Enzymes are biological globular proteins that catalyze the rate of biological reactions. Enzyme kinetics analyzes the rates and factors affecting enzyme activity on substrates. This can be investigated under varying conditions under which the reaction takes place. Factors affecting the rates of the enzymatic reactions include substrates and enzyme concentrations, temperature, pH, and inhibitors. The mechanism of the enzyme activity is that the enzyme binds with the substrate at the active site, forming an enzyme-substrate complex, which later dissociates to form the product and the free enzyme. An enzyme is never used up and does not change the equilibrium level; it only alters the time taken to reach equilibrium. The aim of this experiment is to study the enzyme kinetics of the enzyme pyrophosphatase with acid molybdate. The experiment analyzed the time taken to complete the reaction under different reaction conditions. These conditions, which include temperature, pH and the substrate and enzyme concentration, affect the binding of the substrate to the enzyme and the turnover rate. Inorganic pyrophosphatase, EC 3.6.1.1, catalyzes the hydrolysis of pyrophosphate (PPI) that proceeds as a byproduct in many biochemical syntheses utilizing ATP as the source of energy. The enzyme splits a pyrophosphate molecule to form two phosphate ions in the presence of divalent metal cations and in a highly exergonic reaction. Pyrophosphatase acts as a phosphoryl group donor (Laidler & Bunting, 1973). The enzyme pyrophosphatase has critical roles in the living organism (Tóth et al., 2007; VOLK et al., 1982).
The enzyme plays an essential role in lipid metabolism, DNA metabolism, the formation of bones and neuron growth. Due to its exergonic nature, it is associated with various types of cancers and tumors, such as ovarian, lung and brain cancer. Pyrophosphate acts as a phosphoryl carrier in DNA replication, pyrophosphate is a byproduct of DNA formation to form new DNAs and pyrophosphate (PPI). PPI removes deoxyribonucleosides from DNA to form dNTPs. In fatty acid metabolism, an ATP molecule reacts with fatty acids to form acyl adenylate. Acidified ammonium molybdate is used to quantitatively determine the presence of pyrophosphate in a color reaction. Pyrophosphate is highly reduced by blue molybdous acid and compounds.
Material And Method
In preparation for the standard curve, different phosphate concentrations were used to react with a constant 2.5 ml of molybdate acid for ten minutes for the blue color to form. The intensity of the color was measured at 620 nm. The blank solution was prepared the same way as the reagent solution but lacked phosphate.
In investigating the effect of enzyme concentration, different amounts of pyrophosphate enzyme were added. Magnesium chloride and triethanolamine buffer were used. To investigate the effect of pH, the experiment was carried out under varying pH conditions. Other factors were kept constant, but the buffer solution was not included. To investigate temperature effects, the experiment was carried out at different temperatures while the previous factors were held constant.
Result Presentation
On the Effect of enzyme concentration, after carrying out the experiment and measuring the absorbance, an increasing absorbance with the increasing enzyme concentration over the successive test tubes was noted. Absorbance levels increased with increasing concentration in both sets of experiments. However, data varied slightly between the two sets apart from the 4th test tube. After calculating the rate of reaction with the substrate and enzyme concentration, the rate increased successively from test tube 1 to test tube 5. The graph drawn for the average hydrolysis against the enzyme concentration formed a straight line with linear progression. The trend line was developed after joining the line of best fit and had a positive gradient starting from the origin. There were also anomalous points deviating slightly from the line.
For pH, absorbance increased as the acidic pH increased until an optimum was reached at neutral pH. A further increase into the basic pH range reduced the absorbance steadily. The rate of hydrolysis also increased with increasing pH up to the optimum neutral point, after which a further increase in pH caused a decline in the reaction rate. After a graph of the rate of hydrolysis against pH was drawn, it presented a curve with the optimum rate at the pH 7 mark. There was no trend line developed.
The effect of temperature showed similar results. Absorbance increased with the increase in temperature until an optimum point of 70 degrees was reached. An increase in temperature to 100 degrees reduced the absorbance. Absorbance deviated slightly between the two sets of data. The rate of hydrolysis increased with increasing temperature up to the optimum point but reduced gradually with the increase in temperature. However, the rate at 100 degrees was higher than at 4 degrees Celcius, with both enzymes present at both conditions. The graph drawn with the rate of hydrolysis against pH was optimum at 70 degrees. No trend line developed.
Results
Table 1: Effect on enzyme concentration
Table 2: Effect on ph
Table 3: Effect on temperature
Figure 1
Figure 2: graph on the effect of ph. on enzyme activity
Figure 3: Effect of temperature on the effect of enzyme activity
Discussion
Enzyme activity is affected by the various factors of enzyme concentration, pH and temperature. The amount of enzyme concentration present affects the rate of a catalytic reaction with an increase of the reaction with the increasing substrate concentration and a lowering of the rate with a decrease of enzyme concentration. The enzyme catalyzes the reaction by binding to the substrate’s active site. Increasing the enzyme concentration increases the chances of the enzyme combining with the substrate to form the product and the free enzymes. However, an increase in the enzyme concentration suppressing the substrate concentration can cause a further increase, making the enzyme concentration the limiting factor in the reaction.
Temperature affects the rate of enzyme catalyzing the reaction by interfering with the enzyme. Temperature interferes with the stability of the bonds holding the enzymes together. Temperature also affects the kinetic energy of the reaction molecules, hence affecting the rate of collision and reaction. Different enzymes work best at certain optimum temperatures. An increase in temperature increases the rate of enzymatic reaction until an optimum point, after which the rate lowers. The decrease in temperature also reduces the rate of enzymatic reaction. Since the enzyme is a protein in nature, an increase in temperature beyond the optimum point completely destroys the arrangement and organization of the enzyme structure, hence denaturing it. When temperatures also go below the optimum, it affects the structure of the enzyme, rendering it inactive. However, an increase in temperature will activate the enzyme to normal working.
pH is also a factor affecting the rate of enzymatic reaction. An increase in pH increases the rate of enzymatic reaction until an optimum point is reached, after which a further increase in the pH ionic strength slows the rate of chemical reaction. Each group of enzymes has its optimum pH working condition. Some enzymes work optimally in basic conditions, others in neutral pH, and others in acidic conditions. Ph may affect both the structure and shape of the enzyme and substrate, causing the enzyme not to fit in the active site of the enzyme. This affects the rate of the reaction.
Data from the experiment correspond to the theoretical discussion above. According to Figure 1, enzyme concentration increased with the increase in enzyme concentration. In Figure 2, an increase in pH increased the enzymatic reaction until an optimum pH of 7. According to Figure 3, an increase in temperature increased the rate of chemical reaction until the optimum of 70 degrees. After this, the enzyme becomes denatured, and a further increase in temperature decreases the reaction rate. However, there were some anomalies in the experimental data.
In addressing the anomalies, a more precise method of measurement can be used. Molybdenum compounds highly reduce pyrophosphate compared to the acid itself. The experiment can also be harnessed by the use of modulators, and the effect on substrate concentration and the effect of inhibitors can also be studied.
Conclusion
The experiment was a success in analyzing the effect of pyrophosphatase enzyme with acid molybdenum. The aim of the experiment was to analyze the effect of pyrophosphatase enzymes, and the aim was achieved. Though slight human errors cannot be avoided, the observation of the experiments corresponded well with the expectations.
Appendix
A standard phosphate solution of 1mM /Ml had been prepared, and diluting this solution with different volumes of dilute water resulted in different concentrations of the phosphate solution. Different concentrations can be calculated with the formula.
C1V1=C2V2
| Phosphate volume | Phosphate concentration |
| 0 | 0 |
| 0.20 | 0.2 |
| 0.40 | 0.4 |
| 0.60 | 0.6 |
| 0.80 | 0.8 |
| 1 | 1 |
| Abs 1 | Abs 2 | Average Abs | Range | |
| 0.000 | 0.000 | 0.000 | 0.000 | |
| 0.036 | 0.048 | 0.042 | 0.006 | |
| 0.114 | 0.104 | 0.109 | 0.005 | |
| 0.158 | 0.163 | 0.161 | 0.003 | |
| 0.209 | 0.207 | 0.208 | 0.001 | |
| 0.259 | 0.256 | 0.258 | 0.002 |
The absorbance measured showed different results for the different test tube reactions. The absorbance increased down the tubes for the standard reactions. Data deviated slightly but in a rage of (0-0.002).data was presented in graph and line of best fit formed a straight line, the line developed had a linear equation which can be presented in form of Y=MX+C. The linear equation developed can be presented as shown below.
Y=0.2602x, where y is the x-intercept while x is the gradient.
Figure a: standard curve
Reference
Laidler, K.J. and Bunting, P.S., 1973. The chemical kinetics of enzyme action (Vol. 84). Oxford: Clarendon Press.
Tóth, J., Varga, B., Kovács, M., Málnási-Csizmadia, A., and Vértessy, B.G., 2007. Kinetic mechanism of human dUTPase, an essential nucleotide pyrophosphatase enzyme. Journal of Biological Chemistry, 282(46), pp. 33572-33582.
VOLK, S.E., BAYKOV, A.A., DUZHENKO, V.S. and AVAEVA, S.M., 1982. Kinetic studies on the interactions of two forms of inorganic pyrophosphatase of heart mitochondria with physiological ligands. The FEBS Journal, 125(1), pp. 215-220.
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