Kinetics of Alkaline Phosphatase Student number: 622555 Introduction Enzymes are like a set of tools utilised by living organisms

to speed up and make biochemical reactions more efficient. The normal rate of reaction for most biochemical reactions such as metabolism is far too slow for most living organisms to use, thus this quickening or catalysis is vital for sustaining life. Enzymes are a sophisticated arrangement of proteins designed to carry out a specific function within a reaction. Enzymes do not change during the reaction, nor do they change the reactants or products of the reaction, they solely quicken the reaction. Although enzymes are mostly constructed from protein as mentioned before, they also include components known as cofactors. Cofactors include such things as zinc, potassium or magnesium ions. Coenzymes are another type of cofactor which becomes unattached from the body of the enzyme. Coenzymes can react directly within a chemical reaction. Alkaline phosphatase is a type of enzyme known as a hydrolase enzyme. Alkaline phosphatase is the key to removing phosphate groups or the dephosphorylation of molecules such a nuleotides, alkaloids and proteins. As expected alkaline phosphatase functions better within an alkaline environment. An enzyme has a maximum velocity or Vmax that it will be able to catalyse a reaction. Enzymes are also characterised by their Km or the Michaelis constant. The Michaelis constant is the affinity an enzyme has with a substrate. The Km of an enzyme is the substrate concentration required to make the reaction progress at half of its maximum speed. The purpose of this experiment is to determine the kinetics of alkaline phosphatase. In other words the speed at which it carries out its specific task and how well the substrate binds to the active site of the enzyme. Aim The aim of this experiment is to investigate the kinetics of the enzyme alkaline phosphatase. This will be done by exposing a compatible solution to the enzyme and recording how much product is produced. Method As in hand book. Dilution factors: 0.015M = 10ml of substrate, 0.03M = 2ml substrate + 8ml of buffer, 0.015M = 1ml substrate + 9ml buffer, 0.001M = 0.66ml substrate + 9.34ml buffer, 0.0075M = 0.5ml substrate + 9.5ml of buffer.

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Results Vmax = 0.16666 Km= 0.0066M Discussion By observing Fig 3 it can be determined that the reaction velocity of alkaline phosphatase steadily increases over time as the substrate concentration is increased. Fig 3 shows that reaction velocity increases roughly 40M/sec for every 1000M of substrate. As Km is an affinity an enzyme has with a substrate there are several factors that can be effected by. pH can effect Km by interfering with the efficiency of the enzyme and ultimately denaturing it as can temperature. Substrate concentration will also have an effect on Km as shown in this experiment and also the presence of an inhibitor. The calculated Km value doesnt seem to be consistent with the normal Km value of alkaline phosphatase which is higher. This sample of alkaline phosphatase may have come from E. coli perhaps. To investigate the effects pH would have on the Km an experiment could be set up fairly similar to this experiment. The difference being that the substrate concentration would be fixed and the pH of the solution would be the variable. The same could also be done for temperature with temperature being the variable. If the experiment was repeated using first a competitive inhibitor and then a noncompetitive inhibitor the results would be slightly different. With a competitive inhibitor the enzyme and the inhibitor would be competing for the same active site. This means although Vmax can be reached the concentration of the substrate needs to be higher, in turn making the Km of the enzyme larger. Non-competitive inhibitors however bind onto the enzyme itself, thus decreasing its efficiency. The effect this has on the Km of the enzyme is that it is unchanged. This is because the active site remains the same. The reaction velocity is decreased for all values of [S] with non-competitive inhibitors.