EFFECT OF METAL IONS ON THE ACTIVITY OF ACID PHOSPHATASE EXTRACTED FROM POTATOES

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Abstract

          Acid phosphatase was extracted from potatoes. The purification was not carried out due to lack of the required chemicals and apparatus. However, in the extracted material enough activity of the enzyme was present to carry out the necessary experiments.
          Extract used in 10µl volume gave a measurable activity. Substrate use was para-nitrophenyl phosphatase (PNPP) in concentration ranging from 0-5mM. The Km value obtained was 0.24mM and Vmax value was 0.36 absorbance units.
          With Mg2+, Ca2+, Hg2+, F1- and EDTA enzyme was shown to be inhibited with no change in Km value while the Vmax value changed.

This indicate a noncompetitive mode of inhibition of acid phosphatase extract from potatoes by these elements or metals.

Introduction

DEFINITION OF ENZYME

          “A biomolecule that catalyzes a specific chemical reaction is called an anzym” 
          Ithas does not affect the equillbrium of the catalysed reaction.
          It enhances the rate of reaction by providing a reaction path with a lower activation energy.
          Enzymes are central to every biochemical process. Acting in organized sequences as they catalyze the hundreds of stepwise reactions by which nutrient molecules are degraded, chemical energy is conserved and transformed, and biological macromolecules are made from simple precursors.
MOST ENZYMES ARE PROTEINS
          All enzymes are proteins, with the exception of a small group of catalytic RNA molecule.
          Their catalytic activity depends on the integrity of their native protein conformation. If an enzyme is denatured or dissociate into subunits, catalytic activity is usually lost.
          If an enzyme is denatured or, broken down into its component amino-acids its catalytic activity is always destroyed. Thus the primary, secondary, tertiary and quaternary structures of protein enzymes are essential to their catalytic activity.
          Enzymes like other proteins have molecular weights ranging about 12,000 to over 1 million.
CAFACTOR,
Some enzymes require an additional chemical component for their activity , these are called co-factor. It actually acts as enzyme,s chemical teeth.
COENZYME
          Cofactors may be metal ions such as  Fe2+ , Mg2+, Mn2+or Zn2+. Zn2+ is required for catalytic activity of carboxy - peptidase A.
          Cofactor may be an organic molecules such as NAD+. These metal ions and organic molecules are called as coenzyme.
PROSTHETIC GROUP
          A coenzyme or metal ion that is very tightly or even covalently bound to the enzyme is called a prosthetic group.
HOLOENZYME
          A complete, catalytically active enzyme together with its bound coenzyme and metal ions is called a holoenzyme.
APOENZYME
          The protein part of such an enzyme is called the apoenzyme or apoprotein.
ENZYME ACTS AS CATALYST
          The function of catalyst is to increase the rate of a reaction. Catalyst do not affect reaction equilibria.
          Enzyme s also act as catalyst. In living system uncatalyzed reaction tend to be slow. The reaction which is catalyzed by an enzyme  occurs in the active site of an enzyme.
          The molecule that is bound in the active site of the enzyme is called the substrate. The surface of the active site is lined with amino-acid residues whose substituent groups bind the substrate and catalyze its chemical transformation.
A simple enzymatic reaction might be written as

Where
          E= Enzyme
          S= Substrate
          P= Product
          Any reaction, such as EP, can be described by a reaction coordinate diagram which indicate the energy changes during the reaction.....
Enzymes lower the energy of activation. and enhance the rate of reaction. The enzyme not use up in the process. When the S P reaction is catalyzed by an enzyme, the ES and EP complexes act as intermediates.
CLASSIFICATION OF ENZYMES
          Many enzymes have been suffix “-ase” to the name of their substrate or to a word or phrase describing their activity.
          For example urease catalyzes hydrolysis of urea, DNA polymerase catalyzes the polymerization of nucleotides to form DNA.
Other enzymes, such as pepsin and trypsin have names that do not denote their substrates or reaction.
          Sometimes the same enzyme has two or more names or two different enzymes have the same name. Because of such ambiguities and the every-increasig number of newly discovered enzymes, as system for naming and calssifying enzymes have been adopted by international agreement. This system divides enzymes into six major classes, each with subclasses, based on the type of reaction catalyzed.
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 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 than 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.
ENZYME SPECIFICITY
          It is the ability of enzyme to discriminate between a substrate and a competing molecule. Specificity is easy to distinguish from catalysis, but this distiniction is much more difficult ot make experimentally because catalysis and specificity arise from the same phenomenone. The enzymes have stereospecificity and geomatric specificity.
STEREOSPECIFICITY
          Enzymes are highly specific both in binding chiral substrates and in catalyzing their reaction. This stereo-specificity arises because enzymes, by virtue of their inherent chirality, form asymmetric active sites.
          For example trypsin readily hydrolyzes polypeptides composed of  L-amino acids but not those consisting of  D-amino acids. Likewise , the enzymes involved with glucose metabolism are specific for D-glucose residues.
          Enzymes are absolutely stereospecific in the reactions they catalyze.
GEOMATRIC SPECIFICITY
          Enzymes are quite selective about the identities of the chemical groups on their substrates. It is called geomatric specificity of the enzyme.
          Indeed geomatric specificity is a more stringent requirement than the stereospecificity[1].
ENZYME KINETICS
          The rate of reaction and how it changes in response to changes in experimental parameters is known as enzyme kinetics. This is the oldest approach to understanding enzyme mechanism and remains the most important to day.
SUBSTRATE CONCENTRATION
          The concentration of substrate effects the reaction catalyzed by and enzyme.
          To study the effect of substrate concentration is complicated by the fact that [S] changes during the coarse of a reaction as it is converted to product.
          One simplifying approach in kinetics experiment is to measure the initial rate Vo, when [S] is generally much greater than the concentration of enzyme. Then if the time is sufficiently short following the start of reaction changes in [S] are negligible and [S] can be regarded as a constant.

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