activity Temperature Hydrogen ion concentration (pH) Substrate concentration Enzyme concentration Products of the reaction Presence of activator/inhibitor Allosteric effects Time  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 Q10 Denaturat Enzym ion e activity 0 10 20 30 40 50 © 2007 Paul Billiet ODWS Temperature / °C  Temperature and Enzyme Activity Enzymes are most active at an optimum temperature (usually 37°C in humans) They show little activity at low temperatures Activity is lost at high temperatures as denaturation occurs A few bacteria have enzymes that can withstand very high temperatures up to 100°C Most enzymes however are fully denatured at 70°C  pH and Enzyme Activity Enzymes are most active at optimum pH Amino acids with acidic or basic side-chains have the proper charges when the pH is optimum Activity is lost at low or high pH as tertiary structure is disrupted  Optimum pH for Selected Enzymes Most enzymes of the body have an optimum pH of about 7.4 However, in certain organs, enzymes operate at lower and higher optimum pH values  Enzyme Concentration and Reaction Rate The rate of reaction increases as enzyme concentration increases (at constant substrate concentration) At higher enzyme concentrations, more enzymes are available to catalyze the reaction (more reactions at once) There is a linear relationship between reaction rate and enzyme concentration (at constant substrate concentration) Substrate Concentration and Reaction Rate The rate of reaction increases as substrate concentration increases (at constant enzyme concentration) Maximum activity occurs when the enzyme is saturated (when all enzymes are binding substrate) The relationship between reaction rate and substrate concentration is exponential, and asymptotes (levels off) when the enzyme is saturated  Enzyme 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 Inhibitors  Enzyme Inhibitors Enzyme inhibitors are substances that bind to the enzyme reversibly or irreversibly, decreases the activity of enzyme in a process is known as enzyme inhibition. Enzyme inhibitors are used to gain information about the shape of the active site of the enzyme and amino acids residues in the active site. They are used to gain information about regulation or control of metabolic pathway.  Enzyme Inhibitors They can be used for drug designing. They are important for correcting metabolic imbalance. They are used for designing herbicides, pesticides and for killing pathogens e.g. antibiotics, antivirals, antifungals.  Enzyme Inhibitors - Classification I. On the basis of specificity: 1. Co-enzyme inhibitor: Inhibits co-enzymes only. E.g. cyanide hydrazine, hydroxyl amine inhibits co-enzyme pyridoxal phosphate. 2. Ion-cofactor inhibitor: E.g. fluoride chelate Mg2+ ion of enolase enzyme. 3. Prosthetic group inhibitor: E.g. cyanide inhibit Heme of cytochrome oxidase. 4. Apoenzyme inhibitor: E.g. antibiotics 5. Physiological modulator:  Enzyme Inhibitors - Classification II. On the basis of origin: 1. Natural enzyme inhibitor: E.g. Aflatoxin, – amanitin 2. Artificial enzyme inhibitor (synthetic): E.g. drugs  Enzyme Inhibitors - Classification III. On the basis of reversibility or otherwise 1. Reversible inhibition: The enzyme inhibition in which the enzymatic activity can be regained after removal of inhibitors. - Competitive - Non-competitive - Uncompetitive - Mixed 2. Irreversible inhibition: The enzyme inhibition in which the enzymatic activity cannot be regained after removal of inhibitors.  Enzyme Inhibitors - Competitive inhibition Competitive inhibition Competitive inhibitors are substrate analogues that bind to substrate binding site of enzyme i.e. active site so competition occurs between inhibitor and substrate for binding to enzyme. This type of inhibitor is overcome by increasing the concentration of substrate. The kinetics of reaction is Vmax remains same and Km increases. Enzyme Inhibitors - Competitive inhibition  Enzyme Inhibitors - Competitive inhibition In this reaction, initially inhibitor binds to enzyme but with increase in concentration of substrate causes release of inhibitor. Then, substrate bind enzymes so that the Vmax remains same while Km increases. Example: Succinate dehydrogenase convert succinate to fumarate. Succinate —succinate dehydrogenase————–> Fumarate + NADH +H+  Enzyme Inhibitors - Competitive inhibition Malate is competitive inhibitor of succinate due to structural analogy. Malate + NAD+ —–succinate dehydrogenase———> Oxaloacetate Sulphonamide is competitive inhibitor of PABA during tetrahydrofolate synthesis. Example: Treatment of methanol poisoning: Methanol —–alcohol dehydrogenase————-> Formaldehyde (toxic) Ethanol ——alcohol dehydrogenase———> Acetaldehyde  Enzyme Inhibitors - Non-competitive inhibition Non-competitive inhibition: In this inhibition, there is no competition between substrate and inhibitor because the inhibitor binds to enzyme other than substrate binding site. Since the binding site of substrate and inhibitor to enzyme is different, inhibitor doesn’t affect the affinity of enzyme to substrate. In this case, the inhibition cannot be overcome by increasing substrate concentration.  Enzyme Inhibitors - Non-competitive inhibition The kinetic reaction is that Vmax decreases and Km remains same. This means that substrate concentration has no effect on inhibition. Binding of substrate and inhibitor are equal. The inhibitor changes the conformation of enzyme after binding so that substrate cannot bind to enzyme. This results in decrease of Vmax. Enzyme Inhibitors - Non-competitive inhibition  Enzyme Inhibitors - Non-competitive inhibition Example: Heavy metal poisoning. Hg, Pb etc. distort the -SH group containing enzyme at allosteric site. Doxycycline is non-competitive inhibitor of proteinase enzyme of bacteria. The non-competitive inhibitor can be removed by pH treatment or by hydrolysis. In case of metal poisoning, chelator is used.  Enzyme Inhibitors - Uncompetitive inhibition Uncompetitive inhibitor: This type of inhibition is seen in multi-substrate reaction. It is rare type of inhibition. The process of inhibition is same as non-competitive but it only binds to ES-complex. At first substrate binds to enzyme to form ES-complex. After binding of substrate to active site of enzyme, the binding site for inhibitor forms at allosteric site so that inhibitor bind.  Enzyme Inhibitors - Uncompetitive inhibition The binding of inhibitor distorts the active as well as allosteric site of enzyme, inhibiting catalysis. In this inhibition, Vmax as well as Km both decreases. Examples: Inhibition of lactate dehydrogenase by oxalate. Inhibition of alkaline phosphatase by L-phenylalanine.  Enzyme Inhibitors - Mixed inhibition Mixed inhibition: This type of inhibition is commonly seen in multi-substrate reaction. It is the combination of competitive as well as non-competitive inhibition. The mixed inhibitor can bind to both active site and allosteric site. The kinetics of reaction is that Vmax decreases and Km increases.  Enzyme Inhibitors - Mixed inhibition The Vmax decreases because inhibitor non-competitively binds to allosteric site and distort enzyme. Similarly, Km increases because inhibitor can also bind to active site competition with substrate. This type of inhibition cannot be removed by increasing substrate concentration. Examples: Ketoconazole is mixed inhibitor bind to 5–α reductase enzyme. Pallidium ion is mixed inhibitor of oxidoreductase enzyme. Regulation of Enzyme Activities  Regulation of Enzyme Activities Enzyme activity must be regulated so that the proper levels of products are produced at all times and places This control occurs in several ways: – biosynthesis at the genetic level – covalent modification after biosynthesis – feedback inhibition – Storage as inactive forms called zymogens – regulatory enzymes – allosteric modification A common covalent enzyme modification is the addition or removal of a phosphate group  Under high-energy conditions (high ATP), phosphorylation is favored  Under low-energy conditions (low ATP), dephosphorylation is favored  This regulates the balance between biosynthesis and catabolism  Zymogens Zymogens (proenzymes) are inactive forms of enzymes They are activated by removal of peptide sections For example, proinsulin is converted to insulin by removing a 33-amino acid peptide chain.  Digestive Enzymes Digestive enzymes are produced as zymogens, and are then activated when needed. Most of them are synthesized and stored in the pancreas, and then secreted into the small intestine, where they are activated by removal of small peptide sections. The digestive enzymes must be stored as zymogens otherwise they would damage the pancreas.  Allosteric Enzymes An allosteric enzyme binds a regulator molecule at a site other than the active site (an allosteric site) Regulators can be positive or negative: - a positive regulator enhances the binding of substrate and accelerates the rate of reaction. - a negative regulator prevents the binding of the substrate to the active site and slows down the rate of reaction (non-competitive inhibition)  Feedback Control In feedback control, a product acts as a negative regulator When product concentration is high, it binds to an allosteric site on the first enzyme (E1) in the sequence, and production is stopped When product concentration is low, it dissociates from E1 and production is resumed Feedback control allows products to be formed only when needed Reaction/Enzyme Kinetics  Reaction/Enzyme Kinetics When an enzyme is added to a substrate, the reaction that follows occurs in three stages with distinct kinetics: Phase Concentration of ES Rate of product formation Rapid burst of ES Initially slow, waiting for Pre-steady state complexes form ES to form, then speeds up ES concentration Constant rate of formation, remains constant as it is Steady-state (equilibrium) faster than the pre-steady being formed as quickly as state it breaks down Slow as there are fewer ES Substrate depletes so fewer Post-steady state complexes; slows down as ES complexes form substrate runs out  Reaction/Enzyme Kinetics The pre-steady state phase is very short, as equilibrium is reached within microseconds. Therefore, if you measure the rate in the first few seconds of a reaction, you will be measuring the reaction rate in the steady state. This is the rate used in Michaelis-Menten Kinetics.  Michaelis-Menten Kinetics Michaelis-Menten kinetics is a model of enzyme kinetics which explains how the rate of an enzyme-catalysed reaction depends on the concentration of the enzyme and its substrate. Let’s consider a reaction in which a substrate (S) binds reversibly to an enzyme (E) to form an enzyme- substrate complex (ES), which then reacts irreversibly to form a product (P) and release the enzyme again. S + E ⇌ ES → P + E Two important terms within Michaelis-Menten kinetics are:  Michaelis-Menten Kinetics Two important terms within Michaelis-Menten kinetics are: Vmax – the maximum rate of the reaction, when all the enzyme’s active sites are saturated with substrate. Km (also known as the Michaelis constant) – the substrate concentration at which the reaction rate is 50% of the Vmax. Km is a measure of the affinity an enzyme has for its substrate, as the lower the value of Km, the more efficient the enzyme is at carrying out its function at a lower substrate concentration.  Michaelis-Menten Kinetics The Michaelis-Menten equation for the reaction above is: This equation describes how the initial rate of reaction (V0) is affected by the initial substrate concentration ([S]). It assumes that the reaction is in the steady state, where the ES concentration remains constant.  Michaelis-Menten Kinetics When a graph of substrate concentration against the rate of the reaction is plotted, we can see how the rate of reaction initially increases rapidly in a linear fashion as substrate concentration increases (1st order kinetics). The rate then plateaus, and increasing the substrate concentration has no effect on the reaction velocity, as all enzyme active sites are already saturated with the substrate (0 order kinetics). Michaelis-Menten Kinetics - Graph of the rate of reaction against substrate concentration, demonstrating Michaelis – Menten kinetics, with Vmax and Km highlighted.  Michaelis-Menten Kinetics This plot of the rate of reaction against substrate concentration has the shape of a rectangular hyperbola. However, a more useful representation of Michaelis– Menten kinetics is a graph called a Lineweaver–Burk plot, which plots the inverse of the reaction rate (1/r) against the inverse of the substrate concentration (1/[S]).  Michaelis-Menten Kinetics This produces a straight line, allowing for the easier interpretation of various quantities and values from the graph. For example, the y-intercept of the graph is equivalent to the Vmax. The Lineweaver-Burk plot is also useful when determining the type of enzyme inhibition present by, comparing its effect on Km and Vmax. Michaelis-Menten Kinetics - Different types of enzyme inhibition as shown on a Lineweaver-Burk plot Cofactors, Coenzymes and Prosthetic Groups  Cofactors, Coenzymes and Prosthetic Groups These are small nonprotein organic, inorganic molecules and metal ions that participate directly in substrate binding or catalysis. These extend the repertoire of catalytic capabilities beyond those afforded by the limited number of functional groups present on the aminoacyl side chains of peptides. Enzymes that require a cofactor, coenzyme or prosthetic group but do not have one bound are called apoenzymes or apoproteins. An enzyme together with the cofactor(s) required for activity is called a holoenzyme (or haloenzyme). Cofactors, Coenzymes and Prosthetic Groups The term holoenzyme can also be applied to enzymes that contain multiple protein subunits, such as the DNA polymerases; Here the holoenzyme is the complete complex containing all the subunits needed for activity There are ambiguities in the use of the terms cofactors, coenzymes and prosthetic groups as different authorities define these terms differently.  Prosthetic Groups Constant in their definition is that prosthetic groups are tightly or stably incorporated into the enzyme’s structure by covalent or noncovalent forces. Examples include pyridoxal phosphate, flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), thiamin pyrophosphate, biotin, and the metal ions of Co, Cu, Mg, Mn, and Zn. Metals are the most common prosthetic groups. The roughly one-third of all enzymes that contain tightly bound metal ions are termed metalloenzymes.  Prosthetic Groups Metal ions that participate in redox reactions generally are complexed to prosthetic groups such as heme or iron-sulfur clusters. Metals also may facilitate the binding and orientation of substrates, the formation of covalent bonds with reaction intermediates (Co2+ in coenzyme B12), or interact with substrates to render them more electrophilic (electron-poor) or nucleophilic (electron- rich).  Cofactors Cofactors serve functions similar to those of prosthetic groups but bind in a transient, dissociable manner either to the enzyme or to a substrate such as ATP. Unlike the stably associated prosthetic groups, cofactors therefore must be present in the medium surrounding the enzyme for catalysis to occur. The most common cofactors also are metal ions. Enzymes that require a metal ion cofactor are termed metal-activated enzymes to distinguish them from the metalloenzymes for which metal ions serve as prosthetic groups.  Coenzymes Coenzymes are usually organic molecules that are loosely bound to enzymes and serve as recyclable shuttles-or group transfer agents-that transport many substrates from their point of generation to their point of utilization. Association with the coenzyme also stabilizes substrates such as hydrogen atoms or hydride ions that are unstable in the aqueous environment of the cell. Coenzymes transport chemical groups from one enzyme to another. Examples include NADH, NADPH and ATP.  Coenzymes Some coenzymes, such as riboflavin, thiamine and folic acid, are vitamins, or compounds that cannot be synthesized by the body and must be acquired from the diet. The chemical groups carried include the hydride ion (H−) carried by NAD or NADP+, the phosphate group carried by adenosine triphosphate, the acetyl group carried by coenzyme A, formyl, methenyl or methyl groups carried by folic acid and the methyl group carried by S-adenosylmethionine.  Isoenzyme Isozymes (also known as isoenzymes) are enzymes that differ in amino acid sequence but catalyze the same chemical reaction. These enzymes usually display different kinetic parameters (i.e. different KM values), or different regulatory properties. The existence of isozymes permits the fine-tuning of metabolism to meet the particular needs of a given tissue or developmental stage (for example lactate dehydrogenase (LDH)).  Isoenzyme In biochemistry, isozymes (or isoenzymes) are isoforms (closely related variants) of enzymes. In many cases, they are coded for by homologous genes that have diverged over time. Although, strictly speaking, allozymes represent different alleles of the same gene, and isozymes represent different genes whose products catalyse the same reaction, the two words are usually used interchangeably.  Isoenzyme Isozymes were first described by hunter and Markert (1957) who defined them as different variants of the same enzyme having identical functions and present in the same individual. This definition encompasses (1) enzyme variants that are the product of different genes and thus represent different loci (described as isozymes) (2) enzymes that are the product of different alleles of the same gene (described as allozymes).  Isoenzyme Isozymes are usually the result of gene duplication, but can also arise from polyploidisation or hybridization. Over evolutionary time, if the function of the new variant remains identical to the original, then it is likely that one or the other will be lost as mutations accumulate, resulting in a pseudogene.  Isoenzyme However, if the mutations do not immediately prevent the enzyme from functioning, but instead modify either its function, or its pattern of gene Expression, then the two variants may both be favoured by natural selection and become specialised to different functions. For example, they may be expressed at different stages of development or in different tissues.  Isoenzyme Allozymes may result from point mutations or from insertion-deletion (indel) events that affect the dna coding sequence of the gene. As with any other new mutation, there are three things that may happen to a new allozyme: It is most likely that the new allele will be non- functional — in which case it will probably result in low fitness and be removed from the population by natural selection.  Isoenzyme Alternatively, if the amino acid residue that is changed is in a relatively unimportant part of the enzyme, for example a long way from the active site then the mutation may be selectively neutral and subject to genetic drift. In rare cases the mutation may result in an enzyme that is more efficient, or one that can catalyse a slightly different chemical reaction, in which case the mutation may cause an increase in fitness, and be favoured by natural selection.  Isoenzyme An example of an isozyme is glucokinase, a variant of hexokinase which is not inhibited by glucose 6- phosphate. Its different regulatory features and lower affinity for glucose (compared to other hexokinases), allows it to serve different functions in cells of specific organs, such as control of insulin release by the beta cells of the pancreas, or initiation of glycogen synthesis by liver cells. Both of these processes must only occur when glucose is abundant, or problems occur.  Plasma enzymes In Clinical Diagnosis Plasma enzyme assays can detect abnormal levels of enzymes in the blood. The assay measures units of activity in a sample and so will only measure functional enzyme. If levels of an enzyme are abnormally raised, it may indicate leakage from damaged tissue that the enzyme is normally found in. Abnormally low enzymes may indicate either a non- functional enzyme, produced more slowly than usual or being broken down quickly, owing to maybe genetic defect.  Plasma enzymes In Clinical Diagnosis Enzyme Location Causes of raised Causes of levels low levels Expressed everywhere Acute/chronic tissue Specific isoenzymes: damage, e.g. LDH1 – heart, myocardial infarction. erythrocytes Degree of elevation can 1. Lactate LDH2 – white blood cells indicate the extent of dehydrogenase LDH3 – lung damage LDH4 – white blood cells, Isoenzymes may help kidney, pancreas localise the site of LDH5 – liver, skeletal injury muscle  Plasma enzymes In Clinical Diagnosis Hepatocellular injury Widely distributed (acute/chronic liver but predominantly disease), gall Pregnancy, bladder disease, found in the liver, diabetes, 2. Aspartate kidney failure, heart, skeletal beriberi transaminase (AST) rhabdomyolysis, muscle, kidneys, (vitamin B1 MI (myocardial brain and infarction) deficiency) erythrocytes Degree of elevation can indicate the extent of damage  Plasma enzymes In Clinical Diagnosis Hepatocellular injury (acute/chronic 3. Alanine Widely distributed liver disease), bile transamiase but predominantly duct problems (ALT) in liver More specific marker of hepatic injury than AST  Plasma enzymes In Clinical Diagnosis Greatest Prostate concentration carcinoma, biliary 4. Alkaline in prostate obstruction, high phosphatase Specific isoenzymes bone turnover (ALP) are also found in the (physiological or liver, bone, kidney, pathological) intestine and placenta  Plasma enzymes In Clinical Diagnosis Expressed in various tissues MM – skeletal Specific isoenzymes: muscle dystrophy MM – skeletal musc 5. Creatine MB – myocardial le, heart kinase infarction in last 2- MB – heart 3 days BB – brain, neurons, BB – brain tumour thyroid, kidney, intestine  Plasma enzymes In Clinical Diagnosis Pancreatitis, Exocrine pancreas, infections, DKA, 6. Amylase saliva perforated ulcer, renal failure  Plasma enzymes In Clinical Diagnosis Pancreatitis 7. Lipase Exocrine pancreas (more specific than amylase)  Plasma enzymes In Clinical Diagnosis Widely expressed Specific isoenzymes Diagnosis and 8. Acid in the liver, treatment of phosphatase erythrocytes, platelets prostate cancer and bone  Blood Clotting Enzymes Introduction: Blood clotting is also called coagulation. The process of coagulation starts when some tissue is injured and bleeding is started. Injured tissue releases a clotting factor that stimulates extrinsic and intrinsic pathways. The clotting factor released from damaged tissue also initiates the conversion of inactive zymogen prothrombin into an active enzyme known as thrombin.  Blood Clotting Enzymes Enzyme thrombin catalyzes the conversion of soluble plasma proteins fibrinogen into insoluble fibrous protein fibrin. Fibrin makes a mesh-like structure around the platelet plug made initially and the blood cells are trapped in this mesh to form a clot. After the complete repair of the damaged region, the clot is dissolved by an enzyme plasmin.  Blood Clotting Enzymes Thrombin plays a major role in converting fibrinogen (a glycoprotein complex) to fibrin which functions primarily to occlude blood vessels to stop bleeding. Thrombin is a naturally occurring enzyme, which is responsible for blood clotting.  Blood Clotting Enzymes Serine protease proteins are important enzymes involved in the process of blood coagulation. Blood coagulation is an importance defense mechanism that prevents the host mammal organism from losing excess blood or from forming unwanted blood clot. The process of coagulation can be initiated by both intrinsic factors and extrinsic factors.  Blood Clotting Enzymes A cascade of event is followed which activate these enzymes; normally the enzymes are inactive state a condition called zymogens. Zymogens by their virtual condition of being inactive prevent unwanted blood clotting which may have a far reaching consequence such as thrombosis.  Blood Clotting Enzymes Blood clotting in a series of processes, in which the zymogens’ need to be activated by reacting with its glycoprotein co-factors. Among the serine protease is the thrombin enzyme factor five (v) responsible for clearing clot in the blood. The enzyme is usually present circulating in plasma which is made up of a single monomer chain, it life span can range from 12 to 36 hours. ThankYou