Enzymes are naturally occurring biological catalysts (Klein, 2015) that facilitate the conversion of substrates into products (British Journal of Pharmacology, 2011). They are large, mostly globular proteins. Enzymes increase the rate of biochemical reactions, allowing reactions to occur millions of times faster. Biological reactions that, without enzymes, would take years to complete are too slow to support life, so with the aid of enzymes these reactions can be completed within a matter of milliseconds.
Enzymes are highly sensitive to pH and temperature variations. They are also especially sensitive to different substrates due to their extreme specificity (King, Mulligan, Stansfield, 2013). Each enzyme reacts only with a very small number of substrates. This gave rise to the development of the Lock and Key Model, where the substrate fits the enzyme’s active site just like a key would fit a lock. This Model has since been replaced by the more accurate Induced Fit Model, but it still effectively depicts just how extraordinarily specific enzymes are.
Due to this high specificity, most enzymes are named by adding the suffix “-ase” onto the end of the name of it’s specific substrate. For example, lactase is so named because it catalyses the breakdown of lactose.
Most known enzymes have been classified into six different groups, based on the type of reactions that they catalyse. These six groups include oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases (Encyclopedia Britannica, 2016).
Pyrophosphatase is an enzyme (Enzyme Commission Number: 3.6.1.1) that catalyses the virtually irreversible and highly exergonic hydrolysis of pyrophosphate into two orthophosphates (PO43-) or phosphate ions (Cammack et al., 2006). Pyrophosphatase was the enzyme chosen to be studied in this experiment.
Pyrophosphatase is a very important enzyme for energy metabolism. Firstly, pyrophosphate is produced by the hydrolysis of Adenosine 5’-triphosphate (ATP) into Adenosine 5’-monophosphate (AMP) in cells. Pyrophosphatase then catalyses the hydrolysis of pyrophosphate into the two orthophosphates, which results in energy release (Yi, Sutovsky, Kennedy, Sutovsky, 2012).
The overall aim of this experiment is to determine the effects of enzyme concentration, temperature and pH on pyrophosphatase activity, specifically on the rate or velocity of enzymatic hydrolysis.
The materials and methods used in this experiment were obtained from and followed exactly as in the Biochemistry Lab Manual (Ashby and Beard, 2016). However, the only deviation made from the materials and methods outlined in the Biochemistry Lab Manual was that the concentration of the enzyme used was doubled. This will be explained further in the Discussion section below. This experiment delivered some very interesting results. However, it must first be noted that the results of the experiment included in this laboratory report belong to another group who did the same experiment on the same day. The other group conducted the experiment in a slightly different way, and their results turned out to be much better, in terms of being more consistent. This will be explained further in the Discussion section below.
The first part of this experiment was testing the effects that differing pyrophosphatase concentration had on the velocity of hydrolysis. As seen in Figure 1 above and in Figure 7 in the Appendix, it is clear that there is a positive correlation between the concentration of pyrophosphatase and the velocity of hydrolysis. The highest rate of hydrolysis belonged to the highest concentration of pyrophosphatase, with 0.25 U/mL of pyrophosphatase having a mean velocity of hydrolysis of 0.119 μmol/mL/min. Unsurprisingly, the lower rates of hydrolysis belonged to the lower concentrations of pyrophosphatase, with 0 U/mL and 0.05 U/mL of pyrophosphatase having mean velocities of 0.006 μmol/mL/min and 0.018 μmol/mL/min respectively.
The second part of this experiment was testing the effects that differing substrate pH had on the velocity of hydrolysis. As seen in Figure 2 above and in Figure 8 in the Appendix, the trend as substrate pH increased is somewhat bell-curve-like. The highest rate of hydrolysis belonged to the median substrate pH, with the pyrophosphatase in the substrate that had a pH of 7 having a mean velocity of hydrolysis of 0.244 μmol/mL/min. It seemed the further away the substrate was from a pH of 7, the lower the rate of hydrolysis, with the pyrophosphatases in substrates with pHs of 4 and 8.5 had mean velocities of hydrolysis of 0.049 μmol/mL/min and 0.033 μmol/mL/min respectively.
The third and last part of this experiment was testing the effects that differing temperatures had on the velocity of hydrolysis. As seen in Figure 3 above and in Figure 9 in the Appendix, the trend as substrate temperature increased is quite similar to the trend discussed above concerning substrate pH, in that it is almost bell-curve-like. The highest rate of hydrolysis belonged to the median substrate temperature, with the pyrophosphatase in the substrate that had a temperature of 50 °C having a mean velocity of hydrolysis of 1.178 μmol/mL/min. The results also showed that the more extreme the temperature, the lower the rate of hydrolysis, with the pyrophosphatases in substrates with temperatures of 4 °C and 97 °C had mean velocities of hydrolysis of 0.047 μmol/mL/min and 0.049 μmol/mL/min respectively.
The results of this experiment could almost be said to have been predictable because they are “textbook”; they are perfectly consistent with the results found in the academic literature (Castillo, Hicks, Payne, Lopez, 2000). Of course, it was the results of the other group’s experiment that were “textbook”, not the results from the original experiment. The differences between the results of the other group’s more successful experiment and the results of the experiment conducted originally can be seen in the Appendix, in Figures 4 and 6 respectively, in the absorbance of pyrophosphatase at 620 nm. The reason why the results of the other group’s experiment were better overall was because they doubled the concentration of enzyme that was specified in the Biochemistry Lab Manual. After the enzyme did not behave as expected in the original experiment, the other group doubled its concentration and therefore achieved more consistent results.
The Velocity of Hydrolysis as a Function of Pyrophosphatase Concentration test showed very clearly that enzyme concentration is positively proportionate to the rate of hydrolysis, meaning that if concentration increases, velocity will increase as well. This is clearly depicted in Figure 1. This result was easily predictable because it is simply common sense; the higher the enzyme concentrations in a substrate, the more enzymes there are converting the substrate into product, which therefore produces a more rapid rate of hydrolysis.
The Velocity of Hydrolysis as a Function of pH test also had very clear results. As seen in Figure 2, the pyrophosphatase in the substrate with a pH of 7 had, by far, the highest rate of hydrolysis. This is because 7 is the optimum pH for pyrophosphatase (Kunitz, 1951). There was a dramatic drop in the rate of hydrolysis of pyrophosphatases in substrates in pHs that were very slightly away from 7 (i.e. 8.5). This is because an acidic or basic pH of a substrate can effectively break both intra- and intermolecular bonds within the enzyme, which can eventually denature the enzyme.
The Velocity of Hydrolysis as a Function of Temperature test provided “textbook” results as well. The pyrophosphatase in the substrate with a temperature of 50 °C had, by far, the highest rate of hydrolysis. This is because the optimum temperature for pyrophosphatase is 40 °C (Kunitz, 1951). As the temperature of the substrate increases, the molecules inside have increasing kinetic energy. This results in more collisions between molecules, therefore increasing the rate of hydrolysis. This is why the pyrophosphatases in the cooler substrates have a lower rate of hydrolysis. As seen in Figure 3, the higher the temperature increases above 50 °C, the lower the rate of hydrolysis. This is due to the excess heat giving the molecules too much kinetic energy, which in turn breaks both the intra- and intermolecular bonds in the enzyme, causing its denaturation.