Jessica Wong Analysis of the effect of inhibitors, enzyme concentration, substrate concentration, temperature, and pH on the rate of an alkaline phosphatase catalyzed reaction ABSTRACT The effects of several factors that affect the rate of a reaction catalyzed by the enzyme alkaline phosphatase were tested. The rate-reducing effect of the inhibitors sodium molybdate, sodium phosphate, and phenyl phosphonate was measured. The reaction rate of the complete reaction with alkaline phosphatase and substrate had a rate of 0.2875A/10 min. Sodium molybdate decreased this rate to 0.1833A/10 min through noncompetitive inhibition. The inorganic phosphate decreased the rate of reaction to 0.0458A/10 min through feedback inhibition, and the phenyl phosphonate decreased the rate of reaction to 0.1625A/10 min due to competitive inhibition. The effects of increasing enzyme concentration and substrate concentration were observed. An increase in alkaline phospatase concentration led to an overall increase in rate of reaction. An increase in substrate concentration, pNPP, yielded an initial increase in reaction rate but an eventual leveling off because the enzyme becomes saturated with substrate. The activity of alkaline phosphatase was assayed at pH levels 4.0 to 12.0 and the temperature interval 0°C to 100°C. The optimum pH was 12 and the optimum temperature was around 37°C. These findings suggest that although enzymes speed up the rate of reaction, many other factors influence the rate as well. INTRODUCTION An enzyme, an organic catalyst, speeds the rate of a specific chemical reaction by lowering the activation energy necessary for the reaction without being consumed (Solomon et al., 2005). Enzyme catalyzed reactions are affected by many factors. Inhibitors are substances that decrease the rate of reaction. Enzymes are inhibited through feedback inhibition, competitive inhibition, and noncompetitive inhibition. Feedback inhibition occurs when product formation inhibits the continuation of the reaction (Solomon et al., 2005) Competitive inhibition occurs when the inhibitor and substrate compete for the same active site on the enzyme (Solomon et al., 2005). Noncompetitive inhibition occurs when the inhibitor does not bind to the active site of the enzyme but does bind at another site on the enzyme (Solomon et al., 2005). This experiment will test the hypothesis that the inhibitors sodium molybdate, sodium phosphate, and phenyl phosphonate will slow down the rate of the enzyme-catalyzed reaction. Sodium molybdate is a noncompetitive inhibitor that binds with zinc, a cofactor required by the enzyme alkaline phosphatase. A cofactor is a substance needed for an enzyme to function. The inhibitor sodium phosphate is the inorganic phosphate produced by the enzyme-catalyzed reaction (Davis, et al 2006). It is therefore an example of feedback inhibition. Phenyl phosphonate binds to the enzyme but contains no bonds able to be hydrolyzed (Davis, et al 2006). This competitive inhibitor binds to the active site and prevents the substrate, p-Nitro-Phenyl Phosphate (pNPP) from being broken down. Other factors affecting the rate of reaction are the effect of enzyme and substrate concentration. A substrate is the substance that the enzyme acts on and it is a reactant in the chemical reaction. The substrate in this reaction is p-Nitro-Phenyl Phosphate (pNPP). Products are substances formed in the reaction. The products of this reaction are p-Nitrophenol (pNP) and inorganic phosphate (Pi). An increase in enzyme concentration results in a direct correlation to an increase in reaction rate. If the enzyme concentration is constant, the substrate concentration affects the rate. However, an increase in substrate concentration should result in an initial increase in reaction rate followed by a leveling off due to limited number of active sites. In this instance, the enzyme is the rate limiting factor of the reaction. (Solomon et al., 2005) Enzymes are also affected by pH and temperature. Generally, enzymes have a set of pH values that they are most active and an optimal pH. Enzymes may be affected by a change in pH because the charges are modified (Solomon et al., 2005). pH changes also affect tertiary and quanternary structure due to ionic bonds. The effect of pH will be tested by carrying out the reaction at various pHs, ranging from 4 to 12. Because molecular motion increases as temperature increases, the rates of enzyme-catalyzed reactions increase until the enzyme becomes denatured at high temperatures (Solomon et al., 2005). The effect of temperature will be tested by measuring the rate of reaction at room temperature (37°C), freezing point (0°C), and boiling point (100°C). The rate of reaction should increase until the enzyme becomes denatured. MATERIALS AND METHODS In the first section of this lab the controls and effect of inhibitors were tested on the rate of the enzyme, alkaline phospatase, catalyzed reaction using the Spec 20. The rate of reaction was calculated by measuring the change in absorbance from the Spec 20 over time. For example, for the effect of substrate concentration on the rate of reaction the initial absorbance (A0) was .29 and the aborbance after 10 minutes (A10) was .955. The rate of reaction (∆A/10min) would be calculated by A10 – A0/t10-t0 (.955 - .29)/(10-0) and therefore equal .665A/10 min (Davis, et al 2006). The controls tested were the effect of no enzyme, no substrate, and the complete reaction. The inhibitors tested were molybdate, inorganic phosphate, and phenyl phosphonate. Enzyme concentration was tested by the dilution of the enzyme, alkaline phosphatase, into six different concentrations beginning with undiluted enzyme and ending with no enzyme (Davis, et al 2006). The effect of substrate concentration was tested by using the same dilution method but with the substrate pNPP. To investigate the effect of pH on enzyme activity, the pH of the buffer was varied from a pH of 4 to a pH of 12. The effect of temperature on the rate of reaction was tested by running two sets of reactions, one with the enzyme and one without the use of the enzyme. These reactions were carried out at 0°C, room temperature, 37°C, and 100°C. (Davis, et al 2006). RESULTS Data Sheet 4.1: General features of enzyme-catalyzed reactions Tube No. Volume (mL) of Buffer pNPP Treatment PRODUCT FORMED A after 12 min RATE (A/10 min) 1 4.0 1.0 No enzyme 0.24 0.24A/10 min 2 4.0 0.0 No substrate 0.015 0.0125A/10 min 3 3.0 1.0 Complete 0.345 0.2875A/10 min 4 2.0 1.0 Plus molybdate 0.22 0.1833A/10 min 5 2.0 1.0 Plus inorganic phosphate 0.055 0.0458A/10 min 6 2.0 1.0 Plus phenyl phosphonate 0.195 0.1625A/10 min Data Sheet 4.2: Effect of enzyme concentration upon the rate of enzyme-catalyzed reaction. Tube No: 1 2 3 4 5 6 Concentration 1.0 0.5 0.25 0.12 0.06 None Absorbance (A): 0 min 0.13 0.08 0.061 0.059 0.045 0.032 5 min 0.262 0.145 0.1 0.08 0.071 0.04 10 min 0.39 0.21 0.135 0.11 0.08 0.055 Rate (increased A per 10 min): 0.26A/10 min 0.13A/10 min 0.074A/10 min 0.051A/10 min 0.035A/10 min 0.023A/10 min Data Sheet 4.4: Effect of substrate concentration upon the rate of enzyme-catalyzed reaction Tube No: 1 2 3 4 5 6 Concentration (mM): .4 .2 .1 .05 .025 0 Absorbance (A): 0 min 0.29 0.21 0.18 0.21 0.22 0.052 5 min 0.528 0.458 0.410 0.375 0.368 0.045 10 min 0.955 0.758 0.652 0.564 0.576 0.065 Rate (increased A/10 min) 0.665A/10 min 0.548A/10 min 0.472A/10 min 0.354A/10 min 0.356A/10 min 0.013A/10 min EMBED MSGraph.Chart.8 \s Figure 1: Effect of pH on rate of enzyme-catalyzed reaction Tube No. pH of buffer PRODUCT FORMED A after 6 min RATE A/10 min 1 4 0.62 1.03A/10 min 2 6 0 0A/10 min 3 7 0.009 0.015A/10 min 4 8 0.08 0.133A/10 min 5 10 0.165 0.275A/10 min 6 12 0.123 0.205A/10 min Figure 2: Effect of Temperature on rate of reaction Temperature Tube No. PRODUCT FORMED with Enzyme Tube No. PRODUCT FORMED without Enzyme 0°C (ice) 1+ 0.17 1- 0.06 __ °C (room temp) 2+ 0.34 2- 0.08 37°C (water bath) 3+ 0.50 3- 0.12 100°C (boiling) 4+ 0.145 4- 0.317 According to Data Sheet 4.1, the rate of reaction for the complete reaction (enzyme and substrate) was .2875A/10 min. Tubes 1 and 2 were controls. The reaction rate decreased with all three inhibitors. The rate decreased the most to 0.0458A/10 min when the inorganic phosphate was added. Sodium molybdate decreased the rate of reaction to 0.1833Am/10 min and phenyl phosphonate decreased the rate of reaction to 0.1625A/10 min. Data Sheets 4.1 and 4.2 depict the effect of enzyme concentration on reaction rate. The rate increased linearly as the enzyme concentration increased. Data Sheets 4.4 and 4.5 depict the effect of substrate concentration on reaction rate. A slight increase in substrate concentration initially increased the rate of reaction. However, as the substrate concentration was increased in larger increments, the rate of reaction increased slower once the peak was reached. Figure 1 depicts the effect of pH on rate of reaction. The optimum pH was around 10. The rate of reaction seemed to be higher at basic pHs of 10 and 12 and lower at all other pHs. As shown in Figure 2, the greatest amount of product formed occurred in both enzyme-catalyzed and non enzyme catalyzed reactions at 37°C. Up to 37°C, increasing the temperature increased the amount of product formed in the enzyme-catalyzed reactions. DISCUSSION When testing general features of an enzyme-catalyzed reaction, the first three test tubes were controls. The first control tube still indicated some absorbance which could be due to spontaneous breakdown of substrate. The second tube with no substrate still had minimal absorbance which could be due to marginal buffer absorbance. The fourth tube had the noncompetitive inhibitor sodium molybdate. The sodium molybdate competes with the cofactor zinc on the phosphatase and thus noncompetitively inhibits the enzyme. The inorganic phosphate, sodium phosphate, slowed down the reaction considerably because it reversed the direction of the reaction, causing more substrate to be formed. The competitive inhibitor phenyl phosphonate bound to the active site of the enzyme. However, it does not have oxygen in the phenyl ring so it cannot be hydrolyzed. The prediction for the effect of enzyme concentration was an overall increase in rate of reaction. Experimentally, the increase in enzyme concentration correlated to an increase in reaction rate relatively linearly. The prediction for the effect of an increase in substrate concentration was an initial increase in reaction rate and then a tapering off. The increase in substrate concentration resulted in an immediate increase in reaction rate. However, the rate of reaction quickly slowed as more and more substrate was added. This tapering off of reaction rate is due to the limited availability of the active sites on the enzyme to catalyze the reaction. Because the optimum pH was 10, this could indicate that the enzyme alkaline phosphatase works optimally at basic pHs but only up to a certain point. The rate of reaction increased significantly as the pHs became more acidic and basic until the pH was too basic. Generally, the more basic the pH, the higher the reaction rate. The ionic and hydrogen bonds of a protein with tertiary structure would be affected by a change in pH because the structure of the protein would be changed. In both enzyme-catalyzed and reactions with no enzyme, increasing the temperature increased the amount of product formed. At 100°C, the amount of product formed with the enzyme-catalyzed reaction decreased significantly because the enzyme became denatured. The reaction without the enzyme increased up to 100°C. This could indicate that the substrate breaks down spontaneously at 100°C. However, the enzyme can break down the substrate at room temperature. Overall, the rate of an enzyme catalyzed reaction is not determined by a single factor. Rather, multiple factors including inhibitors, enzyme concentration, substrate concentration, pH, and temperature combine to influence the rate of reaction. Many types of inhibitors affect the enzyme or substrate in different ways to slow down the reaction. Increasing enzyme concentration generally increases reaction rate while increasing substrate concentration only will increase reaction rate to a certain point. Enzymes also work best at optimal pHs and an increase in temperature will result in an increase in reaction rate until the enzyme becomes denatured. LITERATURE CITED Davis, Bill., Diana Martin. General Biology 101: A Laboratory Manual. Rutgers University Press, New Brunswick, NJ, 2006. Solomon, E.P., L.R. Berg, D.W. Martin. Biology, 7th ed. Brooks/Cole-Thompson Learning, California, 2005.