Introduction To Enzymes [PDF]

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INTRODUCTION TO ENZYMOLOGY 3

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'()*m INTRODUCTION TO ENZYMES Enzymes ëm Enzymes are biocatalysts²Catalysts of life ëm G catalyst is defined as a substance that increases the velocity or the rate of a chemical reaction, without itself undergoing any change in the process ëm Enzymes are biocatalysts that are synthesized by living cells. ëm dhey are proteinaceous (exception is the ribozyme, which is an RNG , colloidal, and thermolabile (inactive at 0YC and destroyed at 100YC ëm dhey are specific in action, catalyze all biochemical reactions and are susceptible to many factors like temperature, pH, etc. ëm Examples: urease, carbonic anhydrase, pepsin, rennin, etc. Gntienzymes ëm Gntienzymes are those substances which when injected in the body produces certain molecules, which act as inhibitors and inhibits the function of the enzyme, related to that particular reaction ëm Examples: Gntitrypsin, antirennin, antipepsin, etc. 
strate ëm dhe substrate is a substance upon which an enzyme acts and gets converted into the corresponding product ëm or example, maltose is the substrate over which the enzyme maltase acts to from glucose Maltase Maltose  Glucose

O aracteristics of Enzymes 1m Colloidal nature ëm dhey are of great size ëm dheir molecular weights usually range from 12000 to over a million Daltons

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ëm Hence, they are very large compared to their substrates or the functional group they act upon ëm dhe molecular weight of many enzymes are found to be approximately 6fold (6 is an integer multiple of 17500, which is found to be a unit in most proteins ëm ’n account of their large size, the enzyme molecules possess extremely low rates of diffusion and forms colloidal systems in water ëm Due to their giant size, the enzymes exhibit many colloidal properties such as: i m Diffusion rates are very slow ii m May produce considerable high light scattering. dhey form turbidity in solution known as dyndall effect 2m Catalytic nature ëm G universal feature of all enzymatic reactions is the virtual absence of any side product ëm Gn enzyme is precisely adapted to catalyze a particular reaction. or example, amylase catalyzes the breakdown of starch ëm dhey act catalytically and accelerate the rate of chemical reactions, occurring in the plant and animal tissues ëm dhey normally do not participate in the reaction, or if they do so, at the end of the reaction, they are recovered as such without undergoing any qualitative or quantitative change ëm dhis is why they are capable of catalyzing the transformation of a large quantity of substrate ëm dhus, the catalytic potency of enzymes is extremely great 3m durnover number ëm dhe catalytic power of an enzyme is measured by the turnover number or the molecular activity ëm t is defined as the number of substrate molecules converted into the product in a given unit of time by a single enzyme molecule, when the enzyme is fully saturated with the substrate ëm dhe turnover number of some enzymes are given as follows:

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Enzyme Catalase Carbonic anhydrase Gcetyl cholinesterase -Lactamase umarase Rec G protein

Substrate H2’2 HC’36 Gcetyl choline Benzyl penicillin umarate Gd


Kcat (s61 40,000,000 400,000 140,000 2,000 800 0.4

ëm durnover number is represented as Kcat. dhe constant Kcat is a first order rate constant and its unit is s61

KV


max [E]d

ëm dhe value of the turnover number varies with different enzymes and it depends upon the conditions in which the reaction is taking place ëm dhe turnover number of 40,000,000 s61for carbonic anhydrase is one of the largest known ëm Carbonic anhydrase catalyzes the hydration of carbon dioxide to form carbonic acid: CO2 +

2

Carbonic anhydrase O 

2

CO3

ëm dhis catalyzed reaction is 4×107 times faster than the uncatalyzed one ëm or most enzymes this value falls between 1±104 s61 4m Specificity of enzyme action ëm Enzymes are highly specific in their action when compared with a chemical catalytic reaction; i.e., a particular enzyme attacks only a particular substrate ëm ’ccurrence of thousands of enzymes in the biological system is due to the specific nature of enzymes ëm Enzyme specificity is based on the mode of accepting the substrate and their reaction ëm dhey are i m Stereochemical or optical specificity

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(1 mStereoisomers are those compounds which have the same molecular formula but differ in their structural configuration (2 mdhe enzymes act on only one isomer and thus exhibit specificity towards that particular stereoisomer (3 mExamples: L-amino acid oxidase and D-amino acid oxidase; hexokinase acts only on D-hexoses, arginase acts only on L-Grg and not on D-Grg, amylase acts only on -glycosidic linkages and not on - glycosidic linkages, succinate dehydrogenase dehydrogenates succinate to give fumaric acid and not maleic acid, and so on (4 mStereospecificity is explained by considering 3 distinct regions of the substrate molecule, specifically binding with 3 complementary regions on the surface of the enzyme

Representation of stereospecificity of aƍ, bƍ, cƍ 3 points of attachment of substrate to enzyme (a, b, c

(5 mdhe enzymes belonging to the class of isomerases do not exhibit this stereospecificity since they are specialized in the interconversion of the isomers. or example, alanine racemase is involved in the interconversion of L-ala and D-ala ii m Reaction specificity (1 mEnzymes are specific, in the sense that almost one enzyme catalyzes only one of the various reactions that the substrate can undergo (2 mor example, ’xaloacetate is an important metabolic intermediate. t can undergo several reactions namely, reduction to give maleic acid; decarboxylation to give pyruvate; or accept an amino group to give

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Gsp and so on. Each reaction of ’GG is catalyzed by its own separate enzyme, which catalyzes only that reaction and none other Gspartate dransaminase

Citrate

§

Gcetylation Decarboxylation   ’xaloacetate  CH 3C’’H Decarboxylase


yruvate

Gcetylase

Reductase




Malate

(3 m Glu undergoes several reactions but each reaction is caralyzed by a specific enzyme Gln Glutaminase


§

Gspartate aminotrasferase Glanine aminotransferase  Glu  Gla  -KG Gsp  -KG  Reductase




Malate m

(4 m
artly due to its specificity and partly because they take place at relatively lower temperatures, enzyme catalyzed reactions are much more quantitative iii mSubstrate specificity (1 mdhe extent of substrate specificity varies from one enzyme to the other (2 m t may be either absolute or relative (3 mGbsolute specificity (one to one specificity is seen in some enzymes which are capable of acting on only 1 substrate. or example, urease acts only upon urea (4 mRelative specificity can be further classified into group dependant or bond dependent (5 mGroup substrate specificity (a mGbsolute group specificity or relative group specificity or broad specificity

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(b mSome enzymes catalyze the reaction of a structurally related group of compounds (c mor example, hexokinase adds a phosphate group to all hexoses like glucose, fructose, mannose, etc. (d mGroup specificity is exhibited by proteolytic enzymes with respect to the peptide bond between 2 specific amino acids (e mExamples: (i mdrypsin splits peptide bonds in which the carboxylic group is contributed by either Lys or Grg (ii mChymotrypsin preferentially splits peptide bonds in which the carboxyl group is from an aromatic amino acid (iii dhrombin splits peptide bonds in which the side chain on the carboxyl site of the susceptible peptide bond must be Grg, while the one on the amino group must be Gly (f mdhese enzymes help in the elucidation of the arrangement of the amino acid residues in a protein (6 mBond substrate specificity (a mBond dependent class of enzymes, are very specific in catalyzing bond-dependent reactions (b mExamples: (i m
roteases 
eptide bonds of proteins (ii mGlycosidases  Glycosidic bonds of carbohydrates (iii Esterases  Ester linkages in lipids (iv Exonucleases and endonucleases 
hosphodiester linkages in nucleic acids iv mGeometrical specificity (1 mSome enzymes exhibit specificity towards the cis/trans forms (geometric isomers (2 mor example, fumarase catalyzes the interconversion of fumarate (trans form and malate (cis form

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5m Gmphoteric nature ëm Enzymes are amphoteric. dhey act as both acids and bases ëm dhey migrate in an electric field and the direction of their migration depends upon the charge possessed by them ëm dhe net charge is influenced by their pH value ëm Each enzyme has a fixed value of isoelectric point (p at which it will move in an electric field ëm soelectric field or isoelectric point is the pH value at which the number of cations equals the number of anions ëm dhus, at p , the net electric charge on an enzyme is always 0 ëm But, the total charge on an enzyme molecule (sum of the positive and negative charges at this point is always maximum ëm Hence, enzymes are dipolar ions (or internal salts (or zwitterions ëm Gt p , they exist as  3 ( OO n ëm Gt pH values m p , enzymes will have a net positive charge and will migrate towards the cathode ëm Similarly, at pH ´ p , enzymes will have a net negative charge and will migrate towards the anode

2

Acidic behaviour  OO


2

asic behaviour  OO
   + l

OO +

2




 3



OO + l

6m Solubility ëm dhe solubility of enzymes is markedly influenced by the pH ëm Solubility is lower at p and increases with increasing acidity or alkalinity ëm n either cation or anion, repulsive forces between enzymes are high, since all the molecules possess excess charge of the same site. dhus they will be more soluble ëm Salting-in effect i m Globulins are slightly soluble in water.

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ii m However, their solubility is greatly increased by the addition of neutral salts like NaCl iii mdhis phenomenon is known as salting-in effect iv mdhis is due to the forces of attraction between the salt and protein at low salt concentrations leading to increased solubility ëm Salting-out effect i m
roteins are precipitated from aqueous solutions by high concentrations of neutral salts. ii m dhis is known as the salting-out process iii mdhe salts commonly used for this purpose are sodium sulphate, magnesium salts and phosphates iv mGs the concentration of neutral salts is increased, the solubility increases to maximum and then starts decreasing and finally the proteins get precipitated ëm soelectric precipitation i m Some proteins like casein of mil are readily precipitated at (or near their p . dhis process is described as isoelectric precipitation ii m Salting out of proteins is caused due to the competition for water molecules between the protein and salt at high concentrations 7m ’ptical activity ëm Gll proteins rotate the plane polarized light to the left; i.e., they are laevorotatory 8m Denaturation of enzymes ëm Denaturation is a result of change in conformation or unfolding of the enzyme molecule, i.e., 2Y or 3Y structure of the enzyme is completely lost in the process without any break in the 1Y structure ëm Denaturation is defined as the total loss or randomization of the 3-D structure ëm Denaturation refers to changes in properties of enzymes; i.e., loss of biological activity

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ëm Ggents causing denaturation: i m
hysical agents t includes mechanical action like heat treatment, cooling, freezing operations, rubbing, high hydrostatic pressures, U and ionizing radiations, radioactive and ultrasonic radiations ii m Chemical agents ’rganic solvents (acetone, alcohol , aromatic anions (salicylates , and anionic detergents (SDS are some examples for chemical denaturating agents ëm
roperties of denatured enzymes i m dhey decrease in solubility, and size ii m dhey have altered 3-D shape iii mCessation of biological activity as proteins or hormones iv m ncreased activity of some radicals in the molecule such as the ±SH group of Cys, the -S-S- of cystine and phenolic group of dyr v m Glteration in the surface tension and loss of antigenicity vi mMr and osmotic pressure do not change much vii m Changes in optical rotation in the direction of increased laevorotation ëm Denaturation is of 2 types:

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ëm Reversible denaturation drypsin when exposed to temperatures of 80±90YC gets denatured. However, when cooled to 37YC, the solubility and activity of trypsin is regained ëm rreversible denaturation Boiling of egg results in the loss of tertiary structure and is an irreversible denaturation ëm Significance of denaturation i m Denaturation property of a protein is harmful in clinical laboratories ii m dhe protein-free substance of the blood such as glucose, picrate, and drugs are analyzed by the precipitation of blood proteins by the addition of certain acids 9m Reversibility of a Reaction ëm Enzymes are capable of bringing about reversion in a chemical reaction ëm dhe digestive enzymes catalyze the hydrolytic reactions which are reversible ëm or example, lipase, which catalyzes the synthesis of fat from glycerol and fatty acids, can also hydrolyze them into their component units 2

(

2

OO

15

31 3

+3

2

ipase  O
 

O 2

dripalmitin

Water

O +3

15

31

OO

O

lycerol

Palmitic acid

ëm dhe direction in which the reaction proceeds depends upon many factors like (1 mpH of cell sap (2 m
resence of reacting substance (3 mGccumulation of end products

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Reversibility of action of lipase (from castor on triolein

ëm dhe final equilibrium mixture is the same, whether the reaction starts with the ester, or with its individual components O emical Nat re and Properties of Enzymes ëm Each enzyme has its own tertiary structure and specific conformation, which is very essential for its catalytic activity ëm Chemically, enzymes may be divided into 2 categories ëm Simple protein enzymes §m dhese contain simple proteins only. E.g., urease, amylase, papain, etc. ëm Complex protein enzymes §m dhese contain conjugated proteins, i.e., they have a protein part called apoenzyme and a non-protein part called prosthetic group, associated with the protein unit §m dhe 2 parts together constitute the holoenzyme. E.g., catalase, cytochrome V, etc. ëm Holoenzyme §m dhe functional unit or the active structure of an enzyme (apoenzyme prosthetic group is called holoenzyme

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Holoenzyme

Gpoenzyme

(Gctive enzyme

(
rotein part

 Coenz yme (
rostetic group

ëm Gpoenzyme §m t is the protein part of the enzyme §m t is the inactive form of the enzyme §m t becomes functional only by associating itself with the prosthetic group ëm
rosthetic group §m G prosthetic group is that which is covalently bound to the apoenzyme §m dhey do not dissociate from the protein part of the enzyme and repeatedly participate in enzyme-catalyzed reactions §m E.g., GD, MN, d

,
L
, biotin, etc. ëm Cofactor §m dhey are mainly inorganic metal complexes which are tightly bound to the enzyme §m dhey are highly required for normal conformational structure and function of the enzyme §m dhey act as donors or acceptors in oxidation and reduction reactions        2 2 2 2 2 2 2 §m E.g., Mg , Ca , Cu , Zn , K , e , Mn , Mo , etc. ëm Coenzymes §m dhey are also called co-substrate or second substrate §m dhey are organic, metallo-organic, or inorganic substances which are thermostable and dialyzable, highly required for the normal functioning of the enzyme §m dhey bind covalently or non-covalently to the apoenzyme §m Reactions involving oxidoreductions, group transfers, isomerization, and covalent bond formation require coenzyme §m Coenzymes account for 1% of the entire enzyme molecule §m E.g., NGD, GD, dH, CoG. MN, d

, etc.

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Olassification of Ooenzymes 1. Classification based on chemical characteristics




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2. Based on functional characteristics




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 nctions of Ooenzymes ëm dheir function is usually to accept atoms or groups from a substrate and to transfer them to other molecules ëm dhey are less specific than enzymes and the same coenzyme can act as such in a number of different reactions ëm Coenzymes are also attached to the protein at different but adjacent site, so as to be in a position to accept the atoms or groups, which are removed from the substrate §m M
M
 Hydrogen acceptors in dehydrogenation reactions §m O

 O  dransfer of acetyl/acyl groups used in oxidative decarboxylation of pyruvate, G synthesis and acetylation §m d Decarboxylation of -ketoacids and to carry the ³active aldehyde (R±CH(’H 6  group §m  dransamination and decarboxylation of amino acids §m d
 Carrier of folate, used in the synthesis of purines and pyrimidines §m Ô


 Carbon chain isomerization §m Ô  Carbon dioxide fixation reactions §m


M Hydrogen acceptors in dehydrogenation reactions §m

 Gct as coenzyme in cytochromes, peroxidases and
G synthase complex {ole of Metals in Enzyme Gction ëm dhe activity of many enzymes depend on the presence of many metal ions such      2 2 2 2 as Mg ,Ca , Cu , Zn , K , etc. ëm Depending upon the interaction between the enzyme molecule and the metal ion, the enzymes are classified as metal activated enzymes and metalloenzymes ëm Metal activated enzymes §m n certain enzymes, metals form a loose and easily dissociable complex §m Such enzymes are called metal activated enzymes §m dhe metal ion can be removed by dialysis or any other simple method from the enzymes without causing denaturation (inactivation of apoenzyme    §m or example, Gd
ase±Mg2 and Ca2 , enolase±Mg2 , etc.

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ëm Metalloenzymes §m Some enzymes which are tightly bound to metal ions are called metalloenzymes §m dhese metals cannot be dissociated from the apoenzyme, even after several extensive steps of purification  2 §m or example, Cu2 cytochrome oxidase,phenol oxidase; Zn carbonic  2 2 anhydrase, alcohol dehydrogenase; Mg hexokinase; Ni urease, etc. {ole of Metals in Enzyme Oatalysis ëm dhey help in maintaining or producing, or both, active structural conformation of the enzyme ëm Enable the formation of the E±S complex ëm Metal ions, like protons, can share an electron pair forming a sigma bond ëm dhey can serve as a 3-D template for the orientation of basic groups on the enzyme or substrate ëm Metal ions accept electrons via sigma or pi bonds to activate electrophiles or nucleophiles ëm dhey can also act as nucleophile themselves by donating electrons ëm G metal ion may also ³mask a nucleophile and thus prevent an otherwise likely side reaction ëm dhe coordination sphere of a metal bring together the enzyme and substrate or form chelate, producing distortion in either the enzyme or substrates ëm Stereochemical control of an enzyme catalyzed reaction may be achieved by the ability of the metal coordination sphere to act as a 3-D template to hold reactive groups in a specific steric orientation ëm Metal ions participate in each of the 4 mechanisms by which enzymes accelerate the rates of chemical reactions §m General acid±base catalysis §m Covalent catalysis §m Gpproximation of reactants §m nduction of strain in the enzyme or substrate ëm dhe role of metal ions in some important enzymes are summarized in the following table:

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No 1 2 3 4 5

6 7 8

Enzyme Histidine deaminase Kinases, lyases, pyruvate decarboxylase Carbonic anhydrase Cobamide enzymes
yruvate carboxylase, carboxypeptidase, alcohol dehydrogenase Non-heme iron proteins
yruvate kinase, pyruvate carboxylase, adenyl kinase
hosphotransferase, D-xylose isomerase, heme proteins

{ole of metal ion Masking a nucleophile Gctivation of an electrophile Gctivation of an nucleophile Metal acts as a nucleophile
i electron withdrawal


i electron donation Metal ion gathers and orients ligands Stain effects

dernary Oomplexes wit Metals  nction in Oatalysis ëm or ternary (3 component complexes of catalytic site (E , a metal ion (M , and substrate (S that exhibit 1:1:1 stoichiometry, 4 schemes are possible as follows:

1. E S M (Substrate bridge complex 2. M E S (Enzyme bridge complex 3. E M S (Simple metal bridge complex 4. E M ( yclic metal bridge complex S ëm Gll 4 are possible for metal activated enzymes ëm Metalloenzymes cannot form the E±S±M complex as they retain the metal throughout the purification ëm dhree generalizations are: §m Most, but not all kinases (Gd
: phosphotransferases form substrate bridge complexes of the type: E±nucleotide±M

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§m
hosphotransferases using pyruvate or
E
as substrate, enzymes catalyzing other reactions of
E
and carboxylases form metal bridge complexes §m G given enzyme may form one type of bridge complex with one substrate and a different type with the other ëm Enzyme bridge complexes §m dhe metals in the enzyme bridge complexes are presumed to perform structural roles, maintaining an active conformation (e.g., Gln synthase or to form a metal bridge to a substrate (e.g., pyruvate kinase §m n addition to its structural role, the metal ion in the pyruvate kinase appears to hold one substrate (Gd
in place to activate it

M
yruvate kinase

Gd
Creatine

ëm Substrate bridge complexes §m dhe formation of ternary substrate bridge complexes of nucleoside triphosphates with enzyme, metal and substrate appears to be attributed to the displacement of water from the coordination sphere of the metal by Gd


Gd
46  M(H 2 ’

2 6

26
> >> > Gd
6 M(H 2 ’ 3  3H 2 ’ dhe enzyme then binds, forming the ternary complex:

Gd
6 M(H 2 ’

26 3

 E
> >> > E 6 Gd
6 M(H 2 ’

26 3

§m n phosphotransferase reactions, metal ions are thought to activate the phosphorous atoms and form a rigid, polyphosphate±adenine complex of appropriate conformation in the active quaternary complex ëm Metal bridge complexes §m Crystallographic and sequencing data have established that a histadyl residue is concerned with metal binding at the active site of many proteins (e.g., carboxypeptidase G, alkaline phosphatase (GL
, phospholipase C (
LC , etc..

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§m or binary E±M complexes, the rate limiting step is the departure of water from the coordination sphere of the metal ion §m or many peptidases, activation by metal ions is a slow process requiring many hours §m dhe slow reaction probably is due to the conformational rearrangement of the binary E±M complex to an active conformation E  M(



2

apid O 6  E M(

2

O

6 6

 6 2 O

Rearrangement to active conformation: (2 O



6 6

lo    (2 O

6 6m

§m or metalloproteins, the ternary metal bridge complex must be formed by the combination of the substrate with the binary E±M complex ntracell lar enzymes ëm Enzymes, which are synthesized in a particular cell and catalyze the biochemical reaction of the same cell are known as intracellular enzymes ëm dhey are also known as endoenzymes or metabolic enzymes Extracell lar enzymes ëm Enzymes, which are synthesized in a particular cell and are transported to the target site where they catalyze biochemical reactions, are called extracellular enzymes ëm dhey are also known as exoenzymes Àymase and Àymogen orms ëm dhe zymase form is the active form of an enzyme ëm t acts upon the substrate as such; i.e., without undergoing any prior modifications in its structure ëm ntracellular enzymes belong to the class of zymase enzymes ëm dhe zymogen form is the inactive form of an enzyme. dhey are secreted/exist as inactive precursors ëm dhey are also known as proenzymes or pre-proenzymes

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ëm dhey can be converted into the active form (zymase form by modifications in their peptide bonds ëm or example, nterokinase drypsinogen (inactive  drypsin (active drypsin hymotrypsinogen (inactive  hymotrypsin (active

ëm Here, an intestinal enzyme, enterokinase, converts trypsinogen, a proenzyme secreted by the pancreas, into active trypsin. Similarly, chymotrypsin is synthesized by the exocrine cells of the pancreas in its precursor form viz., chymotrypsinogen. Hydrolysis of this form by trypsin converts it into active chymotrypsin ëm Zymogen secretion is a protective mechanism to prevent the digestion of cell walls and ducts, since it is most frequently found in proteolytic enzymes     ëm dhe process of activation of zymogens by their corresponding active enzymes is called autocatalysis ëm or example, inactive pepsinogen is secreted by the gastric mucosa and is converted to active pepsin, both by the acidity of the gastric juice and by pepsin itself ëm During autocatalysis, a polypeptide is liberated from the proenzyme 

 or Pepsin Pepsinogen  Pepsin  A polypeptide

ëm f a graph of time vs. enzyme activity is plotted, a sigmoid curve is obtained

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Monomeric and M ltimeric Enzymes ëm Enzymes which have only one polypeptide chain in their structre are known as monomeric enzymes ëm Examples: RNase, DNG polymerase ,
E
, pyruvate carboxylase ëm When many different enzyme-catalyzing reaction sites are lovated at different sites of the same molecule, it is known as a multimeric or multienzyme complex ëm dhe complex becomes inactive when it is freactionated into smaller units, each bearing individual enzyme activity ëm dhese complexes pass intermediate products along from enzyme-to-enzyme by transfer reactions ëm Examples: atty acid synthetase, C
S ,
DH,
G synthase Enzyme d rnover ëm dhe requirement for the formation of a single intermediate complex can include the reaction of the type:





2 3 +
> >> >1> >   P  +P 1


rovided 


is

the rate limiting step for the overall reaction

ëm dhe constant Kcat is called the turnover number ëm dhis is obtained from the general expression, max = Kcatm[E0] m

 
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m

ëm t represents the maximum number of substrate molecules which can be converted to products per molecule of the enzyme per unit time ëm or simple reactions, Kcat = K2 ëm or more complicated reactions, Kcat will be a function involving several individual rate constants ëm or a simple reaction, 0 =

K 2 [E 0 ] [S] K m [S]

but E 0  [E] [S] [ES] 

Km [E] [S]


K   0   cat  [E] [S]  Km  ëm Kcat/Km is the catalytic efficiency ëm G high value indicates that the limiting factor for the overall reaction is the frequency of collisions between the E and S molecules ëm G comparison of Kcat/Km for alternative substrates can be used as a measure of specificity of an enzyme soenzymes ( sozymes) ëm Enzymes which exist in tissues in two or more forms and have the same catalytic activity, but are physically, chemically, immunologically, and electrophoretically distinct are known as isoenzymes or isozymes ëm dhey are present in the serum and tissues of mammals, amphibians, birds, insects, plants and unicellular organisms ëm Examples include isozymes of numerous dehyrogenases, several oxidases, transaminases, phosphatases, transphosphorylases, proteolytic enzymes, aldolases, etc.  O  
   ëm dhey catalyze the same reaction, but can be distinguished by physical methods like electrophoresis or immunological techniques m

 
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m

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m

ëm dhe difference between some isozymes are due to differences in the quaternary structure of the enzyme ëm or example, lactate dehydrogenase (LDH exist in 5 isozymic forms ëm dhe isozymic forms of LDH are tetramers, each made up from 2 tyes of units H and M. dhe molecular weight of active LDH is 130,000. ’nly the tetrameric molecule possesses catalytic activity. dhe subunits are expressed in the following 5 ways:m È    1       2        3   isozyme ubunits
      4       5  

ëm Splitting and reconstructing of LDH± 1 or LDH± 5 produces new isozymes ëm Hence, each consist of a single subunit ëm When a mixture of purified LDH± 1 and LDH± 5 is subjected to splitting and reconstitution, LDH± 2, 3,and 4 are also produced Gctive ite ëm t is also called the catalytic site or the substrate site ëm t is the site at which the substrate binds to an enzyme ëm t consists of specific amino acids or groups, which contributes for specificity of the enzyme for a substrate ëm dhey are involved in the formation and breakage of bonds and are known as the catalytic groups Oommon feat res of t e active site 1. t occupies a relatively small portion of the enzyme molecule ëm Most of the amino acid residue in the enzyme are 6
in contact with the substrate

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ëm Nearly all enzymes are made up of more than 100 amino acid residues, which give them a mass of greater than 10 kD, and a diameter of more than 25 Å ëm ’nly a fraction of the amino acids are involved in the active site formation 2. t is a 3-D entity ëm dhe active site of an enzyme is not a point, a line or a plane ëm t is an intricate 3-D form made up of groups that come from different parts of the linear amino acid sequence ëm Residues far apart in the linear sequence ay interact more strongly than adjacent residues in the sequence ëm or example, in lysozyme, the important groups in the active site are contributed by residues numbered 35, 52,62,63,and 101 in a linear sequence of 129 amino acids 3. Substrates are bound to the enzyme by weak bonds ëm E±S complex have an equilibrium constant that range from 10-2 to 10-8 M, corresponding to free energy of interaction ranging from -3 to -12 kcal/mol ëm dhese values can be compared with strengths of covalent bonds which are below -50 to -110 kcal/mol 4. Gctive sites are clefts or crevices ëm n all enzymes, the substrate are bound to clefts or crevices from which water is usually excluded ëm dhe cleft also has several polar residues that are essential for binding and catalysis ëm dhe nonpolar characteristic regions of the cleft enhances the binding ot the substrate ëm dhe cleft creates a microenvironment in which certain polar residues acquire special properties essential for their catalytic role 5. Specificity of binding depends on precisely defined arrangement of atoms in an active site ëm dhe substrate must have matching shape to fit into the site (ischer¶s model m

 
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ëm dhe active site of some enzymes are not rigid and the active site is thus modified by the binding of the substrate ëm dhe active site has a shape complementary to that of the substrate only after the substrate is bound (Koshland¶s model ëm dhe side chain groups like ±C’’H, ±NH2, ±CH2’H, etc.. serve as catalytic groups in the active site ëm dhe crevice creates a microenvironment in which certain polar residues acquire special properties essential for catalysis ëm dhe following figure illustrates the same:

ëm dhere must be at least 3 different points of interaction between the enzyme and the substrate ëm dhese interactions can have either a binding or a catalytic function ëm Binding sites link to specific groups in the substrate, ensuring that the enzyme and substrate molecules are held in a fixed orientation with respect to each other with the reacting group or groups in the vicinity of catalytic sites ëm Consider the following 3-point interaction:

O



















ëm Here, sites Gƍƍ and Gƍƍƍ might represent binding sites of Rƍƍ and Rƍƍƍ

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ëm Gƍ is the catalytic site for a reaction involving Rƍ ëm dhus, even if Rƍ and Rƍƍ are identical, the asymmetry of the E±S complex means that only Rƍ can react, providing binding site ëm Gƍƍƍ is specific for Rƍƍƍ and Rƍƍ can never undergo reaction as it is not brought to the vicinity of Gƍ site even when Rƍ binds to Gƍƍ site dentification/Mapping of Enzyme¶s Gctive ite d 


  
 
 ëm dhe reversible character of the steps involved in enzyme catalyzed reactions makes the determination of each substrate binding site complex ëm Gt a steady state, a constant amount of E±S complex is known to be present, but if an attempt is made to isolate this complex, the effort will be futile because the substrate will dissociate from the enzyme ëm However, if the E±S complex can be trapped in a modified form by some chemical process so that the substrate is no longer able to dissociate from the enzyme, then it may be possible to identify the substrate binding site ëm Consider the following example: Fructose bisphosphate aldolase

AP  lyceraldehyde 3 phosphate
> >> >> >> >> >> >> >> >> >> >> >>> Fructose 1,6 bisphosphate

ëm Horecker et al., (1962 showed that if the reaction mixture was treated with sodium borohydride an inactive complex is formed ëm Upon hydrolysis, ±N±glyceryl lysine was found among the products ëm rom this, it was concluded that DHG
normally binds to the side chain amino group of a lys residue in the enzyme by a Schiff¶s base (±N=CH± linkage ëm dhe substrate in the binding site could be found as follows:

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ëm n general, once an E±S complex has been trapped as an inactive complex, it may be subjected to partial hydrolysis and amino acid sequence on each side of the binding site determined ëm Hence the substrate binding site may be precisely located u
   
   
 ëm Gn alternate way of producing a more stable complex is to replace the natural substrate by an analogue which binds to the same site of the enzyme but is then less rapidly removed ëm or example, the first step in the hydrolysis of nitrophenyl acetate and other acyl esters by chymotrypsin is the rapid splitting of the ester to yield the first product and forms an acyl enzyme ëm dhe subsequent liberation of the acyl group from the enzyme is extremely slow, allowing the structure to be investigated ëm dhe acyl group binds by an ester linkage to the ’H group of serine in the enzyme ëm Enzymes form even stronger linkages with irreversible inhibitors ëm dhus, D
 binds to serine residue at the active site in chymotrypsin

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ëm
artial hydrolysis of each enzyme±inhibitor (E± complex gives a series of peptide fragments which can be separated from each other and analyzed ëm dhe amino acid sequence of any fragment containing D
 must be the primary structure of a part of the active site ëm n this way, it is shown that the amino acid sequence around the essential serine residue of chymotrypsin is ±Gly±Gsp±Gly±Gly±
ro and the serine residue is identified as ser-195. Gn identical sequence is found in chymotrypsin ëm dhe assumption that an irreversible inhibitor binds to the active site of an enzyme is particularly valid when the inhibitor resembles a substrate ëm or example, tosyl (M-toluene sulphonyl L-phenylalanine chloromethyl ketone (d
CK resembles esters which are hydrolyzed by chymotrypsin, but d
CK itself acts as an irreversible inhibitor of this enzyme by alkylating the His-57 residue

ëm Both ser and his are present at the active site ëm Binding of d
CK to the enzyme brings reactive groups to close proximity to his-57 residue and facilitate the formation of a covalent bond     
 
 Gmino acid Enz-GG n m
er His Chymotrypsin ëm His-57 ëm His-199, 12 Cys
apain Cys-25

m

{eagent

pecific {eaction

d
CK odoacetate

Glkylation of histidine

odoacetamide M-ethyl maleimide
CMB

Glkylation nhibition nactivation

 
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Ser Met

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Chymotrypsin Ser-195

D


D
enzyme (inactivation

Chymotrypsin Met-192


hotooxidation

Sulphoxide product

odination or nitration of benzene J-bromophenacyl bromide

nactivation

Carboxypeptidase dyr-248

Gsp and Glu
epsin -carboxylic Lysozyme amino acid Lys RNase Lys 1-e41 drp Lysozyme

Gminomethane sulphonic acid DNB

nactivation by ester linkage with Gsp residue Loss of enzyme activity


hotooxidation


   
  

 ëm f an enzyme is modified by the conversion of a particular amino acid chain to a different form (e.g., by reaction of an irreversible inhibitor and this modification results in a loss of catalytic activity, then it is possible that the amino acid concerned is a component of the active site of the enzyme ëm However, the loss of activity is due to a change in the tertiary structure resulting from a modification to an amino acid residue not present at the active site ëm dhe 2 processes may be distinguished by attempting to carry out the same modification in the presence of excess amount of the substrate or by removing the excess amounts of inhibitor******** Mec anism of Enzyme Gction ëm Maud Menten has proposed a hypothesis on enzyme action which is most acceptable

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ëm Gccording to this hypothesis, the enzyme molecule E first combines with a substrate S to form the ES complex which further dissociates to give the product and the free enzyme ëm Enzyme once dissociated is free to associate with another substrate to form the product

E  S
> >> > ES 
 E  ëm dhe ES complex is an intermediate complex and the bonds involved are weak, non-covalent bonds like hydrogen bonds, ander Waal¶s forces, and hydrophobic interactions ëm Sometimes, 2 substrates can bind to an enzyme and such reactions are known as bisubstrate reactions ëm Most reactions in the biological system are bisubstrate reactions: G  B
> >> >
 

ëm dhese reactions entail the transfer of functional groups such as ammonium group from one substrate to another ëm n oxidation±reduction reactions, electrons are transferred between substrates ëm Bisubstrate reactions are divided into 2 classes viz., §m Sequential displacement reactions (single displacement reactions 8m ’rdered sequential mechanisms (’SM 8m Random sequential mechanism (RSM §m Double displacement reactions (ping-pong reactions

eq ential displacement reactions (single displacement reactions) E  S1
> >> > ES1 ES1  S 2
> >> > ES1S2
> >> > E
2 
1 E
2
> >> > E  
2 ëm Gll the substrates must bind to the enzyme before any product is released ëm G ternary complex of the enzyme and both the substrate forms

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ëm Sequential mechanisms are of 2 types: ëm ’rdered sequential mechanism (’SM §m Enzymes that have NGD or NGDH as a substrate exhibit ’SM  §m LDH reduces pyruvate to lactate, while oxidizing NGDH to NGD §m dhe coenzyme always binds first, and the lactate is always released first §m dhe following is the Cleland notation of ’SM

( A  (Pyruvate
> >> > ( actate ( A 

ëm Random sequential mechanism (RSM §m dhe order of addition of substrates and release of products is random §m t can be illustrated by the formation of phosphocreatine and GD
from Gd
and creatine, catalyzed by creatine kinase §m dhe following is the Cleland notation of the aforementioned reaction

E(Creatine (Gd

> >> > E(
hosphocreatine (GD


§m dhe ternary complexes are formed in this mechanism also ©o
le ©isplacement {eactions (Ping-Pong {eactions) ëm ’ne or more products are released before all the products are released before all substrates bind to the enzyme ëm dhe most important feature is the existence of a substituted enzyme intermediate, in which the enzyme is temporarily modified

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ëm or example, reactions that shuttle amino groups between amino acids and keto acids ëm Gspartate aminotransferase catalyzes the transfer of an amino group from Gsp to -ketoglutarate

ëm Gfter Gsp binds to the enzyme, the enzyme removes aspartate¶s amino group to form the substituted enzyme intermediate ëm dhe first product ’GG departs ëm dhen, the second substrate, -KG binds to the enzyme, accepts the amino group from the modified enzyme, and is then released as the final product viz., glutamine ormation of t e E Oomplex ëm dhe site of formation of the ES complex upon the enzyme is known as the active site ëm t is made up of several amino acids, that come together as a folding of the 2Y and 3Y structure of enzymes ëm So, the active site possess a complete 3-D structure and forms a cleft to accept the substrate ëm ormation of the ES complex has been described by 2 models §m Lock and key model (demplate model by ischer §m nduced fit model by Koshland ëm dhese 2 models are elucidated as follows:    
 
 ëm dhis model was proposed by ischer which states that the V

     
6J J V6  
66
666
 6V 
 

 ëm dhe active site provides a rigid, pre-shaped template, fitting with the shape and size of the substrate

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ëm dhe substrate fits into the enzyme as a key fits into a lock; hence this model is known as the lock and key model ëm dhis model proposes that the substrate binds with a rigid, preexisting site on the enzyme

ëm However, this theory cannot explain the change in the enzyme activity in the presence of allosteric modulators 
  
 ëm Due to the restrictive nature of the lock and key model, another model was proposed by Koshland, known as the induced fit model ëm dhe important feature of this model is the flexibility of the active site ëm Gccording to this, the enzyme doesn¶t possess a rigid, preformed structure of active site to fit in the substrate, but during its binding with the substrate, the substrate induces a conformational change in the active site of the enzyme to attain the final shape for binding ëm Consequently, the enzyme molecule is made to fit completely for the configuration and active centers of the substrate ëm Gt the same time, other amino acid residues may become buried in the interior of the molecule

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ëm dhis model explains several characteristics like: §m Enzymes become inactive on denaturation §m Saturation kinetics §m Competitive inhibition §m Gllosteric modulations

ENÀ ME NOMENO
Gd
{E ëm dhe nomenclature of the enzyme is done as follows: §m dhe first part of the name gives the name of the substrate §m dhe second part indicates the type of reaction catalyzed §m dhe third part is the suffix ³  indicating that it is an enzyme. However, some enzymes do not have this suffix (e.g., trypsin, pepsin, diastase, papain, etc.. ëm Example: GLC’H’L DEHYDR’GENGSE n this enzyme, alcohol is substrate, the type of reaction is dehydrogenation and the suffix ³  is places at the end ëm  
 
 


 §m Carbohydrate Carbohydrases §m
roteins
roteases m

 
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§m Lipids Lipases §m Nucleic acids Nucleases d
 
   
 §m HydrolysisHydrolases §m ’xidation’xidases §m dransaminationdransaminase §m somerization somerases §m DehydrogenationDehydrogenases §m
hosphorylation
hosphorylases  
 
   
 
   
 §m Some enzymes give clue of both the substrates utilized and the type of reaction catalyzed §m Examples: 8m L-glutamate dehydrogenase indicates an enzyme catalyzing a dehydrogenation reaction involving L-glutamic acid 8m Succinate dehydrogenase catalyzes the dehydrogenation of the substrate succinic acid  
   

 §m G few enzymes are named by adding the suffix ³-ase to the name of the substance synthesized viz., rhodonase that irreversibily form hydrocyanic acid and sodium thiosulphate O
      


 §m Based on their chemical composition, enzymes are classified into three categories: 8m Enzyme molecule consisting of protein only. Example: pepsin, trypsin, urease, papain, amylase, etc. 8m Enzyme molecule containing a protein and a cation. Example:  2 carbonic anhydrase (Zn as cation , arginase (Mn2 , tyrosinase  (Cu2 , etc. 8m Enzyme molecules containing a protein and a non-protein organic compound known as prosthetic group. dauber (1950 has subdivided them on the basis of the nature of prosthetic group involved as follows: ëm e porphyrin enzymes: Catalase, cyt V peroxidase , 

 
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ëm lavoprotein enzymes: Gly oxidase, histamine ëm Diphosphothiamin enzymes:
yruvate mutase, carboxylase ëm Enzymes requiring other coenzymes:
hosphorylase, amino acid decarboxylase ëm  
  
 
   
 §m Carbohydrate hydrolyzing enzyme 8m Glycosidases: Cellulase, amylase, sucrose, lactase, maltase 8m -glucuronidase §m
rotein hydrolyzing enzymes 8m
eptide bonds ëm Endopeptidases §m Gnimals
epsin, trypsin, rennin §m
lants
apain, ficin, bromalin ëm Exopeptidases §m Dipeptidase, tripeptidase 8m Non-peptids C±N linkages (Gmidases  ëm Urease, arginase, glutaminase §m Lipid hydrolyzing enzymes 8m Lipases, esterases, lecithinases §m ’ther ester hydrolyzing enzymes 8m
hosphatases, cholinesterases, chlorophyllases, sulphatases, pectinesterases, methylases §m ’xidation±reduction enzymes 8m Hydrases, mutases, oxidases, dehydrogenases, peroxidases §m ’ther miscellaneous enzymes 8m Catalase, carboxylases, carbonic anhydrase, thiaminase, transpeptidase

O
G  OGd ON O ENÀ ME ëm Enzymes are broadly classified into 6 classes, according to the type of reaction catalyzed by them

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ëm dhe 6 classes are: §m ’xidoreductases §m Lyases §m dransferases §m somerases §m Ligases §m Hydrolases ëm Each class is further subdivided into various subclasses and each subclass is divided into various sub-subclasses depending on several criteria involving the catalytic reaction ëm n each sub-subclass a number of enzymes are enrolled, each with their serial number ëm Every enzyme has been given an enzyme code (commission number in 4 numbers, serially standing for class, subclass, sub-subclass, and serial number ëm or example:

’ 

 ëm Enzymes involved in oxidation and reduction reactions are known as oxidoreductases ëm ’xidation is carried out by the removal of electrons from a specific group of substrate or by the addition of oxygen to the specific group ëm Reduction is the opposite change ëm dhis class is divided into subclasses, according to electron donor or acceptor of the substrate ëm or example: §m 1.1.1.1  Glcohol dehydrogenase §m 1.1.2.7  Lactate dehydrogenase §m 1.2.3.4  ’xalate oxidase

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 ëm dhey catalyze reactions of the type G  B
> >> > B  G ëm Enzymes of this class transfer a particular group from one substrate to the other ëm dhese enzymes are further divided into subclass according to the specific group transferred by them ëm or example: §m 2.1.2.1  Glycine hydroxymethyltransferase §m 2.2.1.1  dransketolase §m 2.2.1.2  dransaldolase

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 ëm Enzymes which catalyze cleavage of bonds by the addition of water are called hydrolases ëm dhey catalyze reactions of the type: A X   2 O
> >> > X O  A ëm dhey have different classes according to the bond hydrolyzed ëm or example: §m 3.1.1.1  Carboxyesterase §m 3.1.1.2  Gryl esterase §m 3.2.1.1  -amylase §m 3.1.3.1  GL


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 ëm Lyases cleave a covalent bond of the substrate to convert it into more than one product, but the reaction does not involve any hydrolysis ëm dheir action frequently produce a double bond in one of the product ëm dhey are divided into subclasses according to the atom connected to the bond ëm dhey generally catalyze the breaking of C±C, C±S, and C±N bonds ëm Examples: §m 4.1.1.1 
yruvate decarboxylase §m 4.1.1.22  Histidine decarboxylase §m 4.3.2.1  Grginosuccinate lyase m

 
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ëm somerases convert their substrates to their isomers by intramolecular rearrangement ëm dhey are divided into subclasses according to the type of isomerization ëm Examples: §m 5.1.1.1  Glanine raecimase §m 5.3.1.9  Glucose 6-phosphate isomerase (or phosphohexose isomerase §m 5.4.2.1 
hosphoglycerate mutase

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 ëm Ligases are enzymes that catalyze the formation of a bond between 2 substrates ëm dhey catalyze the formation of bonds between C, ’, N, and S coupled to hydrolysis of high energy compounds like Gd
ëm dhey catalyze reactions of the type:  Y Gd

> >> > 6 Y  GD

i or  Y Gd

> >> > 6 Y  GM


i

ëm dhey are subdivided into classes according to the atom connected by the new bond ëm Examples: §m 6.1.1.1  dyrosine t-RNG ligase §m 6.1.1.2  dryptophan t-RNG ligase §m 6.1.1.3  dhreonine t-RNG ligase

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