Understanding the mechanisms that control the rates enzymatic reactions

Aim of the experiment: Understanding the mechanisms that control the rates enzymatic reactions.

Introduction

Enzymes are biological catalyst and as catalysts they have the function of speeding or lowering the rate of a biological reaction. However, enzymes are considered to be specific in nature since every catalyst has a specific reaction in the body that it affects. There are a number of factors that affects the operation of a catalyst. These factors include temperature, substrate concentration and the PH of the surrounding where the catalysis is taking place. Additionally, the concentration of the enzyme as well as the presence of inhibitors affects the activity of an enzyme. In this experiment the various factors that affect enzymes are studied and their effect to the rate of activity of an enzyme ascertained. The experiment focuses on oxidative reaction.

Biological oxidative reaction catalyzed by an enzyme

Oxidation reaction is an important reaction to living things, this is from the fact that it facilitates the breakdown of food to produce energy with other side reaction products such as CO_2. The enzyme that catalysis oxidative reaction is referred to as oxidative enzyme, with the most common type of enzyme catalyzing this type of reaction in the body being Peroxidases which utilizes hydrogen peroxide to perform the oxidation and oxidases which uses the molecular oxygen to achieve canalization.

The variable in the experiment

PH- This refers to the basicity or acidity of a substance. The pH of the environment where the enzyme is catalyzing the reaction is essential because it determines the effectiveness of the enzyme. It is evident that a change in pH above or below the optimum pH, results to the decrease in the rate of the reaction. This is from the fact that alters the shape of the active sites where substrates are attached for canalization of an experiment.
Concentration of the enzyme: Increasing the concentration of catalyst especially increases the rate of activity of a substance up to a certain level where the rate of the reaction begins to fall again. This is from the fact that all the substrates will be consumed.
Concentration of the substrate: increase in the concentration of the substrate increases the rate of the enzyme catalyzed reaction. However, when it reaches a certain point the reaction falls. The fall in the rate of the reaction can be attributed to the fact that all the active sites of the enzyme will be occupied by the substrate thus the rate remains constant.
Temperature: temperature increases the kinetic energy of molecules and particles. Due to this the collusion of the substrates and the enzyme increases thus increasing the rate of the reaction. Increasing the temperature increases the rate of a reaction but when temperature exists the optimum temperature the rate of reaction quickly reduces because the substrate becomes denatured.
Apparatus and reagents

1. 1% Catechol (substrate)
2. Phos Buff pH 6.0
3. Pot extract (enzyme)
4. hydroquinone
5. HCl
6. NaOH
7. ascorbic acid
8. HgCl2
9. Ethanol
10. Phenyl Thiourea
11. Boiling water

Procedure

Variation of enzyme concentration and substrate concentration
A standard reaction mixture is prepared and then followed by the addition of Catechol, Phos Buff and Pot Extract.  This is followed by a controlled experiment, the control experiment does not contain an enzyme and similar reagents that were added to the standard reagent are added to the mixture and observation made. The second control procedure was to use a mixture that has an enzyme but substrate is not present. The same reagents added to the standard mixture and the mixture which had no enzyme are added and an observation made.
Procedure for using alternative substrate
The second procedure involved using alternative substrates that are related to catechol in structure (phenol and hydroquinone).
Variation in the pH of the acid and the base
To a standard mixture the following reagents Catechol, Phos Buff and Pot Extract are added then followed by the addition of 3 M HCL and 3 M NAOH to a different solution containing the reagents.
The environment where the reaction takes place
To alter the environment of the solution mercury chloride and ascorbic acid are introduced into the solution. Ascorbic acid uses up oxygen in the environment and mercury is a poison to the enzyme.
Modification of the enzyme
Modification of the enzyme is achieved by pretreatment of the enzyme by adding Ethanol, protease and Phenyl Thoreau and the boiling of water. In the boiling of water the sample enzyme and buffer are boiled for ten minutes before the substrate is added.
While using phenyl Thiourea (chelating agent), a pinch for binding the copper is added and allowed to relax for ten minutes before mixing with the other reagents. Protease is an enzyme that hydrolyzes the peptide bonds. After it is added it is left to relax for ten minutes just as for the case of Adding Ethanol.

Observation

The observations tabulated during the experiment are shown in the table below
Part DSubstrate Concentration verses Enzyme Reactivity

Rate of enzyme reactivity	0	0.25	0.8	1.0	2.6	4.0
Substrate concentration	0.355	0.45	0.55	0.65	0.75	0.85

Analysis and discussion

For standard reaction Mixture
From the graphical illustration, the absorbance increases with an increase in time. At optimum level, the rate of reaction is high; then it reduces as time increases beyond the optimum level.
When the experiment was carried out without the action of an enzyme, the absorbance observed was lower as compared to the standard reaction. The resultant solution was clear. There was a steady increase in absorbance as time elapsed after 8 minutes, and the absorbance showed a substantial decrease.
For control experiment with no enzyme
When no enzyme is in action, reaction rate took place normally but with a minimum absorbance.
When the experiment was carried out without substrate, the rate of absorbance was lower as compared to when the reaction took place in the absence of an enzyme. The resulting solution was clear. The absorbance rate increased as time elapsed.
Control experiment with no substrate
With no substrate, the slope of the graph was at its minimum showing that reaction rate was lower, although it increased with an increase in time. The rate of absorbance also tends to increase to an optimum level where the reaction rate is higher, and then it starts reducing thereafter.
Alternative substrates:  These compounds are similar to catechol in structure.
The solution turned clear when phenol was used and reddish when hydroquinone was applied.

Alteration of the enzyme by the addition of alcohol

The graph shows that when 3 ethanol was added, the absorbance increased. In other words, the rate of enzyme reactivity increased due to the presence of a catalyst.
When protease was added to the reaction, the color of the solution became clear showing that oxidation took place. The rate of the reaction also increased steadily to an optimum level, after which it dropped to the level below 1.
By the addition of protease
Phenyl Thiourea is a well-known chelating agent. A pinch was introduced to bind up the copper. The experiment was then allowed for 10 minutes.
The pinch reduced the rate of reaction as it bound up the copper. It oxidized the solution to form a clear coloration. Again, the biological reaction reached its optimum after which there was a steep decrease as observed in the table below.
Part D: substrate concentration verses the enzyme reactivity
The trend from the line graph shows that there is an increase in the rate of an enzyme reactivity following an increase in substrate concentration. From the beginning, there are few substrate but a lot of active enzymes. When the concentration of substrate increases, more enzymes are required to facilitate the rate of biological reaction. Since more enzymes are utilized in the reaction, the rate of chemical reaction adds up. At some points, almost all the enzymes facilitate the reactions. In the process, a fraction of the substrate must wait for enzymes to attach on their active sites. An active site is the point where an enzyme attaches itself to the substrate. After some time, the graph will level up meaning the additional substrates must wait before they are acted upon by the enzymes.
Discussion and conclussion
In the experiment, when no enzymes are present, the chemical reaction will take place but at a slower rate. Enzymes are the body catalysts that speed up or slow down chemical reactions in living organisms.
When the experiment was conducted in the absence of a substrate, the reaction again was slower indicating that substrate concentration speeded up the rate of biological reactions. When added to a chemical reaction, substrate provides an active site where the enzymes can attach themselves to it, thereby increasing general reaction speed.
Phenol and hydroquinone can be used as catalysts for enhancing oxidation which results into color changes in chemical reactions. In the presence of a base, the absorbent rate was higher as compared to acidic medium. In the presence of an acid, the solution turned clear showing that oxidation took place. In sodium hydroxide, there was a yellow coloration. High rate of enzyme concentration leads to high rate of biological reaction as more active sites are utilized. The rate of reaction remains high until all the enzymes are used up. During the biological reaction, the kinetic energy of the molecules increases gradually following an increase in substrate concentration. The final point is reached when there is no further increase in kinetic energy.
As the reaction takes place, it requires time for enzymes to work entirely on the chemical reaction. It is a gradual process for an enzyme to attach itself to the substrate surface. After some period of time, the rate of reaction increases because all enzymes become involved in a chemical process.
When substrate concentration is high, more enzymes are required to facilitate the rate of the biological reaction. Since more enzymes are utilized in the reaction, the rate of chemical reaction shoots. At some points, all the enzymes facilitate the reactions. In the process, a fraction of the substrate must wait for unused enzyme to attach on its active site.
Other factors that affects the rate of enzyme activity include temperature, enzyme concentration, and specificity. At optimum temperature, normally 37⁰C, the enzyme reaction is  high. As temperature goes beyond the optimum range, enzymes are denatured and they will no longer be able to facilitate reactions. When temperature is way below the optimum level, enzymes are inactive; therefore, they are unable to work on body reactions.
The t-test and the correlation co-efficient showed that the relationship between the substrate concentration and the enzymatic reaction is strong.
The actions of ezymes can also be boosted by additing ethanol. The process enables activation of enzymes through opening up their active cites.
In the above experiment, some graphs do not show perfect curves as a resultant inaccuracy of some laboratory measuring tools. To deal with such abnormalities, statistical estimates were used to help in minimising errors. An example of the mathematical approach was used to show reactivity rate and substrate concentration.

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