STUDY ON KINETICS OF ACID PHOSPHATASE EXTRACTED FROM POTATOES

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Abstract

          Acid phosphatase was extracted form potatoes due to back of required chemical and apparatus the purification was hot carried out. However in the extracted material enough activity of the enzyme was present to carry out the necessary experiments 10μl extract was used which gave a measurable activity. P-nitrophenylphosphate (pNPP) was used as a substrate whose concentration ranging from
0 to 5mM.  The km value obtained was 0.28mM and Vmax value was 0.91 absorbance unit.
          With Ni, Cu, Cd, Zn enzyme was shown to be inhibited with a change in km value from 0.25mM to 0.28mM.
This indicates a competitive mode of inhibition of acid phosphatase extracted from potatoes by these elements or metals.



Introduction

Enzymes:
An enzyme is a protein that is synthesized in a living cell and which is capable of catalyzing a specific chemical reaction to yield a specific product.
Most enzymes are proteins:
With the exception of a small group of catalytic RNA molecule, all enzymes are proteins. There catalytic activity depends on the integrity of their native protein confermation.if an enzyme is denatured or dissociation into subunits, catalytic activity is usually lost.
Enzymes like other proteins have molecular weights ranging from about 12,000 to over 1 million Daltons. Some enzyme require no chemical groups for activity other hen their amino acid resides other requires on addition chemical component called as cofactor.
Cofactor:
"One or more inorganic ions, such as Fe, Mg, Mn, or Zn which is required for enzymetic activity is called as cofactor".
Cofactor which essentially acts on the enzymes "chemical teeth".
Coenzymes:
"Complex organic or metalloorganic molecule required for the enzymatic activity is called as coenzyme."
Some enzyme requires both a coenzyme and one or more metal ions for activity. They are often derived from vitamins, organic nutrients required in small amount in the diet.
Prosthetic group:
"A coenzyme or metal ion that is very tightly or even covalently bound to the enzyme protein is called a prosthetic group."
Holoenzyme:
"A complete catalytically active enzyme together with its bound coenzyme and / or metal ion is called a holoenzyme.'
The protein part of such an enzyme is called the apoenzyme or apoprotein.

Enzyme act as catalyst:
Catalyst enhance reaction rate by lowering activation energies.
Enzymes are no exception to the role that catalysts do not affect reaction equilibria. The role of enzyme is to accelerate the interconversion of S and P.  The enzyme is not used up in the process; and the equilibrium point is unaffected. However, the reaction reaches equilibrium much faster when the appropriate enzyme is present because the rate of the reaction is increased.
 There is an energy barrier between S and P, the energy required for the alignment of reacting groups, formation of transient unstable charges, bond rearrangement, and other transformations required for the reaction to proceed in either direction. This is illustrated by "hill" as shown below:

To undergo reaction, the molecule must overcome this barrier and raised to high energy barrier. At the top of this barrier hill is a point at which decay to the S or P state is equally probable. This is called as transition state. The transition state is not chemical specie. The difference between energy levels of the ground state and the transition state is called the Activation energy. High activation energy corresponds to a slower reaction. Reaction rate can e increased by raising the temperature, thereby increasing the number of molecules with sufficient energy to overcome the energy barrier.
Classification of enzymes:
Enzymes are divided into six major classes.
1-Oxidoreductases:
These enzymes catalyze the oxidation and reduction of substances.
2-Transferases:
These enzymes catalyzed the transfer of one carbon carbon group.
3-Hydrolases:
The enzymes catalyze the hydrolysis.
4-Lyases:
These enzymes catalyze the removal of groups from substrate without hydrolysis.
5-Isomerases:
These enzymes catalyze the formation of isomers of the substrate.
6-Ligases:
These enzymes catalyze reactions joining two molecules by forming C=O, C – S , C – N , C – C  bonds.
Enzyme specificity:
Enzymes are very specific in their activity and they react only with the specific substrate. Different types of enzyme specificities are as follows;
1-Absolute specificity:
It includes those enzymes which attack only on a single specific substrate.
2-Group specificity:
It includes those enzymes which act upon a general group of compound.
3-Absolute group specificity:
This type includes those enzymes which attack only on a single substrate of a group.
4-Relative group specificity:
In this type are included those enzymes which attack homologous series of a group.
5-Stereochemical specificity:
This type includes those enzymes which have optical specificity for D or L isomer.
Effects of different factors on enzyme activity:
Enzyme activity is affected by the following factors
(i)       Temperature
(ii)      Inhibitors
(iii)     pH
(iv)     Activators
(v)      Ultraviolet light
Effect of temperature:
The shape of a protein is largely determined by the hydrogen bonding. Temperature changes easily disrupt hydrogen bonds.
Below 35°C the bonds that determine protein shape are not flexible enough to permit the shape changes necessary for substrate to fit into the active site.
When proper shape is lost, the enzyme is, in essence, destroyed; this lose of shape is called as denaturation.
Effect of pH:
Most enzymes have a pH optimum usually between 6 and 8. When pH is low, the hydrogen ions combine with the R groups of the enzyme's active site. Acidic environment can also denature enzyme not adapted to such conditions. The pH optimum of an enzyme is that pH at which the enzyme active site is maximum.
Effect of ultraviolet light:
Ultraviolet light either destroys or modifies the action of enzymes. The rate of destruction is affected by the pH of the medium and is independent of temperature. Rate of destruction is greater for purified enzyme then for impure enzyme.
Effect of activators:
Those enzymes which increase the activity of enzymes are called activators e.g. many enzymes required metal ions for their activation.
Effect of inhibitors:
Substances which decrease the activity of enzyme are called inhibitors e.g. inhibitors of cytochrome oxidase with cyanide[1].
Enzyme kinetics:
Enzyme kinetics is the branch of enzymology which deals with the velocities of reactions.
Kinetic measurements of enzymatically catalyzed reactions are among the most powerful technique for elucidating the catalytic mechanism of enzymes.
Substrate concentration affects the rate of enzyme catalyzed reactions:
During the course of reaction [S] is changing into product. Enzyme kinetics is to measure the initial rate or initial velocity designated Vo, when [S] is generally much greater than the concentration of enzyme [E].
The effect on Vo of varying [S] when enzyme concentration is held constant.

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