Enzymes (UNIT-5) B. pharm

                          

                                          ENZYMES

                    

Introduction-

In 1877, German physiologist Wilhelm Kühne (1837–1900) first used the term enzyme, which comes from Ancient Greek ἔνζυμον (énzymon) 'leavened, in yeast', to describe this process.

The word enzyme was used later to refer to non-living substances such as pepsin, and the word ferment was used to refer to chemical activity produced by living organisms.

Definition

Enzyme, a substance that acts as a catalyst in living organisms, regulating the rate at which chemical reaction proceed without itself being altered in the process.

All enzymes are protein but all proteins are not enzymes.

Catalyst- Any substance that increases the rate of reaction without itself consuming is called catalyst.

Characteristics-

·       Enzymes catalyse all aspects of cell metabolism.

·       This includes the digestion of food, in which large nutrient molecules (such as proteins, carbohydrate, and fats) are broken down into smaller molecules.

·       The conservation and transformation of chemical energy.

·       They work more effectively in presence of air.

·      They are destroyed gradually helping in a reaction.

Properties-

v Act as biological catalysts, speeding up the rates of reactions.

v Transform one form of energy into a much more useful form of energy .

v Do not act alone and typically require helper molecules called cofactors.

v Mostly proteins but some RNA molecules can also act as catalysts.

v They are not depleted and remain unchanged at the end of the reaction.

v Only one enzyme can catalyze a single process.

v Inactivated at temperatures below the boiling point of water.

v Enzyme are proteins in nature although not all proteins are enzyme.

Nomenclature-

IUBMB Classification-  According to International Union of Biochemistry and Molecular Biology an enzymes are classified into six groups.

There are six principal categories and their reactions:-

(1)  Oxidoreductases - Which are involved in electron transfer, enzymes which brings about oxidation and reduction reaction between the substrate.(surface on which an organism Eg-  plant ,fungus lives).

Example- Catalase, ascorbic acid oxidase, alcohol dehydrogenase.

Oxidation- Reactant loses electron during reaction.

Reduction-Reactant accumulates electron during reaction.

(2) Transferases- Which transfer a chemical group from one substance to another.

Example- Hexokinase

            

(3) Hydrolases- This group catalyse hydrolysis Eg- breakdown of chemical bond by addition of water. Eg- Peptidase

Types of hydrolase-

1.    Carbohydrase- They hydrolyse carbohydrate

  Eg- Sucrase, maltase, lactase, Amylase and Cellulose.

2. Esterases- These enzymes hydrolyse the ester

Eg- Lipases, phosphorylase etc.

3. Proteases- They hydrolyses the compound related to proteins.

Eg – Pepsin, renin , trypsin etc.

4. Amidase- they hydrolyse the amide compounds.

Eg- urease, asparaginase, anginose.

5. Nucleases- these enzyme hydrolyse nucleic acid

Eg- Nuclease, nucleosidase, polynucleotides.

(4) Lyases- This group of enzymes catalyse removal of groups from substrate by mechanism other than hydrolysis.

Example- Decarboxylase

 (5) Isomerases- This groups of enzymes catalyses the interconversion of one isomer into another isomer ( Same chemical formula but different arrangements).

1. This groups includes- Isomerases, mutases and epimerases.

 (6) Ligases- This groups of enzymes catalyses joining of two substrates using the energy released in the hydrolysis of a high energy phosphate compound as ATP.   

Eg- Glutamine synthetase.

Enzyme inhibitors- Any substance that can diminish the velocity of an enzymatic reactions is called inhibitors.

The inhibition of enzymes can be classified either as reversible and irreversible or as competitive and non- competitive.

A. Reversible and Irreversible inhibitors-

1.    Reversible inhibitor: The inhibitor bind to enzymes through non- covalent bonds with enzyme and the enzyme inhibition can be reversed if the inhibitor is removed.

a.     Dilution of enzyme- inhibitors complex result in dissociation of the reversible bound inhibitor and recovery enzyme activity.

b.    Reversible inhibition - may be competitive or non- competitive.

B.  Competitive and non- competitive inhibitor

1. Competitive inhibitors- This type of inhibitors occurs when the inhibitors binds reversibly to the active sites where substrate is present. Then both inhibitors and substrate are compete for active sites.

a. Structural similarity between substrate and inhibitors. Eg.- Malonic acid, succinic acid.

b. The combination between enzyme and substrate depends on-

·       Concentration of substrate

·       Concentration of substrate

·       Affinity of both substrate and inhibitor to the active site of enzymes

·       Competitive is reversible and can be removed by adding excess substrate.

Diagram: Competitive inhibitors 

1.    Non- Competitive inhibitors- This type of inhibitors occurs when the inhibitor and substrate bind to different sites on the enzyme.

a.     The inhibitors can bind either free enzyme.

b.    There is no structural similarity between substrate and inhibitors.

c.     The inhibitors bind to a site on the enzyme other than the active site.

    Eg- heavy metals as mercury and oxidizing agent.

d.    Non- competitive inhibition- irreversible.

 

Diagram : Non- competitive inhibitors

Difference between competitive and non- competitive inhibitors—

	Competitive Inhibition	Non- competitive Inhibition
Inhibitor	Structural analogue	No similarity
Site	Active site	Allosteric site
Inhibition	Reversible	Irreversible
Excess substrate	Relives inhibition	Not relieve- Inhibition
Km	Increased	No change
Vmax	No change	Decreased
Significance	Drug action	Toxicological
Conformation	No change	Change

Irreversible inhibitors- The inhibitors bind covalently with the enzymes are inactivate them which is irreversible.

These inhibitors are usually toxic substances the poison enzymes.

Example- The penicillin antibiotics act as irreversible inhibitors of serine- containing enzymes and block the bacterial cell wall. 

REGULATION OF ENZYME ACTIVITY-

Enzyme activity is regulated by many mechanism. This regulation helps to maintain a constant intracellular environment in the face of changes in external factors as change in temperature or presence or absence of water.

 

Mechanisms regulating enzyme activity include-

A.                        Amount of enzyme present in the cell- this is determined by :

1.    Rate of enzyme synthesis- Enzyme (proteins) synthesis from amino acid may be stimulated by certain substances.

 These substances are called inducers and the process called induction.

 Also enzyme synthesis may be inhibited by other substances which are called  repressors and the process is called as repression.

(a)  . Inducers or induction-

·       Inducers may be substrates for enzymes.

·       Inducers may also be compounds similar in structure to the substrates. These compounds are called gratuitous inducers.

·       Inducers usually stimulate DNA (gene) controlling enzyme synthesis.

(b). Repressors, repression-

·       Repressor may be a product of metabolic pathway.

·       It will inhibit DNA (gene) controlling enzyme synthesis. This is called as feedback regulation.

·       As a result of repression-

¨    The metabolic pathways stops.

¨    Metabolic intermediates are removed

¨    Enzyme biosynthesis again occurs. This is called Derepression.

2.    Adaptation- The amount of enzyme present in a cell can be increased or decreased according to the body needs. This process is called as adaptation.

B.    Allosteric regulation of enzyme activity-

1.    Allosteric enzymes generally catalyse the irreversible steps in metabolic pathways.

2.    The term allosteric means “other site”. It indicates that a molecules called effectors ( modifiers or modulators) can bind non – covalently at a site other than active site.

a.     Effector are positive if they stimulate catalytic reaction and negative if they inhibit reaction.

b.    Effector may be the end product of a metabolic pathway. If it inhibit the reaction (negative regulation), it is called feedback inhibition.

3. Classes of allosteric enzymes: There are 2 classes of allosteric enzymes-

(a). Homotropic enzymes- In which the substrate is also the effector. Eg- substrate bind to both active and allosteric site.

(b) Heterotopic enzymes- In which the effector is other substrate than the substrate, oftenly the end product of the pathway. 

COENZYMES :-

The first coenzyme to be discovered was NAD+, which was identified by Arthur Harden William Youndin in 1906.

Coenzyme is defined as an organic molecule that binds to the active site to assist in the catalysis of a reaction.

                                                          OR

According to IUPAC( International Union of pure and Applied Chemistry) Coenzyme is defined as a low molecular weight, non – protein and dialyzable  organic compound that is loosely  attached  and participating in enzymatic reaction is known as coenzyme.

It is commonly made from nucleotide such as adenosine triphosphate.

Coenzymes are second substrates:

v Coenzyme are often regarded as second substrates or co- substrates, since they have affinity with enzyme comparable with the substrate.

v Coenzyme undergo alterations during the enzymatic reactions.

v Coenzyme participate in various reactions involving transfer of atoms or group Eg- hydrogen, aldehyde, keto, amino, acyl, methyl, carbon dioxide

v Coenzyme from B- complex vitamins- Most of the coenzyme are the derivative of water soluble B- complex vitamins.

Example-

v Non-vitamin coenzyme- Not all coenzyme are vitamin derivative .There are some other organic substances, which have no relation with vitamins but functions as coenzymes.

v  They may be considered as non- vitamin coenzyme. Eg- ATP ( Adenosine tri -phosphate),CDP( Cytidine di phosphate), UDP (Uridine Di Phosphate).

Biochemical functions-

Ø The function of coenzyme is to transport groups between enzymes.

Ø Chemical groups include hydride ions which are carried by coenzymes such as NAD (Nicotinamide adenine dinucleotide)

Phosphate groups which are carried by coenzyme such as ATP.

   Acetyl groups which are carried by coenzyme such as coenzyme A.

Ø A coenzymes prepares the active site for catalytic activity.

Ø A coenzyme is necessary helper for enzymes that assist in biochemical transformations.

Ø A Coenzyme is a low molecular weight organic substance without which the enzyme cannot exhibit any reaction. 

Structure of coenzyme-

Coenzyme A (CoASH or CoA) itself is a complex and highly polar molecule, consisting of adenosine 3',5'‑diphosphate linked to 4‑phosphopantothenic acid and hence to β‑mercaptethylamine, which takes a direct part in acyl transfer reactions.

The adenosine 3’,5’‑diphosphate moiety serves as a recognition site as many proteins have nucleotide-binding folds, and it increases the affinity for CoA binding to enzymes.

The genes encoding the enzymes for CoA biosynthesis have been identified, and the structures of many proteins in the pathway have been determined.

Therapeutic and Diagnostic application of enzymes:

Therapeutic application of enzyme-

§  Enzymes are used for aiding digestion. Example- Amylases, Proteases, lipases.

§  They are used as deworming agent. Ex- Papain.

§  They are act as anti- clotting agents like fibrinolytic and thrombolytics ex- Urokinase

§  They act to treat atherosclerosis like serratio. Ex - peptidase.

§  They are used to treat wounds and swelling. Ex – Trypsin, Chymotrypsin,

§  They are used to assist metabolism like hyaluronidase.

§  They are use as surface disinfectants. Ex – Trypsin. 

ENZYME	THERAPEUTIC USE
Asparaginase	Acute lymphatic leukaemia
Streptokinase	Lyse intracellular clot
Urokinase	Lyse intracellular clot
Plasminogen	Plasmin /clot lysis
Streptokinase	DNAase applied locally
Hyaluronidase	Enhance local anesthesia
Pancreatic (lipase & trypsin)	Pancreatic insufficiency
Papain	Anti – inflammatory
Alpha anti trypsin	Emphysema

Diagnostic Application-

v They are also used in the diagnosis purpose. Ex – glucose oxidase/ peroxidase to detect the level of glucose.

v Liver disease: SGPT (Serum glutamate pyruvate transaminase) gama – glutamyl -transpeptidase.

v Heart attacks: Aspartate aminotransferase(AST)

v Myocardial infarction : Creatine phosphokinase.

v Uric acid :  Uricase.

Enzyme for diagnostic purpose	Estimation
Urease	Urea
Uricase	Uric acid
Glucose oxidase	Glucose
Peroxidase	Glucose/cholesterol
Hexokinase	Glucose
Cholesterol oxidase	Cholesterol
Lipase	Triglyceride
Horse radish peroxidase	Elisa
Alkaline phosphate	Elisa
Restriction endonuclease	Southern blot
Reverse transcriptase	Polymerase chain reaction

 Isoenzymes- 

The multiple forms of an enzyme catalysing the same reaction are isoenzymes or isozymes.

They, however differ in their physical and chemical properties which include the structure , electrophoretic and immunological properties, Km and Vmax value, pH optimum to inhibitors and degree of denaturation.

Among the isoenzymes, lactate dehydrogenase (LDH) has been the most thoroughly investigated.

Diagnostic importance of LDH:-

v Isoenzyme of LDH have immense value in the diagnosis of heart and liver related disorder.

v In healthy individuals, the activity of LDH2 is higher than the LDH1 in serum.

v In the case of myocardial infarction, LDH1 is much greater than LDH2 and this happens with in 12 to 24 hours after infarction.

v Increased activity of LDH5 in serum is an indicator of liver diseases.

v LDH activity in RBC is 80- 100 times more than that in the serum.