1-Introduction to Enzymes

8/8/2019 1-Introduction to Enzymes

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1- Introduction to enzymes

Enzymes are mainly proteins (complex structured proteins) that functionas biological catalysts, and the diversity in their protein structure allowdifferent enzymes to be specialized in their activity.

substrateenzyme product+enzyme

A catalyst is a chemical that:- Increase the rate of the reaction ,by lowering the activation energy

{The amount of energy required for the reaction to proceed} Not consumed by the reaction they catalyze nor alter the equilibrium of

these reactions, i.e.{ the reaction can proceed without the catalyst but ata slower rate }.

Enzyme activity can be affected by other molecules:-

Note: cofactors that cannot be removed from enzymes are calledprosthetic groups , which are linked tightly to enzymes by covalent bondOR loosely by H-bond.

Activity of enzymes can also be affected by: ph, temperature,concentration of the substrate.

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Inhibitors activators

Molecules that decreases The

Enzymes activity Ex. Drugs &

Poisons.

Molecules that increases The enzyme activity

Ex. Cofactors (inorganic substances)as Ca ++ , Mg ++ ,Mn ++ ,Cu ++ and Zn ++ {canremoved by dialysis

or may not attach tightly to enzyme to giveapoenzymes} & coenzymes (organicsubstances)

{derived from soluble vitamins in H 2Osucniacin

And riboflavin }.


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Enzymes can be classified according to their structure to :

Enzymes composed of Mainly protein structure, ex. Enzymes catalyzingthe hydrolytic reactions of gastric and intestinal tracts

Enzymes with additional substances. {Cofactors &coenzymes}.

Properties of enzymes :-

1-increase the rate of reactions.

2-dont change the physical activity to give the end result.

3-reactants can work without enzymes, but enzymes fasten the reaction.

4-reactants must have sufficient energy for the reaction to proceed, whichis not needed for all the reaction molecules.

5- The heat increases also the rate of the reaction.

6- Enzymes are essential for the synthesis, breathing, digestion andmetabolic reactions.

7-enymes exhibits a high degree of specificity for their substrate.

8- The biological cell may contain up to 3000 enzymes.

9-they r globular proteins range from 62 a.a. {4-oxalocrotonatetautomerase monomer} to 2500 a. a. {animal fatty acid synthase }

10-like all proteins, made of long linear chain of a.a. that fold to produce3D. Structure. Each with specific sequence and properties.

11-activity is determined by the 3-dimensional structure of their proteinsamino acid.

12-some enzymes are larger than its substrate and only small portion of enzyme {3-4 amino acids} is involved in catalysis, this portion called theactive site.

13-can be denatured & deactivated by heat or chemical denaturants,according to enzyme this process can be reversible or irreversible.

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14-they lower the barriers that normally prevent the chemical reactionfrom occurring or slow them down by decreasing the required activationenergy. Thus, in presence of enzyme the reaction proceed in a faster rate.

15-theyre usually named with the suffix ase at the end of their substratename. EX 1. sucrosesucr ase.

EX 2. lactoselact ase.

The lock and key model

Because both substrate and enzyme possesses specific geometric shapesthat they fits into one another, this is referred to as { the lock and keymodel }, but this model failed to explain the transition state of enzymereaction.

The induced fit model (Modification of lock and keymodel)

As the enzyme is flexible structure, the active site continually reshaped

when interacts with the substrate. The amino acid side chain which form the active site are molded into the

exact positions that enable the enzyme to function correctly. Sometimes, the substrate also changes its shape slightly as it enters the

active site, ex. Glycosidase .

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2- SPECIFICITY The ability of an enzyme to catalyze the specific reaction and no others isperhaps its most significant property.However specificity varies from one enzyme to another, so we have thefollowing types of enzyme specificity.

1) Absolute specificityCatalyze only one substrate not the other.Ex 1. Urase acts only on ureaEx 2. Fumerase acts only on fumrateOOC.CH=CH.COO FUMERASE OOC.CHOH.CH.COO

L.Fumerate H 2 O L.malate

2) Broad specificity The enzyme is specific to one type of bond possessed by a group of chemically related substances.Ex 1. Pancreatic lipase hydrolysis alpha ester bonds (at position 1 and 3) of triglycerides.Ex 2. Salivary amylase attack alpha 1:4 glucosidic bonds of starch.Ex 3 . Polypeptidase.

3) Group specificity4


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A- Act on specific chemical group such as glycosidases on glycosides , pepsin and trypsin on peptide bonds

and esterases on esters. ii- Lytic enzymes exhibit a higher order of group specificity; e.g

carboxypeptidases and aminopeptidases split off amino acids one at

a time from the carboxy or amino terminal end of polypeptide chains.B- Linkage specificity is related to group specificity and it act on aparticular type of chemical compound regardless of the rest of themolecular structure.

4) Optical specificity or Stereochemical specificity

The enzyme is specific for only one isomer of the substrate.Ex. enzymes oxidizing glucose act on D-forms of glucose only.

Because of high specificity and accuracy the enzymes are involved in thecopying and expression of the genome.

These enzymes have Proof-reading mechanism.Ex. DNA polymerase catalyzes a reaction in first step and thenchecks that the product is correct in a second step. This two-step processresults in average error rates of less than I error in 100 6 reactions in high-

fidelity mammalian polymerases.It also found Proof-reading mechanism in RNA polymerase, aminoacyltRNA synthetases and ribosemes.

Some enzymes that produce second metabolites are described aspromiscuous, as they can act on relatively broad range of differentsubstrate and it is important for the evolution of new biosyntheticpathways.

3- NAMING OF ENZYMES

1. Many enzymes have more than one name.

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Among the first identified and named were the digestive enzyme thatcatalyzes the hydrolysis of the glycoside, ester and amide bond of carbohydrates, lipids and proteins.

2. Enzymes were named for the substrates on which they acted by addingthe suffix - ase to the substrate such as proteinase , lipase.

3. Enzymes given the name of chemical reaction such as oxidases ,decarboxylases and dehydrogenases.

4. The same enzyme being identified with two different names. One at thenormal physiological reaction and other at artifical conditions.Ex 1 . Glucose isomerase industrially to convert glucose into thesweetner fructose.EX 2 . Xylose isomerase in vivo.

5. Naming depends on the types and mechanisms of chemical reactions.

a) Reactions and enzymes that catalyze them divided into 6 classes eachwith 4-13 subclasses.b) The enzyme names has 2 parts , the first is the substrate, second the

ending asec) Each enzyme has a systemic code number (E.C). This number

characterized the reaction type. The Class,Subclass,Sub subclass thenthe specific name.

E.C 2.7.1.1 denotes

Class 2 (transferase)Subclass 7 (transfer of phosphate)

Sub subclass 1 (an alcohol function as the phosphate acceptor)

The enzyme name (hexokinase)

E.C 3.4.11.4 (tripeptide aminopeptidases

Class 3 (hydrolases)

Subclass 4 (hydrolases act on peptide bonds)

Sub subclass 11 (hydrolases that cleave off the amino-terminal aminoacidfrom a polypeptide)

The enzymes are those that cleave off the amino-terminal end from atripeptide.

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4- CLASSIFICATION OF ENZYMES

Although an international committee has established a uniform namingsystem for enzymes, the names that are in common use do not follow acompletely consistent pattern. With the exception of some older enzymenames (such as pepsin, trypsin), all enzymes names end with the suffix-ase. Classes of enzymes are named according to their job category.

1) Oxidoreductases

Are biological oxidation and reduction between 2 substrate and there forare essential in the process metabolism energy releasing, respiration andfermentation.

Ex. Lactic to pyruvic acid

CH 3 -CH-COOH LDH+NAD CH 3 -C-COOH + NADHLactic acid Lactate dehydrogenase Pyruvic acid

2) Transferases

These enzymes transfer group from one substance,the donor, to another

the acceptor (e.g amino group, pohsphate group etc).Ex. Transaminases transfer amino groups.

Glutamic acid pyruvic acid -

ketoglutaric acid alanine3) Hydrolases

Catalyze hydrolysis reactions when a molecule is split into two or moresmaller molecules by addition of water.

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Ex. phosphatase catalyze dephosphorylation {removal of phosphategroup}

Glucose + ADP glucose-6-phosphate + ADP +

4) Lyases

Catalyze the cleavage of C-C, C-O, C-S and C-N bonds bymechanism other than hydrolysis leaving double bond.

Ex. L.malate Lyase L.fumarate

OOC.CHOH.CH.COO -H2O+Lyase OOC.CH=CH.COO

5) Isomerases

Catalyze atomic rearrangement within a molecule to give another shapeor structure.

Ex. Rotamase and protein disulfide isomerase which can assist apeptidechain to fold into a correct 3D structure.

b) D-glucose L-glucose

6) Ligases

Catalyze the reaction which joins two molecules and formation of differentbonds by condensation reaction coupled to ATP cleavage; C-C, C-N, C-Sand C-O.

5-Coenzymes

Definition

Some enzymes are purely protein in nature and depend for activity ontheir structure, while certain enzymes requires for their function, one ormore non-protein part, these are termed as Coenzymes, Cofactors orprosthetic groups.

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If such a compound is firmly attached to the enzyme protein then itscalled Coenzyme.

Properties of coenzymes:

They are organic molecules that cooperate in the catalytic action of the

enzyme.

Ex . NADH, NADPH & ATP (that are released from the enzymes active siteduring the reaction.

Coenzymes can be considered as a special class of substrate, or asecond substrate since they are chemically changes as a consequenceof enzyme action.

Organic prosthetic groups can be covalently attached to enzyme,

Ex . Thiamine pyrophosphate in enzyme (pyruvate dehydrogenase) They can be used for many different enzymes which mean theyre widely

used and spread in the biological cell reactions.Ex . About 700 enzymes can use the Coenzyme NADH.

They can increase OR decrease the enzyme activity. Certain coenzymes exist in Free State in solutions and contact the enzyme

protein, ONLY at the time of reaction. Theyre continuously regenerated which means that even small amounts

of coenzymes are used very intensively.Ex. Human body turns over its own weight in ATP each day. Some of those coenzymes are Vitamins, which cant be synthesized by the

body, but should be acquired from the diet.Ex . Riboflavin (B), Thiamine (B) and Folic acid.(Small amount of those vitamins are produced by the body, EX. Vita. K and Nicotinic acid)

Different types of Coenzymes:1. Nicotinamide dinucleotidesNADH, NADPH and their reduced forms are involved in manydehydrogenase reactions in mitochondrion, cytosol, and endoplasmicreticulum of the cell.

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Theyre water soluble and diffuse freely away from the enzyme after theoxidation or reduction reaction is completed to join anotherdehydrogenase reaction.

The general form of reactions involving NAD is:RH2+ NADNADH + H

2. Flavin nucleotide coenzymesFAD is another class of dehydrogenases coenzymes known as(Flavoproteins); its Flavin part is derived from the vit.B (riboflavin).Unlike the NAD, they remain tightly bound to the protein part of thecoenzyme throughout the reaction which means that FAD never leavesthe enzyme.

3. Adenine nucleotide coenzymesSuch as ATP, ADP and AMP which are influencing the direction of flow of metabolic pathway.ATP often joins as a donor of phosphate group to other molecules.

4. Thiamine pyrophosphate Thiamine (B) is the precursor of thiamine pyrophosphate, is thecoenzyme for some important oxidation-decarboxylation reactionsEx . Pyruvate dehydrogenase and Oxoglutarate dehyrogenase.

5. Coenzyme AComplex group contains sulphahydryl (--SH) group.

This group reacts with carboxyl group.

6. Biotin coenzymeCoenzyme for carboxylation reactions.Ex. Pyruvate carboxylase.

7. Ascorpic acid (vit.C)It prevents poor wound healing in scurvy because it involves in thehydroxylation of prolin.

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6- Mechanism of enzymes

Activation energy:

The amount of energy required for the reaction to proceed.

Enzymes act in several ways, all by lowering the G: Lowering the activation energy by stabilizing the transition state:

Straining the shape of substrate, by binding its transition stateconformation, the enzyme distort the bound substrate into their transitionstate form, this reduce the amount of energy required to complete thetransition.

Lowering the energy of the transition state, without distortingthe substrate:By creating an environment with opposite charge distribution to that of the transition state.

Providing an alternative pathway:For example: temporarily reacting with the substrate to form ESintermediate complex.

Reducing the reaction entropy change:Entropy is a measure of the number of ways in which a system may be arranged, often taken to be a measure of 'disorder' (thehigher the entropy, the higher the disorder).

By bringing the substrates together in the correct orientation to react, thisentropic effect involves the destabilization of the ground state.

By increasing the temperature: This helps the enzyme to function and develop the end product faster,although too much temperature may distort the enzymes shape and willnever regain its shape until temperature comes back to normal.

Transition state stabilization: Enzymes can stabilize its transition state more than the transition state of

uncatalyzed reactions. Most effective way to reach large stabilization energy is to use

electrostatic effects, by having fixed polar environment which orientedtoward the charge distribution of the transition state.

Dynamics and function:

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Internal dynamics is the movement of parts of the enzymes structure, forexample:

Individual amino acid. Group of amino acids. Entire protein domain.

These movements are connected to the enzymes mechanism,whether small and fast movement, or large and slow movement,depending on the type of the reaction.

7-Enzymes Regulation

Many of enzyme reactions occur in cells involving synthesis, degradation

or interconversion.These reactions regulate by 2 ways

1) Regulation of amount of enzyme Long termregulation

A) Enzyme induction

The inducer is a substrate or sometimes hormone, produce a signal tospecific gene of DNA, this gene starts to synthesis the required enzyme.

Ex 1 . Enzymes in the liver called cytochrome P450 oxidases, which areimportant in drug metabolism. Induction or inhibition of these enzymescan cause drug interactions.

Ex 2 . If a new substrate is made available to the cell; it may induce thesynthesis of the enzymes needed to cope with it.

Yeast cells, for example, do not ordinarily metabolize lactose and nolactase can be detected in them. However, if grown in a mediumcontaining lactose, they soon begin synthesizing lactase by transcribingand translating the necessary gene(s) and so can begin to metabolizethe sugar.

B) Enzyme repression

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The repressors which may be a product or hormone affect DNA to stopsynthesis of specific protein which is an enzyme.

Ex. If ample quantities of an amino acid are already available to the cellfrom its extracellular fluid; synthesis of the enzymes that would enablethe cell to produce that amino acid for itself is shut down.

2) Regulation of catalytic efficiency short termregulation

A) Allosteric modification

In the case if feedback inhibition and precursor activation, the activity of the enzyme is being regulated by a molecule which is not its substrate.

In these cases, the regulator molecule binds to the enzyme at a differentsite than the one to which the substrate binds.

When the regulator binds to its site, it alters the shape of the enzyme sothat its activity is changed.

B) Feedback inhibition (F.B.I.)

The end product(s) of a metabolic pathway are often inhibitors for one of the first enzymes of the pathway (usually the first irreversible step, calledcommitted step), thus regulating the amount of end product made by thepathways.

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The amount of the end product produced is regulated by its ownconcentration.

Negative feedback mechanism can effectively adjust the rate of synthesisof intermediate metabolites according to the demands of the cells.

This helps allocate materials and energy economically, and prevents themanufacture of excess end products.

The control of enzymatic action helps to maintain a stable internalenvironment in living organisms.

C) Inactive precursors ( zymogens )

The enzymes are produced in inactive form and when it reaches the siteof reaction it is activated.

Ex 1 . Chymotrypsin, a digestive protease, is produced in inactive form aschymotrypsinogen in the pancreas and transported in this form to thestomach where it is activated.

This stops the enzyme from digesting the pancreas or other tissues beforeit enters the gut.

Ex 2 . Some enzymes may become activated when localized to a different

environment (e.g. from a reducing (cytoplasm) to an oxidising (periplasm)environment, high pH to low pH etc).For example, ( hemagglutinin ) in theinfluenza virus is activated by a conformational change caused by theacidic conditions, these occur when it is taken up inside its host cell andenters the lysosome.

Ex 3 . Pepsin is synthesized within the chief cells (in gastric glands) as aninactive precursor, pepsinogen. Only when exposed to the low pH outsidethe cell is the inhibiting portion of the molecule removed and activepepsin produced.

D) Regulatory protein

The activity of an enzyme is regulated by calmodulin which is a proteinvery rich in glutamic and aspartic amino acid that form a Ca +2 binding site.

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A Ca +2 bind to calmodulin induce a co-formational change that convert itfrom inactive to active form.

Activated calmodulin bind to many enzymes and other proteins in the celland modify their activity either by stimulation or inhibition.

E) Covalent modificationSome key enzymes exist in forms, phosphorylated and a non-phosphorylated form, which differ greatly in enzyme activity. The totalactivity of the enzyme depends on the ration of these 2 forms.

Protein kinase makes phosphorylation.

Phosphatase makes dephosphrylation.

Phosphorylation activate some enzymes and inactive other responsible forthe opposite reaction.

8- Factors affecting the activity (velocity) of enzyme

1) Concentration of substrate and enzyme:

Rate of the reaction increase when the concentration of substrate or theenzyme concentration increases.

If the enzyme concentration is constant the rate of the reaction willincrease up to maximum rate and will not increase upon further additionof the substrate.

As the substrate concentration increase more substrate molecule fills theactive site for longer period of time and more products.

Enzyme rate

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Conc. Of sub. And enzyme

2) Temperature:

When the temperature increase the rate of the reaction will increase evenwithout enzyme but with low rate .But when add enzyme it acceleratesthe reaction, because it removes barriers.

Enzymes work at optimal temperature. Under or above this temperaturethe reaction is altered. For most animal enzymes the optimumtemperature is between 38 oc-40 oc .

Like most chemical reactions, the rate of an enzyme-catalyzed reactionincreases as the temperature is raised. A ten degree Centigrade rise intemperature will increase the activity of most enzymes by 50 to 100%.Variations in reaction temperature as small as 1 or 2 degrees mayintroduce changes of 10 to 20% in the results. In the case of enzymaticreactions, this is complicated by the fact that many enzymes areadversely affected by high temperatures. As shown in Figure, the reactionrate increases with temperature to a maximum level, then abruptlydeclines with further increase of temperature. Because most animalenzymes rapidly become denatured at temperatures above 40C, mostenzyme determinations are carried out somewhat below thattemperature.

Over a period of time, enzymes will be deactivated at even moderatetemperatures. Storage of enzymes at 5C or below is generally the most

suitable. Some enzymes lose their activity when frozen .Activation velocity

Plato

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Inactivation

Activation temperature

3)PH:

Enzymes are affected by changes in PH The most favorable pH value - thepoint where the enzyme is most active - is known as the optimum pH andis between 2-10 PH

The PH optimum of enzyme usually reflects the PH of the body fluid inwhich the enzyme is found.

Extremely high or low pH values generally result in complete loss of activity for most enzymes. PH is also a factor in the stability of enzymes.As with activity, for each enzyme there is also a region of pH optimalstability.The optimum pH value will vary greatly from one enzyme toanother, as shown in this table :

Enzyme PH Optimum

Lipase (pancreas) 8.0

Lipase (stomach) 4.0 - 5.0

Lipase (castor oil) 4.7

Pepsin 1.5 - 1.6

Trypsin 7.8 - 8.7

9-Enzyme InhibitionEnzymes can be inhibited by many substances and reduce the initialvelocity of such enzymes. Accordingly there are 2 types of enzymesinhibition.

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1) Irreversible inhibitors

React with the enzyme and form a covalent adducts with the protein. Theinactivation is irreversible. These compounds include eflornithine a drugused to treat the parasitic disease sleeping sickness . Penicillin and Aspirialso act in this manner. With these drugs, the compound is bound in theactive site and the enzyme then converts the inhibitor into an activatedform that reacts irreversibly with one or more amino acid residues.

2) Reversible inhibitors

Reversible inhibitors bind and dissociate with their enzyme in equilibriumprocess. It can be classified as

a) Competitive inhibition

b) Non competitive inhibitionc) Uncompetitive inhibitiond) Mixed inhibition

It classified according to where on the enzyme the inhibitor binds, or theorder with which it binds, relative to substrate.

A) Competitive inhibition

Competitive inhibitors compete with substrate for an enzymes active site,lowering the enzymes likelihood of binding substrate and slowing theobserved reaction velocity.

Competitive inhibitors bind reversibly to the enzyme, preventing thebinding of substrate. On the other hand, binding of substrate preventsbinding of the inhibitor. Substrate and inhibitor compete for the enzyme.

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B) Non competitive inhibition

Can bind to the enzyme at the same time as the substrate, i.e. they never bind to the active site. Both the EI and EIS complexes are enzymaticallyinactive. Because the inhibitor cannot be driven from the enzyme byhigher substrate concentration (in contrast to competitive inhibition), theapparent V max changes. But because the substrate can still bind to theenzyme, the K m stays the same.

Occur in cells when specific molecule bind to an enzyme site other thanthe active site called Allosteric site causing a shift in the enzymeconfiguration which prevent the substrate from binding to the active site.

C) Uncompetitive inhibition

In uncompetitive inhibition the inhibitor cannot bind to the free enzyme,but only to the ES-complex. The EIS-complex thus formed is enzymatically

inactive. This type of inhibition is rare, but may occur in multimericenzymes.

D) Mixed inhibition

This type of inhibition resembles the non-competitive, except that the EIS-complex has residual enzymatic activity.

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In many organisms inhibitors may act as part of a feedback mechanism. If an enzyme produces too much of one substance in the organism, thatsubstance may act as an inhibitor for the enzyme at the beginning of thepathway that produces it, causing production of the substance to slowdown or stop when there is sufficient amount. This is a form of negative

feedback. Enzymes which are subject to this form of regulation are oftenmultimeric and have Allosteric binding sites for regulatory substances.

Their substrate/velocity plots are not hyperbolar, but sigmoidal (S-shaped).

Uses of inhibitors:

Since inhibitors modulate the function of enzymes they are often used asdrugs and poisons.

Drugs A common example of an inhibitor that is used as a drug isaspirin. This inhibits the COX-1 and COX-2 enzymes that produce theinflammation messenger prostaglandin, thus suppressing pain andinflammation.

Poisons the poison cyanide is an irreversible enzyme inhibitor thatcombines with the copper and iron in the active site of the enzymecytochrome c oxidase and blocks cellular respiration.

10- m easurement of enzymes activity

To measure the amount of the enzyme in a sample of tissue extract orother biologic fluid, the rate of the reaction catalyzed by the enzyme inthe sample is measured.

The measured rate is proportional to the quantity of enzyme present. Thisrate is compared with rate catalyzed by a known quantity of the highlypurified enzyme.

Enzyme units :

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Enzyme units are expressed in micromoles (10 -6 mol), nanomoles (10 -9

mol) or picomoles (10 -12 mol) of substrate reacting or product produced 1minute.

11-Intracellular distribution of enzymes The enzymes of glycolysis are located in the cytoplasm, where enzymes of the citric acid cycle are in the mitochondria.

The distribution of enzymes among sub cellular organelles may be studiedfraction of cell homogenates by high-speed centrifugation. The enzymecontent of each fraction is then examined.

IsozymeIt is the presence of enzyme in different forms but all have the samereaction differ from each other in some chemical or physical properties.

EX 1 . Malate m-dehydrogenase oxaloacetate

Malate dehydrogenase is a generic name that includes all proteins(enzymes) which catalyze the oxidation of malate to oxaloactate.

Ex 2 .Lactate dehydrogenase ( LDH ) enzyme presents in blood in a form of 5fractions (isozymes) LDH1, LDH2, LDH3, LDH4, and LDH5.

Proenzymes

Many proteins are manufactured and secreted from cells in the form of

inactive precursor or proteins known as "proproteins". When the proteinsare enzymes, they are termed proenzymes or zymogens.

Conversion of a proprotein to mature protein involves a process known aslimited selective proteolysis.

Ex 1 . Insulin proinsulin

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Ex 2 . Pepsin pepsinogen

12-Enzymes in the Diagnosis of Pathology

The measurement of the serum levels of numerous enzymes has beenshown to be of diagnostic significance. This is because the presence of these enzymes in the serum indicates that tissue or cellular damage hasoccurred resulting in the release of intracellular components into theblood. Hence, when a physician indicates that he/she is going to assay forliver enzymes, the purpose is to ascertain the potential for liver celldamage. Commonly assayed enzymes are the amino transferases: alaninetransaminase , ALT (sometimes still referred to as serum glutamate-pyruvate aminotransferase , SGPT ) and aspartate aminotransferase, AST

(also referred to as serum glutamate-oxaloacetate aminotransferase,SGOT ); lactate dehydrogenase , LDH ; creatine kinase, CK (also calledcreatine phosphokinase, CPK ); gamma-glutamyl transpeptidase, GGT .Other enzymes are assayed under a variety of different clinical situationsbut they will not be covered here.

The typical liver enzymes measured are AST and ALT . ALT is particularlydiagnostic of liver involvement as this enzyme is found predominantly inhepatocytes. When assaying for both ALT and AST the ratio of the level of these two enzymes can also be diagnostic. Normally in liver disease or

damage that is not of viral origin the ratio of ALT/AST is less than 1.However, with viral hepatitis the ALT/AST ratio will be greater than 1.Measurement of AST is useful not only for liver involvement but also forheart disease or damage. The level of AST elevation in the serum isdirectly proportional to the number of cells involved as well as on the timefollowing injury that the AST assay was performed. Following injury, levelsof AST rise within 8 hours and peak 2436 hours later. Within 37 daysthe level of AST should return to pre-injury levels, provided a continuousinsult is not present or further injury occurs. Although measurement of AST is not, in and of itself, diagnostic for myocardial infarction, takentogether with LDH and CK measurements (see below) the level of ASTuseful for timing of the infarct.

The measurement of LDH is especially diagnostic for myocardial infarctionbecause this enzyme exists in 5 closely related, but slightly differentforms (isozymes). The 5 types and their normal distribution and levels innon-disease/injury are listed below:

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LDH 1 Found in heart and red-blood cells and is 17% 27% of the normalserum total.LDH 2 Found in heart and red-blood cells and is 27% 37% of the normalserum total. LDH 3 Found in a variety of organs and is 18% 25% of the normal

serum total.LDH 4 Found in a variety of organs and is 3% 8% of the normal serumtotal.LDH 5 Found in liver and skeletal muscle and is 0% 5% of the normalserum total.Following a myocardial infarct the serum levels of LDH rise within 24-48hours reaching a peak by 23 days and return to normal in 5-10 days.Especially diagnostic is a comparison of the LDH-1/LDH-2 ratio. Normally,this ration is less than 1. A reversal of this ration is referred to as aflipped LDH . Following an acute myocardial infarct the flipped LDH ratiowill appear in 1224 hours and is definitely present by 48 hours in over80% of patients. Also important is the fact that persons suffering chestpain due to angina only will not likely have altered LDH levels.Diagnostic and prognostic value of specific enzymes

The determination of the activity of the following enzymes can provide thephysician with valuable diagnostic evidence.1- Lipase

The plasma lipase level may be low in liver disease, vitamin A deficiency,some malignancies and diabetes mellitus. It may be elevated in acutepancreatitis and pancreatic carcinoma.

2- Amylase

The plasma amylase may below in liver disease and increased in acutepancreatitis and diabetes mellitus.3- Trypsin

Elevated level of trypsin in plasma occurs during acute pancreaticdisease. The elevation is more sensitive and considers indicator of pancreatic disease than plasma amylase or lipase.4- Cholinesterase

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In general, low levels are found in patients ill with liver diseasemalnutrition, acute infection disease and anemia. High levels occur innephritic syndrome.Since the content of cholinesterase in young red blood cells isconsiderably higher than in the adult red blood cells, the cholinesterasetiter of erythrocytes in peripheral blood gives an indication of hematopoietic activity.5- Alkaline phosphatase

The level of enzymes capable of catalyzing the hydrolysis of variousphosphate esterase at alkaline PH (alkaline phosphatase activity) may beincreased in hyper parathyrodisim and osteoblastic sarcoma.Isozymes of alkaline phosphatase are present in body fluid. These includespecific isozymes originating from bone, liver, placenta and intestine.Measurement of specific alkaline phosphatase isozymes may therefore

improve the diagnostic value of this test.6. Acid phosphatase

The level of enzymes capable of catalyzing the hydrolysis of variousphosphate esters at acidic PH (acid phosphatase activity) may be elevatedin the metastatic prostatic carcinoma.7. Transaminases

Two transaminases are of clinical interesta) Glutamic oxaloacetic transaminases ( GOT ) which catalyzes the transferof the amino group of aspartic acid to alpha-ketoglutaric acid, formingglutamic and oxalo acetic acids.b) Glutamic pyruvic transaminase ( GPT ) transfers the amino group of alanine to alpha-ketoglutaric acid, forming glutamic and pyruvic acids.Liver tissue rich in both transaminases are elevated in sera than GOT .While both transaminases are elevated in sera of patients with acutehepatic disease, GPT , which is only elevated by cardiac necrosis, is amore specific indicator of liver damage.

8. Lactate dehydrogenaseIn myocardial infarction, the concentration of serum lactatedehydrogenase ( LDH ) rises within 24hrs after the infract and return to thenormal range within 5-6 days. High levels of LDH also occur in patientswith acute and chronic leukemia in relapse.9. LDH isozymes

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1-Introduction to Enzymes

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