7.3.         Co-factor and Co-enzyme


Co-factors are mostly non protein organic inorganic chemical, compound or metalic ion that is required for catalysis of the reaction. Co-factors helps in catalysis of reactions. So they are also know as "Helper molecules". Co-factors binds with enzyme and help during the catalysis of reactions. Without cofactor binding, enzyme is known as Apoenzyme, after binding of co-factor with apoenzyme it is known as Holoenzyme, which is the active form of an enzyme. Mostly co-factors are metal ions and co-factors bind at active site of the enzyme if it is not available to be bound the active site, function does not occur. Co-factors are devided into coenzymes and prosthetic groups and prosthetic groups refer to tightly bound. Co-factor as metal ions are trace elements such as Fe+3, Manganese (Mn+2), Cobalt (CO+2), Copper (CU+2), Zinc (Zn+2), Selemiun (Se+2), and Molybdenum (MO+5)

Co-enzymes :

Coenzymes are small, organic, non-protein molecules that assist the catalysis of reactions. Coenzymes are loosely bound to the enzyme and play role in group transfer seactions such as ATP and coenzyme A and oxidation-reduction reaction, such as NAD+ and coenzyme Q10. Most of the coenzymes are derivatives of vitamins.          

Enzyme Cofactor

Some inorganic elements that serve as cofactors for enzymes


7.4.         Nomenclature and classification of enzymes

Various systems have been evolved to name and classify the enzymes over the period of times.

  1. On the basis of substrate, Duclaux (1883) named the enzymes by adding the suffix -ase in the name of the substrate catalyzed. For example, enzymes acting upon carbohydrates were named as carbohydrases, upon proteins as proteinases, upon lipids as lipases, upon nucleic acids as nucleases. Lactase (acting upon lactose) maltase (acting upon maltose), sucrase (upon sucrose), etc.
  2. Enzymes nomenculture is also based upon the type of reaction catalysed. Hydrolases (catalyzing hydrolysis), isomerases (isomerization), oxidases (oxidation), dehydrogenases (dehydrogenation), transaminases (transamination), transaldolases (transaldolation), transketolases (transketolation), phosphorylases (phosphorylation) etc.
  3. Enzymes nomenculture  based upon substrate acted upon and type of reaction catalyzed. The names of some enzymes give clue of both the substrate utilized and the type of reaction catalyzed. For example, the enzyme succinic dehydrogenase catalyzes the dehydrogenation of the substrate succinic acid.
  4. Enzymes nomenculture  based upon substance that is synthesized. Fumarase that forms fumarate  from  malate. Hexokinase and Glucokinase are different enzymes working on same substrate and carried out same type of reaction.

To address the unambiguity and uniformity in identification of enzymes a nomenclature system based on type of chemical reaction and there mechanicm is given by International Union of Biochemistry (IUB).

The major features of this system of classification of enzymes are as follows :

  • Enzymes are grouped into six major classes each with 4 to 13 subclasses.
  • Each enzyme name has 2 parts—the first part is the name of the substrate(s) and the second part which ends in the suffix -ase, indicates the type of reaction catalyzed.
  • Additional information regarding the nature of the reaction, if needed, is given in parenthesis. For example, the enzyme malate dehydrogenase catalyzes the following reaction :

L-malate + NAD+  \rightarrow    Pyruvate + CO2 + NADH + H+

This enzyme has now been designated as L-malate : NAD oxidoreductase (decarboxylation).

  • In the IUB system, each enzyme has a unique name and code number that reflect the type of reaction catalyzed and the substrates involved. The enzyme code (EC) number having the four digits characterized the enzymes. The four digits characterized the class, sub-class, sub-sub class and serial no of a particular enzyme. The first numbers Indicate the major class to which the enzyme belongs. The second number indicates the type of grouped involved. The third number indicates the reaction more precisely indicating substrates on which the group acts. The fourth number indicates serial number of the enzyme.

Thus E.C. represents class 2 (a transferase), subclass 7 (transfer of phosphate), sub-subclass 1 (an alcohol group as phosphate acceptor). The final digit denotes the enzyme, hexokinase or ATP: D-hexose-6-phosphotransferase. This enzyme catalyzes the transfer of phosphate from ATP to the hydroxyl group on carbon 6 of glucose.


ATP + D-Hexose    \rightarrow    ADP + Hexose-6-phosphate

Oxidoreductases (Redox)

Oxidation-reduction reactions are very common in biochemical pathways and are catalyzed by enzymes called oxidoreductases. In oxidation-reduction reaction  one substrate gains electrons and becomes reduced, and another substrate loses electrons and becomes oxidized. Example is dehydrogenase, hydroxylases and oxidases. Most hydroxylases and oxidases require metal ions, such as Fe+2, for electron transfer.

Ared + Box   ---------------\rightarrow    A ox + Bred


Transferases transfer a functional group from one molecule to another. If the transferred group is a high-energy phosphate, the enzyme is a kinase; if the transferred group is a carbohydrate residue, the enzyme is a glycosyltransferase; if it is a fatty acyl group, the enzyme is an acyltransferase. A common feature of these reactions is that the group being transferred exists as a good leaving group on the donor molecule. Another subset of group transfer reactions consists of transaminations.

A-B + C  ---------------\rightarrow   A + B-C


In hydrolysis reactions, C-O, C-N, or C-S bonds are cleaved by the addition of H2O in the form of OH and H to the atoms forming the bond. The enzyme class names specify the group being cleaved (e.g., the enzyme commonly named chymotrypsin is a protease, a hydrolase that cleaves peptide bonds in proteins).

A-B + H2O  ---------------\rightarrow   A-H + B-OH


The lyase class of enzymes cleaving C-C, C-O, and C-N bonds by means other than hydrolysis or oxidation. Some of the enzymes catalyzing C-C bond cleavage are called aldolases, decarboxylase, thiolases. dehydratases and many synthases.

A=B + HX ---------------\rightarrow  A-X + B-H


Enzymes rearranging the bond structure of a compound are called isomerases, whereas enzymes catalyzing movement of a phosphate from one atom to another are called mutases.

AB ---------------\rightarrow  BA


Ligases synthesize C-C, C-S, C-O, and C-N bonds in reactions coupled to the cleavage of a high-energy phosphate bond in ATP or another nucleotide.

A+B + ATP ---------------\rightarrow A-B + ADP

Example are Carboxylases and synthetases

7.5.         Characteristics of enzymes

The enzymes possess many outstanding characteristics. These are enumerated below :

  1. Colloidal Nature : Enzyme are made up of proteins and proteins are  macromolecule. Thus enzyme molecules are of giant size as compared to their substrate. Because of the size the rate of diffusion of enzyme is low and  exist in colloidal form. Being colloidal in nature, the enzymes are nondialyzable although some contain dialyzable or dissociable component in the form of coenzyme.
  2. Catalytic Nature or Effectiveness :  Enzymes are highly effective. An universal feature of all enzymatic reactions is the virtual absence of any side products.
  3. Specificity of Enzyme Action : Enzymes are highly specific for both substrate as well as the reaction they catalyze. With few exceptions, the enzymes are highly specific in their action. Their specificity lies in the fact that they may act 
  • on one specific type of substrate molecule or
  • on a group of structurally-related compounds or
  • on only one of the two optical isomers of a compound or (d) on only one of the two geometrical isomers.

7.6.         Measures of enzyme activity    

Enzyme assay are laboratory methods to calculate the enzymatic activity under given condition. Enzyme activity is a measures of the quantity of active enzyme present in solution.

(i)   Specific Activity : Specific Activity of an enzymes is activity an enzyme per milligram of total protein. It is expressed as \mu mole of substrate converted into product per minute per milligram of enzyme under optimum condition (\mu mol min–1 mg–1). It is a measurement of purity of an enzyme in mixture of protein.

(ii)  Turn over number or Kcat is the number of substrate molecule converted into product per enzyme molecule per second.

Mechanism of an enzyme reaction :-

1.            Covalent Catalysis

A nucleophile (electron-rich group with a strong tendency to donate electrons to an electron-deficient nucleus) on the enzyme displaces a leaving group on the substrate. The enzyme-substrate bond is then hydrolyzed to form product and free enzyme.

2.            Acid-base Catalysis

e.g. Lysozyme cleaves the glycosidic bond between C1 of N-acetylmuramic acid (NAM)  and C4 of Nacteylglucosamine (NAG) of bacterial cell wall polysaccharides. Glu35 of lysozyme donates a proton to the oxygen of the polysaccharide glycosidic bond thereby hydrolyzing the bond.

3.            Proximity

An enzyme may bind with two reactants and doing so, increase their proximity. Reaction rate is related to the number of collisions of correct orientation. When an enzyme binds with its substrates it insures that their orientation is precisely that required for reactivity.

4.            Molecular distortion

The enzyme active site undergoes a conformational change upon binding with substrate, distorting the substrate into a conformation resembling the transition state species.

7.7.         Mechanism of enzymes action -

According to Michaelis and Menten enzymes hypothesis, enzyme molecules bind to substrates to form enzyme-substrate complex with a rate constant k and further dissociated to form product and enzyme back. After the dissociation physical and chemical properties of enzymes remain unchanged.

A simple enzymatic reaction can be written as

E + S  \leftrightharpoons ES  \leftrightharpoons  EP  \leftrightharpoons   E + S

Where E, S, and P represent the enzyme, substrate, and product; ES and EP are transient complexes of the enzyme with the substrate and with the product.

There are few terms related to enzyme catalyzed reaction

a-            Active site is the site of the enzyme where the substrate binds. After binding form enzyme substrate complex is formed. Enzyme substrate complex involves weak non covalent interaction such as hydrogen bond, hydrophobic interactions, ionic interactions and vanderwaals forces.

b-            The ground state and the excited state – the ground state is the condition of the substrate when it is not able to convert into product and when energy is provided it comes in excited state and converted into products

c-            Activation energy - Minimum energy required to excite the substrate and convert into product is called as activation energy. The larger the activation energy, the slower the reaction will be. This is because only a few substrate molecules have sufficient energy to overcome the activation energy barrier. Most biological reactions have large activation energy, so they without enzymes they happen far too slowly to be useful. Enzymes catalyzed the reactions by lowering the activation energy.

7.8.         Mechanism of lowering of activation energy by enzyme

Now LET'S TALK about how enzymes lower the activation energy. There are a number of mechanisms by which this activation energy of catalyzed reaction is  decreased. The enzyme binds with substrate in correct orientation, bring the substrates closer to each other. In this way the binding energy between enzyme and substrate is used to reduce the activation energy. After binding with substrate/substrates enzymes bring them in proper orientation and formed product.

d-            Transition state – It is the most unstable state of substrate and it has highest level of energy which is needed to break the bond for a particular reaction without enzymes and is able to convert into products.

An energy diagram helps to understand what is meant by "lowering the activation energy.  An activation energy is shown much less than the energy required for achieving the unanalyzed transition state.

7.9.         Enzyme substrate complex models

7.9.1.     Lock and key model : It was proposed by Emil Fischer, according to this model each enzyme have a rigid active site which is specific for a substrate. Substrate fits into the active site of an enzyme as the key fits into the lock, thus called as lock and key model.

7.9.2.     Induced fit model : It was proposed by Koshland, according to this model active site of enzyme are flexible they can be change according to the shape of the substrate that is active site does not possess a rigid structure.

7.10.      Factors that Affect the Rate of Enzyme Reactions

7.10.1.   Temperature:

Enzymes function optimally at a particular temperatures. As temperature increases, kinetic energy increases and collision increase and more molecules now have sufficient energy to overcome the activation energy. The enzyme reaction rate increases with an increase in temperature but as the temperature rises above optimum temperature, the denaturation of enzyme starts. Once it is denatured, the enzyme’s three dimensional structure is lost. The enzyme’s shape changes, therefore the three dimensional shape of its active site changes as it cannot bind to the substrate anymore and the enzyme cannot function furthermore. Therefore, at higher temperatures the enzyme’s reaction rate decreases.

The optimal temperature for an enzyme is the temperature at which the enzyme “works best” and the rate of chemical reaction is highest. The “optimal temperature” for most of the enzymes in the body is 98.6 degrees F (also known as ~37 degrees C).

The increase in rate with temperature can be quantified as the Q10, which is the relative increase for a 10°C rise in temperature. Q10 is usually around two for enzyme-catalysed reactions (i.e. the rate doubles every 10°C) and usually less than two for non-enzyme reactions.

7.10.2.   Effect of pH

Enzymes function optimally at a certain pH. They are extremely sensitive to changes in acidity and works within quite narrow pH range. Changes in pH can make new bonds with substrate and break the existing bond within the enzyme. This leads to change the shape of the enzyme. If the pH is too low (too acidic) or too high (too basic), the enzyme becomes denatured. Enzymes is made up of amino acid. The charge on protein depends upon pH. Change in pH change the charge on enzyme which affects its binding with substrate and also changes the existing three dimensional structure of enzyme. The chemical bonds within the enzyme are rearranged. As the enzyme’s shape changes, the three dimensional shape of its active site changes and the active site cannot bind to the substrate anymore. Thus, the enzyme cannot function anymore. The “optimal pH” for most of the enzymes in the body is ~pH7.4. However there are exceptions, such as the digestive enzymes of human stomach function at pH of 3-4.

7.10.3.   Effect of enzyme concentration

When substrate [S] is not limiting then under these conditions an increase in enzyme [E] has a direct effect on the rate of reaction. A plot of rate of reaction [v] versus [E] results a straight line.

7.10.4.   Substrate concentration :

The rate of reaction is also depended upon substrate concentration. However it depends upon the total number of active site present on enzyme. At a particular enzyme concentration increase in the substrate concentration increase the rate of reaction in the begining but once all the active site of a given enzymes is saturated by substrate there is no further increase in the rate of reaction. The maximum rate of reaction is known as Vmax. i.e. the rate of reaction is saturated as there is no change in the rate of reaction on further increase in substrate concentration. The Vmax is directly proportional to the total enzyme concentration (E).

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