Rates of Reaction- Hydrolysis of Urea by Urease

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The Effects of Temperature and pH on the Hydrolysis of Urea by Urease

By Justine Hyu

Abstract: The relationship and effect of both temperature and pH, on the enzyme urease was investigated. This was accomplished by initiating the hydrolysis of urea by urease in different variables in order to show changing enzyme activity. Several theories which involved the optimum conditions of urease were explored during the experiment, and in effect were highly involved with the modelling of the experiment. Many expected results were obtained, some of which applied to the researched theories. However, although this experiment was functional there, overall, improvements and adjustments could be made to enhance accuracy.

Aim:  The aim of this experiment is to investigate the optimum temperature and pH of the enzyme urease, and to show the diverse effects of both variables.

Hypothesis:  As the temperature increases, urease enzyme activity will rapidly increase until reaching its maximum potential of 50˚C, where the rate of reaction is at a peak. After this point the enzyme will denature and become inefficient. Urease will be less active in acidic and basic environments but will work most efficiently in more neutral surroundings; meaning that urease will be less active in a solution of a low pH and high pH, but at a neutral pH of 7 it will function at its best.

Introduction and Background Information:

An enzyme is a biological catalyst which speeds up the rate of reaction without being consumed in the reaction; without being changed or broken down in any way. Enzymes are extremely necessary in life as they speed up reactions to up to 10 billion times. They are made of proteins which are organic molecules made from amino acids and are involved with all the chemical activity in a cell. Each enzyme is specific and catalyses one type of reaction. This is due to the ‘lock and key’ theory where only specific substances are able to bind to the enzyme’s active site. However a chemical bond is not formed between the enzyme’s active site and the substrate, so after the reaction has occurred the product is released and the enzyme remains unchanged. Another theory is ‘induced fit’ where the enzyme and substrate interact with each other and the enzyme changes the shape of the active site to fit the substrate. There are factors which affect the rate of the enzyme reaction, such as temperature, pH, and amount of the substrate and enzyme concentration. Each enzyme has an optimum temperature and pH, at which the enzyme works most efficiently and the reaction time is minimal. These optimum conditions, may be, but are not always related to the specific environment it is found in.

Temperature and pH are extremely important factors to consider as they both have a substantial effect on the efficiency and rate of reaction of enzymes. As temperature increases, molecules of the substrate and enzyme begin to move more rapidly and therefore, there is an improved chance of the molecules of substrate randomly colliding with the enzyme’s active site. As a result enzyme activity; and therefore, the rate of reactions are also increased. The temperature, at which point the enzyme activity is at its peak, is called the optimum temperature. After this temperature is exceeded, enzyme activity will decrease and become inefficient. This is due to a denaturing of, or in other words a deformation of the enzyme’s active site. As enzyme activity is very much based on the ‘lock and key’ function, the substrate cannot ‘fit’ properly with the enzyme and so activity is reduced.

An enzyme is similarly affected by the pH. However this is more specifically related to the environment a particular enzyme is found in. Some enzymes prefer an acidic environment; others prefer a basic or neutral environment. Similarly, after the pH of the environment exceeds the optimum pH, the enzyme will also begin to denature. However when the enzyme begins to denature due to a change in pH, this is likely to be caused by the alteration of the ionization of particular organic groups of the amino acids, and therefore, hydrogen bonding within the enzyme may change; causing the active site to change.

‘pH’ simply means ‘power of hydrogen’, and is a measurement of the concentration of hydrogen ions H⁺ in a solution. It is usually on a scale of 0 to 14 but a negative pH is possible. A pH of 0 to 6 is considered as acidic and has a high concentration of hydrogen ions H⁺ and a low concentration of hydroxide ions OH¯, a pH of 7 is neither acidic nor basic and is considered neutral as the concentration of H⁺ and OH¯ ions are the same. Basic solutions have a higher concentration of OH¯ ions than H⁺ ions and cover a range of pH from 8 to 14. In order to make a solution more acidic hydrochloric acid HCl is used as it contains H⁺ ions and to make a solution more basic sodium hydroxide NaOH is used as it contains OH¯ ions. To achieve a neutral pH7, equal amounts of HCl and NaOH are combined, or distilled water can be utilised.

The investigated enzyme is urease, a protein catalyst; an enzyme found in soil and many plants (particularly abundant in jack bean and soy bean). The active site of urease is a crystalline structure which has a hydroxide group (OH¯) which bridges two water molecules (H₂O), and most importantly contains two nickel ions (Ni) which are necessary for the reaction to occur.  A picture of the enzyme’s active site 1.1 can be found in the appendix

Urease lowers the activation energy required to trigger the hydrolysis, or the decomposition of a substrate called urea. Urea is a common soluble organic compound CO(NH₂)₂ often found in some moulds and fungi, and plants, and is common in all soils. It is also a product of mammalian metabolism, where the liver converts ammonia into urea and secretes it out of the body through urine. Urea is usually abundant in soil, and is a part of an important process known as ammonification, which is a necessary natural process important for vegetation. Ammonification occurs when the enzyme urease catalyses the hydrolysis of urea, producing carbamic acid and ammonia which then spontaneously decomposes into the final product of ammonia NH₃ and carbon dioxide CO₂.

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Below is a balanced chemical equation of the ammonification of urea, and in the appendix 1.2 is a diagram of the reaction and how the enzyme urease functions.

 The hydrolysis of urea is instantaneous important as it releases ammonia and carbon dioxide. Carbon dioxide is important for the photosynthesis of plants, which supplies oxygen back into the environment. Ammonia is essential as it is a main source of Nitrogen for vegetation. Nitrogen provides energy, protein and nutrients for vegetation and therefore influences how plants take form and function within. Urea is slightly acidic but ...

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