Effect of Enzyme Concentration, Temperature and pH on Enzymes - Lab Report Example

Effect of enzyme concentration, temperature and pH on enzymes Introduction An enzyme is a biological molecule that speedsup biochemical reactions. Enzymes work on specific substrates since different substrates produce different products without being consumed in the chemical reactions. Specificity of an enzyme is determined by characteristics of the R-groups of the amino acid residues that form the active site of an enzyme. Enzymes are important in maintenance of normal bodily functions and absence or deficiency may lead to metabolic disorders such as Krabbe disease or Phenylketonuria (PKU). In addition, several disease states have been linked to excessive enzymatic activity. Enzymes are protein in nature and may require a non-protein component called a cofactor to function. An apoenzyme is an inactive enzyme and the active enzyme including the cofactor is referred to as a holoenzyme. A prosthetic group is a cofactor that is bund tightly to an enzyme and may be difficult to remove without damaging the enzyme (Palmer & Bonner, 2007). Enzymes interact with substrates in their active sites as either lock-and-key or induced fit interaction model therefore promoting their transition state to form products. In this regard, the activation energy required is decreased resulting to increase in the rate of biochemical reaction. Enzyme lower the activation energy by altering the transition state of the substrate and releasing free energy due to formation of non-covalent and covalent bonds at the active site (Cornish-Bowden, 2013). Activation energy is expended to overcome activation barrier that is the energy involved in the path of conversion of substrate to product. The activation energy produces transient unstable charges that are present in the high energy transition state. It creates a barrier that prevents complex molecules from spontaneously reverting back to simpler forms. The transition state is the unstable chemical state with high free energy that can decay to either substrate or product. So as to convert substrate to product, enzymes may form and cleave bonds (Berg, et al., 2002). Enzyme activity is the rate at which an amount of enzyme can transform a substrate to a product at a particular temperature. The amount of enzyme activity in a solution is equivalent to the total number of units in the solution. The enzyme with the highest activation energy is the rate limiting step (Klein, 2012). Factors that influence enzymatic activity include enzyme concentration, temperature, pH, inhibitors, and substrate concentration. Pyrophosphatase increases the rate of hydrolysis of pyrophosphatase into two molecules of organic phosphate. Hydrolysis of adenosine triphosphate generates pyrophosphate in cells. This experiment is aimed to find out how pyrophosphatase activity is affected by enzyme concentration, temperature and pH. Materials and methods The procedure for the experiment were obtained from the Unit Laboratory Manual for Biochemistry. No deviations were made to the specified procedures (Ashby & Prior, 2014). Results (400 words) The enzyme activity of pyrophosphatase was investigated under different conditions of enzyme concentration, pH and temperature. The results were prepared in Microsoft Excel and the data presented as Tables 1, 2, 3 and graphs as Figures 1, 2, 3. Table 1 is a representation of the effect of enzyme concentration at 0.0, 0.25, 0.50, 0.1 and 0.125 U/ml on enzyme activity. Table 1: Effect of enzyme concentration on enzyme activity In Figure 1 below, the average rate of hydrolysis of inorganic pyrophosphate (PPi) is plotted against enzyme concentration. The effect of enzyme concentration on reaction rate was at a constant rate at a constant level of substrate. At enzyme concentration of 0.100U/ml and 0.125U/ml was the highest reaction velocity. Figure 1: Effect of enzyme concentration on enzyme activity Table 2: Effect of pH on enzyme activity According to Table 2, the optimum pH for the reaction was 7.000 with an average rate of hydrolysis at 0.850 µmol/mL/min. The lowest rate 0.090 µmol/mL/min was at 10.500. Figure 2: Effect of pH on enzyme activity Figure 2 above shows that the rate of hydrolysis is optimum at a pH of 7.000. The rate of hydrolysis was lowest at pH 10.50. Temperature (⁰C) Abs1 (620nm) Abs2 (620nm) [PO4] 1 (mM) [PO4] 2 (mM) Amt Pi 1 (µmol) Amt Pi 2 (µmol) Amt PPi 1 (µmol) Amt PPi 2 (µmol) Rate of Hydolysis 1 (µmol/mL/min) Rate of Hydrolysis 2 (µmol/mL/min) Average Rate of Hydrolysis (µmol/mL/min) Range Room temp. 0.120 0.128 0.639 0.682 0.639 0.682 0.320 0.341 0.160 0.170 0.165 0.005 Ice bath 0.081 0.074 0.432 0.394 0.432 0.394 0.216 0.197 0.108 0.099 0.103 0.005 30⁰ 0.232 0.222 1.236 1.183 1.236 1.183 0.618 0.591 0.309 0.296 0.302 0.007 50⁰ 0.595 0.709 3.170 3.777 3.170 3.777 1.585 1.889 0.792 0.944 0.868 0.076 70⁰ 0.726 0.859 3.868 4.576 3.868 4.576 1.934 2.288 0.967 1.144 1.056 0.089 Boiling (no enzyme) 0.011 0.027 0.059 0.144 0.059 0.144 0.029 0.072 0.015 0.036 0.025 0.011 Boiling (enzyme) 0.874 0.886 4.656 4.720 4.656 4.720 2.328 2.360 1.164 1.180 1.172 0.008 Table 3: Effect of temperature on enzyme activity The effect of temperature variation at room temperature, ice bath, 30⁰C, 50⁰, 70⁰ while boiling with enzyme and no enzyme on enzyme activity. The range for the rate of hydrolysis 1 and 2 was between 0.005 and 0.089. Figure 3: Effect of temperature on enzyme activity Discussion (400 words) From the findings, it can be established that enzyme concentration, pH and temperature affect enzyme activity. The average rate of hydrolysis increased with the enzyme concentration. Absence of enzymes at 0.000 U/ml had the lowest reaction velocity since there was no catalysis to lower the activation energy. An increased number of enzyme pyrophosphatase molecules in the solution will induce the breaking of covalent bonds in the substrate molecules. The substrate will react faster forming the product quicker. As the concentration increases, the rate of reaction increases due to the lowered activation energy. Reaction rates can be enhanced by increasing the numbers of enzyme-substrate complexes which promotes the formation of more products (Stockbridge & Wolfenden, 2011). Findings indicate that pyrophosphatase works optimally at a neutral pH of 7.000. At this point, the amino acid responsible for enzyme activity is at its specific ionization state. Extreme low and high pH environments lowers the enzyme activity (Rodrigues, et al., 2013). This is because changes in pH affect the properties of both the enzyme and the substrate characteristics making them unable to bind in the active sites. Extreme pH causes variations in the protein structure of the enzyme. The pH also influences the ionization states of the substrate and amino acid residues that are involved in the catalytic activity of the enzyme. The weak base and acid amino acid groups could be affected chemically by changes in pH (Voet & Pratt , 1999). In most of the chemical reactions, increase in temperatures leads to increase in the rate of reaction. The enzyme activity was lowest and there was no significant reaction at ice bath because at the ice bath temperature since the temperatures are low and below optimum. With increase in temperature the rate of reaction increases (Daniel & Danson, 2013). At 40 ⁰C, the hydrogen bond, Van der Waals and hydrophobic interactions are broken At boiling temperature, the rate of enzyme pyrophosphatase was high indicating that the enzyme is thermostable thus cannot be degraded by temperature. However, enzymes in the body work at an average temperature of 37⁰C. The thermostable inorganic pyrophosphatase is applicable in removal of pyrophosphate produced during sequencing reactions (Park, et al., 2010). A boiling point with no enzymes there was a slight rate of reaction due to kinetic energy that produced collisions to enhance little reaction. Possible error to this experiment could be caused by inaccurate measurement or timing. Errors could be minimized by being more precise in measurements. Conclusion Temperature, pH, and enzymatic concentration affect the rate at which enzymatic factors affect the rate at which enzymatic reactions. Increase in substrate concentration increases the rate of enzyme function. Pyrophosphatase activity is highest at a pH of 7.000. The highest rate of velocity was at 100 ⁰C because the enzyme is thermostable. Knowledge in factors that affect enzyme activity such as for pyrophosphatase is useful in PCR applications in preparation of appropriate buffers for the reactions. Bibliography Ashby, R. & Prior, R., 2014. Laboratory Manual for Biochemistry (6530) and Biochemistry G (6480), University of Canberra: Faculty of Education, Science, Technology and Mathematics. Berg, J. M., Tymoczko, J. L. & Stryer, L., 2002. Enzymes Are Powerful and Highly Specific Catalysts. In: Biochemistry. s.l.:W. H Freeman and Company, pp. 95-105. Cornish-Bowden, A., 2013. Fundamentals of Enzyme Kinetics. 4th ed. s.l.:Weiheim Wiley. Daniel, R. M. & Danson, M. J., 2013. Temperature and catalytic activity of enzymes: A fresh understanding. FEBS Letters, 587(17), pp. 2738-2743. Klein, D., 2012. Organic Chemistry, s.l.: John Wiley and Sons. Palmer, T. & Bonner, P. L., 2007. Enzymes: biochemistry, biotechnology and chemical chemistry. 2nd ed. Oxford: Woodhead Publishing. Park, S. Y. et al., 2010. Facilitation of polymerase chain reaction with thermostable inorganic pyrophosphatase from hyperthermophilic archaeon Pyrococcus horikoshii. Applied Microbiology Biotechnology, 85(3), pp. 807-12. Rodrigues, R. C. et al., 2013. Modifying enzyme activity and selectivity by immobilisation. Chemical Society Reviews, Volume 42, pp. 6290-6307. Stockbridge, R. B. & Wolfenden, R., 2011. Enancement of the Rate of Pyrophosphate Hydrolysis by Non-Enzymatic Caalysts and by Inorganic Phosphatase. The Journal of Biological Chemistry, 286(21), pp. 1-103. Voet , J. G. & Pratt , W. L., 1999. Fundamentals of Chemistry. s.l.:John Wiley. Appendix [PO4] mM Abs1 (620nm) Abs2 (620nm) Average Abs Range 0 0.000 0.000 0.000 0.000 0.2 0.024 0.008 0.016 0.008 0.4 0.089 0.090 0.090 0.001 0.6 0.130 0.133 0.132 0.002 0.8 0.135 0.144 0.140 0.004 1 0.184 0.183 0.184 0.001 Read More