Pharmacy Readers: ENZYME

The non-protein substance is termed as cofactors. Commonly encountered cofactors include metal ions (Zn2+, Fe2+) and organic molecules (coenzyme)

The cofactor can be subdivided as follows: --------

Organic (coenzyme):

Tightly bound (prosthetic group)

Loosely bound (second substrate)

Inorganic (metal ions):

Tightly bound (metallo enzymes)

Loosely bound (ion activators)

Inorgenic Elements as Cofactors3

Ions

Enzymes

Fe2+, Fe3+

- cytochrome oxidase

- catalase

- peroxidase

- ferredoxin

Cu2+

cytochrome oxidase

Zn2+

- carbonic anhydrase

- alcohol dehydrogenase

- carboxypeptidase

Mg2+

- phosphohydrolases

- phosphotransferases

Mn2+

Arginase

phosphotransferases

K+

- Pyruvate kinase

- Urenase

- Dinitrogenase

- Glutathione peroxidase

Na+

- plasma membrane ATPase

COENZYME2:

The non-protein, organic, low molecular weight and dialyzable substance associated with enzyme function are known as coenzyme.

Characteristics:

Coenzymes are heat stable and non-protein organic molecules.

Generally derived from water soluble vitamins, exception of vitamin C.

They may be regarded as second substrate or co-substrate.

They are less specific than enzyme.

They participate in------

Hydride (H-) and electron transfer reactions. E.g. NAD+, NADP+, FMN etc

Group transfer reactions. E.g. CoA-sh, Thiamin-pyrophate, cobamide (B12) coenzymes.

The coenzymes are attached to the protein at a different but adjacent site so as to be in a position to accept the atoms or groups which are removed from the substrate.

Functions: Their function is usually to accept atoms or groups from a substrate and to transfer them to other molecules.

Coenzyme

Functions performed.

NAD+, NADP+

Hydrogen transfer

FAD, FMN

Thiamine pyrophate

Acetyl group transfer

Pyridoxal phosphate

Amino group transfer

Biotin

Carboxyl group transfer.

ISO-ENZYME2:

Iso-enzymes are enzyme variants. They are enzymes which perform the same catalytic action but which are present in multiple molecular forms within the same animal species.

Occurrence: Iso-enzymes are present in the serum and tissues of mammals, amphibians, birds, insects, plants and unicellular organisms.

Examples: Iso-enzymes of lactate dehydrogenase. Iso-enzymes of Creatine phosphokinase. Iso-enzymes alkaline phosphatase.

Characteristics:

Iso-enzymes synthesized from different genes. E.g. malate dehydrogenase of cytosol is different from that found in mitochondria.

Difference carbohydrate content in glycoprotein enzymes may be responsible for the formation of iso-enzyme. E.g. alkaline phosphatase.

They catalyze the same reaction but they can be distinguished by physical methods such as electrophoresis or by immunological methods.

The difference between some iso-enzymes is due to differences in the quarternary structure of the enzyme. E.g. lactate dehydrogenase exists in five iso-enzymic forms.

Question: Compare between Enzyme and Coenzyme.

Answer:

Compare between Enzyme and Coenzyme1

Enzyme

Coenzyme

Enzymes may protein catalysts that increase the rate of reactions.

Coenzymes are low molecular weight, heat stable, dialyzable organic compounds.

Enzyme does not undergo alteration during reaction.

Coenzymes undergo alteration during the enzymatic reaction which is later, regenerated.

Enzyme increases the rate of reaction.

Coenzymes function with the enzyme in the catalytic process.

Do not act as carrier of atom or functional group.

Some coenzymes act as transient carrier of specific atom or functional group.

Enzyme’s protein part is apoenzyme that is required for enzymic activity.

Coenzymes are covalently bound to the apoenzyme and function at or near the active site in catalysis.

Enzymes are protein in nature with high molecular weight.

Coenzymes are non-protein in nature and low molecular weight.

Enzyme may be regarded as apoenzyme which refers to the protein portion of the holoenxzyme (enzyme + cofactor)

Coenzymes often regarded as the second substrates or co-substrate.

They are heat and pH labile and non-dialyzable.

They are heat and pH stable and dialyzable.

Essentially all biochemical reactions are enzyme catalyzed.

They play an essential role in performing catalytic activity of enzyme.

They are naturally produced by the body.

These are vitamins derivatives.

Enzymes can decrease the free energy of activation of a reaction.

They cannot do so without enzyme.

Example:

a) Oxidoreductases

b) Transferases

c) Hydrolases

Example:

a) Thiamine pyrophosphate(TPP)

b) Flavin adenine dinucleotide (FAD)

C) Tetrahydrofolate (FH4)

Q. Compare between Enzyme and Catalyst:

Question: Compare and Contrast between Enzyme and Hormone.

Answer:

Compare and Contrast between Enzyme and Hormone1.

Enzyme

Hormones

Enzymes may be defined as biocatalysts synthesized by living cells. They are protein in nature (exception RNA acting as ribosome), colloidal and thermoliable in character, and specific in their action.

Hormones are conventionally defined as organic substances, produced in small amounts by specific tissues (endocrine glands), secreted into the blood stream to control the metabolic and biologic activities in the target cell.

These are biological catalyst not chemical messenger.

These are chemical messenger.

Enzyme increases the rate of reaction by acting as a catalyst.

Hormones participate in the reaction.

Act directly.

Act either directly or through second messenger.

Remain unchanged after the reaction.

Change their structure after the reaction.

They only catalyze the reaction.

Growth, health and welfare are their function.

For enzyme action no receptor is required.

Definite receptor is required for hormonal action.

They cannot regulate morphogenesis.

Generally regulate morphogenesis, especially secondary sex character.

Examples:

-Oxidoreductases

-Transferases

-Hydrolases

Examples:

-Insulin,

-Glucagon,

-T3, T4,

Question: Classify Enzyme with Examples.

Answer:

There are several ways of naming enzyme:

Trivial names4: Here many enzymes have been named by adding the suffix

-ase to the name of the substrate i.e. the molecule on which the enzyme exerts catalytic action. For example:

Urease catalyzes hydrolysis of urea to ammonia and CO2.

Arginase catalyzes the hydrolysis of arginine to ornithine and urea.

Enzymes are sometimes considered under two broad categories:

(a) Intra-cellular enzymes: They are functional within cells where they are synthesized.

(b) Extra-cellular enzymes: These enzymes are active outside the cell. All the digestive enzymes belong to this group.

Systematic names1: The International Union of Biochemistry and Molecular Biology (IUBMB) established a system of nomenclature in which enzymes are classified into six major groups, each with numerous subgroups. This systematic scheme classifies enzymes on the basis of the reaction catalyzed.

Oxidoreductases: Enzymes involved in oxidation-reduction reactions. Examples: Alcohol dehydrogenase, Cytochrome oxidase, Lactate dehydrogenase and L & D-amino acid oxidases.

Subclasses:

Oxidases: Use O2 as an electron acceptor but does not incorporation into the substrate.

Dehydrogenases: Use molecules other than O2 as electron acceptor. (e.g. NAD+)

Oxygenases: Directly incorporate oxygen into substrate.

Per-oxidases: Use H2O2 as an electron acceptor.

Transferases: Enzymes that catalyze the transfer of functional groups. Examples: Hexokinase, Transaminase, Transmethylases and Phosphorylase.

Subclasses:

Methyltransferases: Transfer one carbon unit between substrate.

Aminotransferases: Transfer NH2 from amino acids to keto-acids.

Kinases: Transfer PO3- from ATP to a substrate.

Phosphorylases: Transfer PO3- from inorganic phosphate to a substrate.

Hydrolases: Enzymes that bring about hydrolysis of various compounds. Examples: Lipase, Choline esterase, Acid & alkaline phosphatases and Urease.

Subclasses:

Phosphatases: Remove PO3- from a substrate.

Phosphodiesterases: Cleave phosphodiester bonds such as those in nucleic acids.

Proteases: Cleave amide bonds such as those in protein.

Lyases: Enzymes specialized in the addition or removal of water, ammonia and CO2 etc. Examples: Aldolase, Fumarase and Histidase.

Subclasses:

Decarboxylases: Produce CO2 via elimination reactions.

Aldolases: Produce aldehydes via elimination reactions.

Synthases: Link two molecules with out involvement of ATP.

Isomerases: Enzymes involved in all the isomerization reactions. Examples: Triose phosphate isomerase, Retinol isomerase and Phosphohexose isomerase.

Subclasses:

Racemose: Interconvert L & D stereoisomers. .

Mutoses: Transfer groups between atoms within a molecule.

Ligases: Enzymes catalyzing the synthetic reactions (Greek: Ligate – to bind) where two molecules are joined together and ATP is used. Examples: Glutamine synthetase, Acetyl CoA carboxylase and Succinate thiokinase.

Subclasses:

Carboxylases: Use CO2 as a substrate.

Synthetases: Link two molecules via an ATP-dependent reaction.

Question: Discuss the Properties of enzymes.

Answer:

Enzymes are protein catalysts that increase the velocity of a chemical reaction and are no consumed during the reaction they catalyze. Enzymes may possess the following properties2:

Active site: Enzyme molecules contain a special pocket or cleft called the active site. The active site contains amino acid residue that create a three dimensional surface complementary to the substrate. The active site binds the substrate, forming an enzyme-substrate (ES) complex. ES converted to enzyme-product (EP), which subsequently dissociates to enzyme and product.

Catalytic efficiency: Most enzyme catalyzed reactions are highly efficient, proceeding from 103 to 108 times faster than un-catalyzed reactions. Typically each enzyme molecule is capable of transforming 100 to 1000 substrate molecules into product each second. The number of molecules of substrate converted to product per enzyme molecule per second is called the turnover number.

Specificity: Enzymes are highly specific, interacting with one or a few specific substrates and catalyzing only one type of chemical reaction.

Cofactors: Some enzymes associate with a non-protein cofactor that is needed for enzymic activity. Commonly encountered cofactors include metal ions (for example: Zn2+, Fe2+) and organic molecules, known as coenzymes, that are often derivatives of vitamins (for example: NAD+, FAD).

The functional unit of the enzyme is known as holoenzyme which is often made up of apoenzyme (the protein part) and a coenzyme (non-protein organic part). A prosthetic group is a tightly bound coenzyme that does not dissociate from the enzyme (for example: the biotin of carboxylases)

Regulation: Enzyme activity can be regulated that is enzymes can be activated or inhibited so that the rate of product formation responds to the needs of the cell.

Location within the cell: Many enzymes are localized in specific organelles within the cell. Such compartmentalization serves:-----

To isolate the reaction substrate or product form other competing reactions

To provide a favorable environment for the reaction

To organize the thousands of enzymes present in the cell into purposeful pathways.

Question: Describe briefly How Enzyme catalyze a Biochemical Reaction.

Answer:

Catalysis is the prime function of enzymes. The prime requisite for enzyme catalysis is that the substrate (S) must combine with the enzyme (E) at the active site to form enzyme–substrate complex (ES) which ultimately results in the product formation.

The mechanism of enzyme action can be viewed from two different perspectives:

Energy changes occurring during the reaction.

How enhance the rate of reaction.

Energy changes occurring during the reaction2:

The energy required by the reactants to undergo the reaction is known as activation energy. The reactants when heated attain the activation energy, the enzyme reduces the activation energy and this causes the reaction to proceed at a lower temperature.

For example: Figure shows the changes in energy during the conversion of a molecule of reactant A to product B through the transition state.

This peak of energy represents the transition state in which a high energy intermediate is formed during the conversion of reactant to product.

Enhancement the rate of reaction1:

The enhancement in the rate of the reaction is mainly due to four processes:

Acid-base catalysis

Substrate strain

Covalent catalysis

Entropy effects

Acid-base catalysis: Role of acids and bases is quite important in enzymology. For example: At the physiological PH, histidine is the most important amino acid , the protonated form of which functions as an acid and its corresponding conjugate as a base.

Substrate strain: During the course of strain induction the energy level of the substrate is raised, leading to a transition state. The mechanism of lysozyme (an enzyme of tears that cleaves (-1,4 glycosidic bonds) action is believed to be due to a combination of substrate strain and acid-base catalysis.

Covalent catalysis: In the covalent catalysis, the negatively charged (nucleophilic) or positively charged (electrophilic) group is present at the active site of the enzyme. This group attacks the substrate that results in the covalent binding of the substrate to the enzyme.

Entropy effect: Entropy is a term used in thermodynamics. It is defined as the extent of disorder in a system. The enzyme brings about a decrease in the entropy of the reactants. This enables the reactants to come closer to the enzyme and thus increase the rate of reaction.

In the actual catalysis of the enzymes, more than one of the processes-acid-base catalysis, substrate strain, covalent catalysis and entropy are simultaneously operative. This will help the substrate(s) to attain a transition state leading to the formation of products.

Question: Discuss the Factors modifying the Enzymes Actions.

Answer:

The contact between the enzyme and the substrate is the most essential pre-requisite for enzyme activity. The important factors that influence the velocity of the reaction is discussed here under1.

Concentration of enzyme: As the concentration of the enzyme is increased, the velocity of the reaction proportionately increases. This property of enzyme is made use in determining the serum enzymes for the diagnosis of diseases.

Concentration of substrate: If the concentration of a substrate is increased, while all other conditions are kept constant, the rate of an enzyme catalyzed reaction increases to a maximum value and no further.

Most enzymes show Michaelis-Menten kinetics and a rectangular hyperbola is obtained when velocity is plotted against the substrate concentration.

Three distinct phases of the reaction are observed in the graph: -------

At low substrate concentration, the velocity of the reaction is directly proportional to the substrate level (part A in graph).

In the second phase (part B), the substrate concentration is not directly proportional to the enzyme activity.

In the third and final phase (part C), the reaction is independent of the substrate concentration.

Effect of temperature: Velocity of an enzyme reaction increases with increase in temperature up to a maximum and then declines. A bell-shaped curve is usually observed.

The optimum temperature for most of the enzymes is between 400C-450C. However, a few enzymes (e.g. muscle adenylate kinase) are active at 1000 C. Some plant enzymes like urease have optimum activity around 600C. This may be due to very stable structure and conformation of enzymes.

In general when the enzymes are exposed to a temperature above 500C, denaturation leading to derangement in the native (tertiary) structure of the protein and active site. Majority of the enzymes become inactive at higher temperature (above 700C).

Effect of PH: Each enzyme has an optimal PH at which the velocity is maximum. Below and above this PH, the enzyme activity is much lower and at extreme PH, the enzyme become totally inactive. Most of the enzymes of higher organisms show optimum activity around neutral PH (6-8). There are, however, many exceptions like pepsin (1-2), acid phosphatase (4-5) and alkaline phosphatase (10-11). Enzymes from fungi and plants are most active in acidic PH (4-6).

Effect of product concentration: The accumulation of reaction products generally decreases the enzyme velocity. For certain enzymes, the products combine with the active site of enzyme and form a loose complex and, thus, inhibit the enzyme activity.

Effect of activators: Some of the enzymes require certain inorganic metallic cations like Mg2+, Mn2+, Ca2+, Co2+, anions are also needed for enzyme activity. Rarely, Cl- for amylase. Metals functions as activators of enzyme velocity through various mechanisms-combining with the substrate, formation of ES metal complex, direct participation in the reaction and bringing a conformational change in the enzyme.

Effect of time: Under ideal and optimal conditions (like PH, Temperature etc), the time required for an enzyme reaction is less. Variations in the time of the reaction are generally related to the alterations in PH and temperature.

Effect of light and radiation: Exposure of enzymes to ultra violate, beta, gamma and X-rays inactivates certain enzymes due to the formation of peroxides. E.g. UV rays inhibit salivary amylase activity.

Positive and Negative modifiers:

Small molecules modifiers which decrease catalytic activity are termed negative modifiers. For example: Hg, Ag and Au etc.

Small molecules modifiers which increase or stimulate catalytic activity are termed positive modifiers. For example: Fe, Mg and Cu etc.

Question: Describe in-details Michaelis-Menten of an enzyme catalyze reaction with Lineweaver-Burk modification.

Deduce the Mechaelis-Menten equation for enzyme action. What are the various Linear transforms of its?

Answer:

Michaelis-Menten equation4:

Mechaelis and Menten proposed that an enzyme-catalyzed reaction involved the reversible formation of an enzyme-substrate complex, which then broke down to from free and one or more products. Their postulate can be depicted thus: -------

Assumption 1: [S] is very large compared to [E], so that when all E is bound in the form ES, there is still an excess of S. By the Law of mass action, the rate of formation of ES from E + S is proportional to [E] ( [S]. or

Assumption 2: Conditions are such that there is very little accumulation of P, so that the formation of ES from E + P is negligible. The rate of ES is given by: ----------

Assumption 3: The rate of breakdown of ES very rapidly equals the rate of formation of ES. This is the steady state assumption: -----------

or,

But, [E] = [e] – [ES], where [e]is the total enzyme concentration; therefore: -----

Rearranging this equation yields, successively,

Or, finally,

Multiplying both sides in this equation by K3, we get: -----------

But, the initial velocity of an enzyme-catalyzed reaction V0, is of course equal to the rate of breakdown of the enzyme-substrate complex ES. So that we can write the first order equation: --

Dividing the numerator and the denominator by K1[S]yields: -----------

Because K1, K

2 and K3 are constants, the expression (K2+K3)/K1, is a constant, which may be written as Km. therefore: -----------

When the substrate concentration is so high that essentially all the enzyme in the system is present as the ES complex, i.e. when the enzyme is saturated, we reach the maximum initial velocity Vmax, given by: -------------

Thus, the expression for the velocity of reaction may be rewritten as: --------

This is the Michaelis-Menten equation, the rate equation for a one-substrate enzyme-catalyzed reaction. It relates the initial velocity, the maximum velocity and the initial substrate concentration through the Michaelis-Menten constant.

When, V0 = ½ Vmax, then

If we divide by Vmax, we obtain: ---------

On, rearranging, this becomes: --------

Therefore, Km is equal to the substrate concentration at which the velocity is half maximal. The Michaelis constant, Km, is not a true dissociation constant, but it does provide a measure of the affinity of an enzyme for its substrate. The lower the value of Km, the greater the affinity of the enzyme for substrate-enzyme complex formation.

The approximate value of Km is obtained graphically from a plot of initial velocity vs. initial substrate concentration, which has the form of a rectangular hyperbola3.

At very low substrate concentrations, the initial velocity V0 is nearly proportional to [S]; i.e. the reaction shows essentially first-order behavior. At very high substrate concentrations the reaction rate approaches Vmax asymptotically and is essentially zero order, i.e. nearly independent of substrate concentration3.

Linear transforms4:

Because it is difficult to estimate Vmax from the position of an asymptote, as in the plot of a rectangular hyperbola. Linear transforms of the Michaelis-Menten equations are often used.

Lineweaver-Burk transform4: This transformation is derived simply by taking the reciprocal of both sides of the Michaelis-Menten equation: -----

This equation is the Lineweaver-Burk equation which is similar to Y = ax + b. When 1/V0 is plotted against 1/[S], a straight line is obtained. This line will have a slop = Km/Vmax, the Y intercept = 1/Vmax and the X intercept = Km/Vmax.

Eadie-Hofstee transform4: This transformation is obtained by multiplying both sides of equation by Vmax and rearranging to yield: -------------

A plot of V0 against V0/[S], called the Eadie-Hofstee equation. Where the Y intercept = Vmax, the X intercept = Vmax/Km and the slop = - Km.

Question: describe the different types of enzyme inhibition and explained it with graphical representation.

Answer:

Enzyme Inhibition1:

Enzyme inhibitor is defined as a substance which binds with the enzyme and brings about a decrease in catalytic activity of that enzyme. The inhibitor may be organic or inorganic in nature.

Types1:

There are three broad categories of enzyme inhibition: ---------

Reversible inhibition

Irreversible inhibition

Allosteric inhibition.

Reversible inhibition1:

Here the inhibitor binds non-covalently with enzyme and the enzyme inhibition can be reversed if the inhibitor is removed. The reversible inhibition is further sub-divided into: ----

Competitive inhibition

Non-competitive inhibition

Un-competitive inhibition.

Competitive inhibition: The inhibitor competes with substrate and binds at the active site of the enzyme but does not undergo any catalysis. As long as the competitive inhibitor holds the active site, the enzyme is not available for the substrate to bind. During the reaction, ES and EI complexes are formed as shown below: --------

Features:

Inhibitor closely resembles the real substrate, usually known as substrate analogue.

Such inhibition of enzyme activity is reversible i.e. the inhibition could be overcome by a high substrate concentration.

In competitive inhibition, the Km value increases whereas Vmax remains unchanged.

The relative concentration of the substrate and inhibitor and their respective affinity with the enzyme determines the degree of competitive inhibition.

The Michaelis-Menten form of the inhibitor equation is4: -------

The Lineweaver-Burk transform of the Michaelis-Menten form of the inhibitor equation is4: -----

Example3: Inhibition of succinate dehydrogenase (SDH) by malonic acid, glutaric acid and oxalic acid in the oxidation of succinate to fumarate. These acids have structural similarity with succinic acid and compete with the substrate for binding at the active site of SDH. Among the above compounds malonic acid is the most potent competitive inhibitor of SDH.

Clinical significance5: Competitive inhibition is used therapeutically to treat patients who have ingested methanol (CH3OH). CH3OH is converted to formal dehyde (HCHO) by the action of enzyme alcohol dehydrogenase. HCHO damages many tissues and blindness is a common effect. Ethanol (CH3CH2OH) competes effectively with CH3OH as a substrate for alcohol dehydrogenase.

Non-competitive inhibition: The inhibitor binds at a site other than the active site on the enzyme surface. This binding impairs the enzyme function, possibly due to the distortion of the enzyme conformation. The inhibitor generally binds with the enzyme as well as the ES complex. The overall relation in non-competitive inhibition is represented below: ------

Features:

The inhibitor has no structural resemblance with the substrate.

There usually exists a strong affinity for the inhibitor to bind at the second site.

The inhibitor does not interfere with the enzyme-substrate binding.

Here, the Km value is unchanged while Vmax is lowered.

The Michaelis-Menten form of the inhibitor equation is: -------

The Lineweaver-Burk transform of the Michaelis-Menten form of the inhibitor equation is: -----

Example: Heavy metal ions (Ag+, Pb2+, Hg2+ etc.) can non-competitively inhibit the enzymes by binding with cysteinyl sulfhydryl groups. The general reaction for Hg2+ is shown in below: ---

Un-competitive inhibition: This is the third class of reversible inhibition which, however, is not very common. In this case, the inhibitor does not bind with enzyme but only binds with enzyme-substrate complex.

Features:

Un-competitive inhibitor decreases both Km and Vmax value of the enzyme.

This inhibition is rare in one substrate reactions but common in two substrate reactions.

Irreversible inhibition1:

The inhibitors bind covalently with the enzymes and inactivate them, which is irreversible. This inhibition will not be reversed by dialysis unless the linkage is chemically labile like that of an ester or thioester.

Features:

These inhibitors are usually toxic substances which may be present naturally or man made.

The Vmax is lowered but the Km is unchanged.

Example5: The thiol (-SH) group of the active site in glyceraldehyde

3-phosphate dehydrogenase is reacts with p-chloromercuribenzoate to form a mercuribenzoate adduct of the enzyme. Such adducts are not reversed by dialysis or by addition of substrate.

Clinical significance1:

The nerve gas (diisopropyl fluorophosphate) inhibits acetylcholine esterase, the enzyme essential for nerve conduction and paralyses the vital body functions.

Penicillin antibiotics irreversibly inhibit serine containing enzymes of bacterial cell wall synthesis.

Allosteric inhibition1:

Some of the enzymes possess additional sites, known as allosteric sites (Greek: allo - other) besides the active sites. The allosteric sites are unique places on the enzyme molecule.

Certain substances referred to as allosteric modulators bind at the allosteric site and regulate the enzyme activity. If a negative allosteric effector binds at the allosteric site called inhibitor site and brings about a conformational change in the active site of the enzyme, leading to the inhibition of the catalytic activity. `

Example1: Carbamoyl phosphate undergoes a sequence of reactions for synthesis of the end product, CTP. When CTP accumulates, it allosterically inhibits the enzyme aspartate transcarbamoylase by a feedback mechanism.

Question: Name some drugs that act enzyme inhibitors.

Answer:

Sulfa Drugs6: Sulfanilamide is an antibacterial agent. It competes with p-aminobenzoic acid (PABA), which is required for bacterial growth. Bacteria cannot absorb folic acid but must synthesize it. Since sulfanilamide is a structural analog of PABA, the bacterial dihydropteroate synthetase is tricked into making an intermediate containing sulfanilamide that cannot be converted to folate.

Methotrexate6: For the biosynthesis of purines and pyrimidines, folic acid is essential because it serves as a coenzyme in transfer of one carbon units from various amino acid donors. Methotrexate is a structural analog of folate and competes with dihydrofolate for dihydrofolate reductase. Thus inhibit the synthesis of thymidine monophosphate because of failure of the one carbon transfer reaction.

Other antimetabolites6:

Fluorouracil is a thymine analog in which the ring bound methyl is substituted by fluorine. The deoxynucleotide of this compound is an irreversible inhibitor of thymidylate synthetase.

6-mercaptopurine is an analog of hypoxanthine, adenine and guanine. It is converted to the 6-mercaptopurine nucleotide in cells. This nucleotide is a broad spectrum antimetabolite because of its competition in reactions involving adenine and guanine nucleotides.

Inhibitor

Target enzyme

Effect

Aspirin

Cyclo-oxygenase

Anti-inflammatory agent

Penicillin

Transpeptidase

Anti bacterial drug

5-Fluorouracil

Thymidylate synthetase

Anti cancer drug

Allopurinol

Zanthine oxidase

Antihypertensive drug

Pargyline

MAO

Antihypertensive drug

Omeprazole

H+/K+ ATPase proton pump

Ulcer therapy

Viagra

Phosphodiesterase

Treatment of impotency

Enalapril

Angiotensine converting enzyme (ACE)

Antihypertensive

Rofecoxib

COX-2

Anti-inflammatory drug.

Question: discuss the importance of enzyme in clinical diagnosis.

Answer:

For the right diagnosis of a particular disease, it is always better to estimate a few serum enzymes, instead of a single enzyme. Examples of enzyme patterns in important diseases are given here1: -------

Creatine phosphokinase (CPK), Aspartate transaminase (AST) and Lactate dehydrogenase (LDH) ------ are important in the diagnosis of myocardial infarction.

Alanine transaminase, Aspartate transaminase, Lactate dehydrogenase and Isocitrate dehydrogenase ----- are useful for the diagnosis of liver dysfunction due to viral hepatitis, toxic hepatitis, cirrhosis and hepatic necrosis.

Alkaline phosphatase and 5’-Nucleotidase --------------------------------------------------------- markedly increase in

intra-hepatic and extra-hepatic cholestasis.

Serum-(-glutamyl transpeptidase ------------------ is useful in the diagnosis of alcoholic liver diseases.

Serum levels of Creatine phosphokinase, Aldolase and Aspartate transaminase is increased in muscular dystrophies. Of these, Creatine phosphokinase is the most reliable indicator of muscular diseases, followed by Aldolase.

Increase in the serum acid phosphatase (Tartarate labile) is highly specific for the detection of prostatic carcinoma.

Increase in certain enzymes like Lactate dehydrogenase (LDH), Alkaline phosphatase and transaminase may be associated with malignancy in any part of the body.

(-Glucuronidase estimation in urine is useful in detecting the cancers of urinary bladder, pancreas etc.

Alkaline phosphatase and (-Glutamyl transpeptidase are important for the follow up in the treatment of cancers.

Serum amylase and lipase are increased in acute pancreatitis.

Alkaline phosphatase in rickets and hyperparathyroidism.

Increase in (2-Alkaline phosphatase suggests hepatitis whereas pre (-Alkaline phosphatase indicates bone diseases.

Certain enzymes are utilized as therapeutic agents. Streptokinase in used to dissolve blood clots in circulation while Asparaginase is employed in the treatment of leukemia.

REFERENCE

Biochemistry (1st Ed.) by Dr. U. Satyanarayana

Lippincott’s Illustrated Reviews Biochemistry (2nd Ed.) by Pamela C. Champe and Richard A. Harey.

Biochemistry (2nd Ed.) by Albert L. Lehninger

NMS Biochemistry (3rd Ed.) by Ian D.K. Halerston.

Biochemistry by Stayer.

Text book of Biochemistry with Clinical correlations (4th Ed.) by Thomas M. Deblin.

Prepared by Md. Badrul Alam (Prince)

PAGE

NUMPAGES

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Where,

E = Free enzyme

S = Substrate

ES = Enzyme-substrate complex

P = Product

K1 = Rate constant for the formation of ES

K2 = Rate constant for the dissociation of ES to E + S.

K3 = Rate constant for the dissociation of ES to E + P

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Where,

Km = Michaelis constant.

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Vmax

½Vmax

Substrate concentration [S]

Fig: Effect of substrate concentration on the rate of an enzyme-catalyzed reaction.

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Slop=Km

/Vmax

1/S

1/Vmax

1/Km

1/V0

Fig: The Lineweaver-Burk transform of the Michaelis-Menten equation.

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Slop= - Km

V/[S]

Vmax/Km

Vmax

Fig: The Eadie-Hofstee transform of the Michaelis-Menten equation.

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Vmax

½Vmax

[S]

Km’

+ I

1/V

1/Vmax

- 1/Km

- 1/Km’

+ I

1/[S]

Fig: Effect of competitive inhibitor on enzyme velocity.

(A) Velocity vs. Substrate plot. (B) Lineweaver-Burk plot.

(A)

(B)

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Fig: Effect of non-competitive inhibitor on enzyme velocity.

(A) Velocity vs. Substrate plot. (B) Lineweaver-Burk plot.

Vmax

½Vmax

[S]

+ I

(A)

Vmax

½Vmax

1/V

1/Vmax

-1/Km

+ I

1/[S]

(B)

1/Vmax

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1/V

+ I

1/[S]

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Fig: Effect of un-competitive inhibitor on enzyme velocity.

Lineweaver-Burk plot.

Hyperbolic curve

Sigmoidal curve

Substrate conc.

Fig: Effect of substrate concentration on allosteric enzyme in comparison with normal enzyme

Carbamoyl phosphate + Aspartate

Aspartate transcarbamoylase

Carbamoyl aspartate + Pi

Cytidine triphosphate (CTP)

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Fig: Effect of substrate concentration on the rate of an enzyme-catalyzed reaction.

Substrate concentration [S]

½Vmax

Vmax

Optimum

Temperature 0C

Fig: Effect of temperature on enzyme velocity.

Fig: Effect of PH on enzyme velocity.

Optimum pH

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Activation energy

Without enzyme

Energy change in reaction

Mountain

Tunnel

Activation energy

With enzyme

Pharmacy Readers

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Wednesday, November 5, 2014

ENZYME

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