A complete Enzyme Info : Nomenclature, Enzyme Commission, Co-enzyme, Mechanism, Application

Enzymes

Enzyme

Definition of Enzyme

It is defined as a biocatalyst synthesized by living cells. they are proteins in nature (exception RNA acting as ribozyme), colloidal & thermolabile in character, & specific in their action. OR

The enzyme is a remarkable & highly specialized protein.

Enzymes are catalyzed by biological systems & are often much more efficient as compared to synthetic catalysts.


History of enzyme

Catalyst (the Greek word means to dissolve) → Introduced by Berzelius (1836)

  • Kühne (1878) used the enzyme (the Greek word means- yeast)
  • In 1883- Buchner isolated of enzyme system from the cell-free extract of yeast.
  • In 1926, Iames Sumner- 1st achieved the isolation & crystallization of the enzyme. urease jack beam & identified it as a protein.

Nomenclature

  • Suffix ‘ase’ is added to substrate enzyme are comes under 2 categories.
  • 1)Intracellular enzyme- which functions within the cell.
  • 2)Extracellular enzyme- acting outside the cell eg. Digestive enzyme
  • IUB:- International union of biochemistry applied
  •  EC: Enzyme commission in 1961.

Enzyme Commission  (EC)

  • This class is divided into many classes.
  • 4 digit EC number is assigned to each enzyme. representing 1st class 1st digit, subclass. 2nd digit. subclass- 3rd digit, sub-sub-sub class 4th digit.
  • 2,3 digit describes reaction 4th digit use to distinguish between differentiation enzymes of some function
  1. Alcohol dehydrogenase

eg. Alcohol, NAD+ Oxidorectase E.C 1.1.1.1

1)Hexokinase

ATO:b-hexose 6- phosphotransferase E.C 2.7.1.1


Classification of Enzymes

In 1964 IUB classified enzymes into 6 classes

enzyme classification


Co-factor

The catalytic activity of many enzymes depends on the presence of small, non-protein molecules called co-factor.

Co-factor is non-coating molecules that carry out a chemical reaction.

Types

  • Organic co-factor
  • Inorganic

1)Organic co-factor

These are organic molecules.

Glycogen phosphorylase requires the small organic molecule pyridoxine phosphate.

Types:-

a)Prosthetic group:-

Tightly bound with the organic cofactor.

eg. flavins, heme group.

b)Co-enzymes:-

Co-factor that are small organic molecules are called co-enzymes.

Co-enzymes can be defined as non-protein, organic, low molecular weight substances associated with enzyme activity.

eg. NAD+, NADP, FAD, FMN

NAD contains Niacin, FAD contains riboflavin.

Co-enzyme derived from vitamin.

It acts by donating or accepting H atoms of e-

Co-enzyme that only transiently associate with enzyme is called co-substrate.

2)In-organic co-factor

These are inorganic molecules required for the proper activity of enzymes.

eg. Carbonic anhydrase requires Zn2+ for ita activity on eg metabolic ion cofactor

Hexokinase has cofactor Mg2+


Co-enzyme

Silent features of co-enzyme

They are heat-stable & low mol. weight substances.

They combine loosely with enzyme molecules, separated easily by dialysis.

When the reaction is completed co-enzyme released from the apoenzyme goes to some other reaction site.

According to their participation in the reaction

Chemical characteristic

1)Co- enzyme-containing aromatic heteroatom eg: ATP

2) Co-enzyme containing non-aromatic heteroatom eg: Biotin

3) No-heteroatom eg: sugar phosphate

4)Based on nutritional characteristics. eg. co-enzyme A, NAD, NADP.


3)Function of co-enzyme

  • Biological activity of co-enzyme
  • Co-enzyme is a low mol. wt organic sub without which the enzyme.cannot exhibit any chemical reaction.
  • One molecule of co-enzyme able to comment large no. of substrate molecule with help of any.
  • NAD (Nicotinamide adenine dinucleotide)
  • This is a co-enzyme synthesis from nicotinamide a member of the Vit B complex.
  • The reversible reaction of lactate to pyruvate is catalyzed by the enzyme. lactate hydrogenase but the actual transfer of H is taking place on co-enzyme NAD+ eg. ATP.
  • ATP is considered to be the energy currency in the body during the oxidation of food, stuff, energy is released apart.

Haloenzyme

Active enzyme with its non-protein component.

Apoenzyme

Inactive enzyme without its non-protein past.

Active site

  • Enzymes are big in size compared to substrates which are relatively smaller.
  • Evidently, a small portion of the huge enzyme molecule is directly involved in the substance the substrate-binding & catalysis.
  • The active site (active center) of an enzyme. represents as the small region at which the substrate (s) binds & participate in the catalysis.

Salient features of the active site

  • The existence of an active site is due to the tertiary structure of protein resulting in 3-dimensional native conformation.
  • The active site is made up of amino acids (known as catalytic residues) which are far from each other in the linear sequence of the amino acids (primary structure of the protein) for instance, the enzyme lysozyme has 129 amino acids. the active site formed by the contribution of amino acid residues numbered 35,52,62,63&101.
  • Active sites are regarded as cleft or crevices or pockets occupying a small region in big enzyme molecules.
  • The active site is not rigid in structure & shape. it is rather flexible to promote the specific substrate binding.
  • Generally, the active site possesses a substrate-binding site & a catalytic site the latter is for the catalysis of the specific reaction.
  • The co-enzyme or co-factor on which some enzyme depends are present as a part of the catalytic site.
  • The substrate (s) binds to the active site by weak non-covalent bonds.
  • Enzymes are specific in their function due to the existence of active sites.
  • The commonly found amino acids at the active sites are serine, aspartate, histidine, cysteine, lysine, arginine, glutamate, tyrosine, etc.
  • Among these amino acids, serine is the most frequently found.
  • The substrate [s] binds the enzyme [E] at the active site to form an enzyme-substrate complex the product [P] is released after the catalysis & the enzyme is available for reuse
  • E +  S → ES → E + P

Mechanism of enzyme action

  • Catalysis is the prime function of enzymes the nature of catalysis taking place in the biological system is, similar to that of non-biological catalysis.
  • For any chemical reaction to occur the reactant has to be in an activated state or transition state.

Enzyme lower activation energy

  • The energy required by the reactants to undergo the reaction is known as activation energy.
  • The reactant when heated attain the activation energy. the catalyst (or the enzyme in the biological system) reduces the activation energy & this caused the reaction to proceed at a low temperature.
  • The enzyme does not alter the equilibrium constant, they only enhance the velocity of the reaction.
  • The role of catalyst or enzyme is comparable with a tunnel made in the mountain to reduce the barrier as illustrated in below fig.

Enzyme-Substrate complex formation

  • The prime requisite for enzyme catalysis is that the substrate (s) must combine with an enzyme at the active site to form an enzyme-substrate (ES) complex. which ultimately results in product formation.

E + S ↔ ES →E + P

  • A few theories have been put forth to explain the mechanism of ES complex formation.

Lock & Key model : (Fischer’s template theory)

  • This theory was proposed by German biochemist Emil Fischer.
  • This is in fact the 1st model proposed to explain an enzyme-catalyzed reaction.
  • According to this model, the structure or conformation of the enzyme is rigid
  • The substrate fits the binding site (active site) just as a key fits into the proper lock or a hand into the proper glove.
  • Thus the active site of an enzyme is a rigid & pre-shaped template where only a specific substrate can bind.
  • This model does not give any scope for the flexible nature of enzymes, hence the model totally fails to explain many facts of enzymatic reaction.
  • The most important being the effect of allosteric modulators.

Induced fit theory (Koshland’s model)

  • Koshland, in 19158 proposed a more acceptable & realistic model for enzyme substance complex formation.
  • As per this model the active site is not rigid & pre-shaped.
  • The essential features of the substrate-binding site are present at the nascent active site.
  • The interaction of the substrate with the enzyme. induces a fit or conformation changes in enzyme resulting in the formation of a strong substrate binding site.
  • Further due to induced fit. the appropriate amino acid of the enzyme is repositioned to form the active site & brings about catalysis.
  • The induced-fit model has sufficient experiment evidence from x-ray diffraction studies Koshland’s model also explains the action of allosteric modulators & competitive inhibition on enzymes.

Substrate strain theory

  • In this model, the substrate is strained due to the induced conformation change in the enzyme.
  • It is also possible that when the substrate binds to the preformed active site, the enzyme induced a strain on the substrate.

Mechanism of enzyme catalysis

  • The formation of an ES complex is very crucial for the catalysis to occur & for product formation.
  • It is estimated that an enzyme-catalyzed reaction proceeds 100 to 1012 times faster than a non-catalyzed reaction.
  • The enhancement in the rate of reaction is mainly due to 4 processes.
    • i.acid base catalysis
    • ii.substrate strain
    • iii.covalent catalysis
    • iv.entropy effect

Acid-base catalysis

  • Mostly undertaken by oxidoreductase enzyme.
  • Mostly at the reactive site, histidine is present which acts as both proton donor & proton acceptor.

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 β-1,4 glycosidic bonds) action is believed to be due to a combination of substrate strain & acid-base catalysis.

Covalent catalysis

  • Enzyme form covalent linkages with substrate forming transition.ES complex with very low activation energy.
  • The enzyme is released unaltered after the completion of the reaction.

Entropy effect

  • Entropy is a term used in thermodynamic.
  • It is defined as the extent of disorder in a system.
  • The enzyme brings about  ↓ the entropy of the reactants.
  • This enables the reactant to come closer to the enzyme & thus ↑ the rate of reaction.
  • In the actual catalysis of the enzyme, more than one of the processes, acid-base catalysis, substrate stain, covalent catalysis & entropy are simultaneously operative.
  • This will help the substrate (s) to attain & transition state leading to the formation of products.

Thermodynamic changes in the mechanism of enzyme action

  • All chemical reactions have energy barriers between reactions & products.
  • The difference in transitional state & substrate is called the activational barrier.
  • Only a few substances cross the activational barriers & change into products, that is why the rate of uncatalyzed reaction is much low.
  • Enzymes provide an alternate pathway of conversion of substrate into product.
  • Enzyme accelerates reaction rate by forming transitional state having low activational energy.
  • Hence reaction rate is ↑ many folds in the presence of the enzyme.
  • The total energy of the system remains the same & the equilibrium state is not distributed.

Michaelis – menten constant (km)

  • The enzyme (E) & substrate (s) combine with each other to form an unstable ES complex & the formation of the product.

  • Here,

K1, K2, K3 = Represents velocity constant or rate constant for the respective reaction as indicated by the arrow.

Km= Michaelis-Menten constant or Bring’s & Haldane’s constant

Km=(k2+K3)/k1

The following equation was obtained after suitable algebraic manipulation.

V=(Vmax [S])/(Km+[S])…………(1)

where,

V= measured velocity

Vmax= maximum velocity

S= substrate conc.

Km= michaelis -menten constant

Let us assume the measure velocity Vo is equal to 1/2 Vmax, than equation (1) may be substituted as,

1⁄2  Vmax=  (Vmax [S])/(Km+[S])

Km +[S]=  (2Vmax [S])/Vmax

Km + s = 2 [S]

Km = S

  • In these case K stands for constant ‘m’ stand for michaelis menten
  • Km is defined as substrate conc express in mohr’s (moles/l) to produced half maximum velocity in an enzyme catalysed reaction.
  • It is a representative for measuring these strength of ES complex.
  • Low Km value indicates strong affinity between enzyme & substrate.
  • High Km value indicates weak affinity between enzyme & substrate.
  • For majority of enzyme Km value are in the range of KM=10-5 to 10-2 moles.
  • Km value is not depend upon concentration of enzyme.
  • It indicate that half of the enzyme molecules i.e. 50% are bound with sub.molecules when sub.concentration equal to Km value.

K1,K2,K3 = velocity constant/rate constant for respective reaction.

Km= michaelis-menten constant

Km=(k2+K3)/k1

the following equation obtained after suitable algebric manipulation

Vo=(Vmax [S])/(Km+[S])…………(1)

Vo= measured velocity

Vmax= maximum velocity

S= substrate conc

let assume the major velocity Vo=1/2 Vmax,

the equation (1) may be substituted as,

1⁄2  Vmax=  (Vmax [S])/(Km+[S])

Km +[S]=  (2Vmax [S])/Vmax

Km + [S] = 2[s]

Km= 2S – S

Km = S mohr’s (moles/1)

Km is a constant & characteristic feature of a given enzyme.

It is representative for measuring the strength of the ES complex.

Double -reciprocal plot

For the determination of Km value, the substrate saturation curve is not very accurate since Vmax is approached asymptotically.

By taking the reciprocal of equation (1) a straight line graphic representation is obtained.

Vo= Vmax [S]/ Km + [S]…….(1)

1/Vo= Km +[S]/Vmax [S]

1/Vo= Km/ Vmax [S] + [S]/Vmax [S]

1/Vo= Km/ Vmax[S] + 1/ Vmax

1/Vo = Km/Vmax × 1/[S] + 1/Vmax……(2)

this equation compare with y= mx + c

C= 1/Vmax, y=1/Vo, m= Km/Vmax, X= 1/s

m= slope, c=y intercept

equation (2) is similar to straight line equation

y= mx + c

where m= slope

m= Km/Vmax

Therefore, the graph is plotted between reciprocal of velocity 1/Vo versus reciprocal of substrate concentration.


Factors affecting enzyme activity.

  1. Concentration of enzyme
  2. concentration of substrate
  3. effect of temperature
  4. effect of PH
  5. effect of product concentration
  6. effect of activators
  7. effect of time
  8. effect of light & radiation

Concentration of enzyme

  • If the concentration of enzyme increased then the rate of reaction increased.
  • as the concentration of enzyme increased velocity of reaction proportionally.
  • These properties of the enzyme are used in determining serum of enzyme for diagnosis of disease.

The concentration of substrate

  • Increased substrate concentration gradually increased the velocity of enzyme reaction.
  • The rectangular hyperbola is obtained when velocity is plotted against substrate concentration.
  • 3 distinct phases of reaction are observed in the graph.
  1. Linear graph
  2. Curve
  3. Almost unseen

Km & double reciprocal plot are points included in it.

coenzyme can be defined as non-protein, organic, low molecular weight substances associated with enzyme activity

eg. NAD+

Concentration of enzyme

  • ↑ then the rate of reaction ↑
  • The concentration of enzyme ↑, the velocity of reaction ↑
  •  These properties of an enzyme are used to determine serum of enzyme for diagnosis of disease.

The concentration of substrate

  • ↑ than ↑ the velocity of reaction
  • Rectangular hyperbola is obtained velocity is plotted against substrate concentration.
  • 3 distinct phases of reaction are observed in the graph
  • Linear graph
  • Curve
  • Almost unseen
  • Km & double reciprocal plot these points including in it.

Effect of temperature

  • ↑ then the velocity of reaction ↑ up to maximum & then declines
  • A bell shape curve is observed.
  • ↑ in temperature results in higher activation energy of the molecules & more molecular substrate collision & interaction for the reaction proceed faster.
  • The optimum temperature for most of the enzymes is between 35º – 45º c.
  • Some enzymes like venom phosphokinase active at 100ºc & plant enzyme urease are active at 60ºc.
  • Their activity is due to stable structure & conformation.

Effect of pH

  • ↑ in pH influence enzyme activity
  • Each enzyme has an optimum pH at which velocity maximum.
  • Most enzymes exhibit optimal activity at pH 5 to 9.
  • Optimum value causes ionization of enzyme which results in denaturation of the enzyme.
  • High & low pH value enzyme activity is much less.
  • Extreme pH enzymes become totally inactive.
  • Due to ionization of enzyme which results in denaturation of the enzyme.

Effect of product concentration

  • The accumulation of reaction products ↓ the enzyme activity.
  • For certain enzyme, the product combines with the active site of enzyme & form loose complex & thus inhibit the enzyme activity.

Effect of activators

  • ↑ enzyme activity
  • Two categories of enzyme activators
  • 1. metal activated enzyme- metal not held tightly eg. ATPase, Enolax.
  • 2.metalloenzymes- metals are held tightly eg. alcohol, dehydrogenase.

Effect of time

  • The time required for an enzyme reaction  is less
  • Variation in time of reaction are generally related to alteration in pH & temp

Effect of light & radiations

  • Exposure of enzymes to UV, β, gamma, x-ray inactivate certain enzymes due to the formation of peroxides.
  • UV rays inhibit salivary amylase activity.

 

Reversible Inhibition

  • In this type, inhibitors bind non-covalently with enzymes & the inhibitions can be reversed by removal of inhibitors.
  • Sometimes the effect of reversible inhibitors can also be reversed by ↓ a concentration of inhibitors.

a) Competitive inhibition

  • The inhibitor competes with substrate & binds at the active site of the enzyme but does not undergo any catalysis.
  • The inhibitors (I) closely resemble the real substrate (s) are regarded as substrate analogs.
  • In these competitive inhibition ES & EI complex forms.

E + S ⇔ ES → E + P

E + I ⇔ EI

  • The main feature of these initial is that they can be overcome by ↑ the concentration of substrate.

  • In competitive inhibition, an intercept of 1/v is the same in the presence & absence of inhibitors.
  • But the slope of lines differ.
  • This suggests that Vmax is not altered by competitive inhibition & Km value ↑
  • The slope of the line inactivates the strength of binding competitive inhibitors.
Non-competitive inhibition
  • The inhibitor binds to enzyme & enzyme-substrate complex. this is also called mixed inhibition.
  • This type of inhibitor has no structural similarity to a substrate.
  •  Inhibitor bind at the site other than active site on the enzyme surface.
  • This binding impairs the enzyme function.
  • Usually exist a strong affinity for inhibitors to binds at the second site.
  • Enzyme catalysis is prevented due to distortion in the enzyme conformation.
  • E + S ⇔ ES → E + P
  • E + I + S ⇔ EI + S
  • ES + I ⇔ ESI

Irreversible inhibition
  • Inhibitor bind covalently with enzyme & inactive them
  • It is irreversible
  • Inhibitors are toxic, poisonous sub eg. Iodoacetate, which is an irreversible inhibitor of the enzyme papain, glyceraldehyde. 3 p-dehydrogenase.
  • Iodoacetate combines with sulphydryl group at the active site & inactive them.
  • Penicillin act as irreversible inhibitors of enzyme in bacterial cell wall synthesis.
Allosteric inhibition
  • In these in which enzymes contain an additional site known as an allosteric site.
  • In greek Allo means other beside the active side.
  • Such enzyme is known as allosteric enzyme & the type of inhibition is allosteric inhibition.
  • The allosteric enzyme binds with allosteric inhibitor & binds to the allosteric site, inhibit enzyme function.

Marker enzyme

  • Marker enzymes are not found everywhere but are confined to specific types of organelle subcomponents or cells.
  • Their detection can indicate the presence of a source whereas absence means lack of that source.
  • Marker enzymes are one of the cell biomarkers use to characterize a cell type.
  • They are also used in the isolation of target cellular components.
  • Eg. succinate dehydrogenase is a marker enzyme for mitochondria where as acid phosphatase.
  • use: Diagnosis-
  • 1. Cardiac disease
  • 2.Muscle disease
  • 3.Bone disease
  • 4.Hepatic disease

Application of enzyme

  1. Enzymes are therapeutic agents
  2. Making lactose-free production for patients suffering from lactose in tolerance (lactose breaks to glucose & galactose.
  3. To remove the toxic substances.
  4. In preparation of medicines
  5. Streptokinase prepared from streptococcus useful for dissolving blood clots.
  6. Streptokinase activates plasma, plasminogen to plasmin, plasmin attacks fibrin to convert into soluble products.
  7. Tumour cells depend on the aspargine of hosts plasma for their multiplication.
  8. Asparginase administration reduces aspargine.
  9. Enzymes are analytical reagent some enzyme is useful in clinical laboratory or measurement of substrate drugs & activity of other enzymes.
  10. Biomolecules compounds for example glucose, urea., uric acid, cholesterol can be estimated by enzymatic procedures.
  11. eg. Estimation of plasma glucose by glucose oxidase & peroxidase method.

Enzyme induction & repression

  • A process in which a molecule (eg a drug) induces (i.e. initiates or enhances) the expression of an enzyme.
  • An enzyme inducer is a type of drug which binds to an enzyme and increases its metabolic activity.
  • Regulated by exposure to the drug and environmental chemicals leading to an increased rate of metabolism eg. Iac operon.
  • Enzymes that are susceptible to induction are said to be inducible.
  • Enzymes that are susceptible to induction are said to be inducible.
  • Enzyme induction can increase the metabolic clearance of a concomitantly administered drug, resulting in reduced efficacy, which may comprise may comprise the therapeutic effectiveness of a drug
  • Effectors can associates with the operator & alter the configuration so that the binding of the polymerase occurs less efficiently or not at all. this effect is known as repression.
  • Feedback inhibition
  • Prevens wasting of energy.

Importance of enzyme induction

  • Essential to understanding various reactions that occur inside the body.
  • Important to analyze drug reactions.
  • Study toxicity.
  • Causes less or over production of hormones.

Usefulness of enzyme induction

  • Certain inducers have been used therapeutically for hyperbilirubinemia in children.
  • Some inducers stimulate the conversion of drugs into more polar metabolites.
  • Inducer molecule merges with repressor- induction.
  • Repressor binds to operator-repression.r