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The effect on rate of an enzyme catalysed reaction by different objectives which include effect of substrate, temperature, ph and effect of a competitive inhibitor phosphate ions. This is determined by the reaction of hydrolysis by p-nitrophenylphosphate (PNP) as a substrate by the enzyme phosphatase. Abstract The hydrolysis of p-nitrophenyl phosphate has been studied in human red blood cells. To see if hydrolysis was related to the functioning of the sodium pump. Acid phosphatase catalysis’s the hydrolysis of p-nitophenyl phosphate under four different objectives ph, temperature, substrate inhibition and a competitive inhibitor.
The phosphatase and PNP were placed in test tubes with different concentration of each but under different objectives. The concentration of the enzyme and substrate were the same for each experiment. It was shown that the Vmax was 0. 286 and the Km was 0. 0114. As the valve of Km is small this shows that there is a high enzyme substrate affinity. There were some anomalous results obtained within experiment 2 and experiment 3 these may be due to experimental or human errors. Introduction Enzymes are biological catalysts that speed up the rate of reaction between substances without themselves being consumed in the reaction.
The catalytic thrive of an enzyme comes from the addition of a substrate binding to a specific region on the enzyme called the active site. May factors oppose on the effect of an enzyme these include temperature, ph, concentration of substrate and competition. All of these factors are investigated in the experiment of PNP with the enzyme phosphatase. Looking at the effect of concentration of substrate for this reaction you would expect that at a constant concentration of phosphatase the reaction rate increases with increasing PNP concentration until it reaches a maximum velocity.
At the maximum velocity it suggests all the enzyme phosphatase active sites are occupied by PNP and therefore the reaction rate cannot increase. The diagram below illustrates the theory above. Previous work within industry with enzymes and substrate formed the bases of the prediction above. [http://resources. edb. gov. hk/biology/english/images/health/substrate_concentration. jpg] The substrate concentration against the rate of the reaction can be illustrated by the Michaelis-Menten plot. Vmax is the maximum rate at which the enzyme can catalyse a reaction.
Km is the Michaelis constant which is the concentration of substrate that gives half the Vmax. However the Lineweaver-Burk plot gives a more concise value of Km: The graph above illustrate that as the temperature is increased, the reaction rate of the enzyme also increases however it shows that it reaches a optimum temperature where the reaction proceeds at its maximum. Above this temperature the reaction decreases. This is due to the bonds within the enzyme breaking changing the 3d shape of the enzyme and therefore denaturing.
Taking this theory in to account with the research being carried out it would be expected that at the optimum temperature of around 37? c the acid phosphatase will start to denature and the rate of reaction above this temperature will decrease. Many enzymes are sensitive to ph and they all have a specific range of activity. All enzymes have an optimum ph and effects the enzymes 3 dimensional shape by breaking its weak non covalent bonds such as hydrogen bonds and ionic bonds causing the enzyme to denature. Acid phosphatase optimum ph will be below 7 it would work best within the range of 4. -6. 0. Enzyme inhibitors are substances which interfere with the catalytic action of the enzyme. They slow down or completely stop the reaction. These include reversible and non reversible. Reversible inhibitors are further more be classified as competitive and non competitive. A competitive inhibitor closely resembles the substrate. The inhibitor competes form the same active site as the substrate. The inhibitor interacts with the enzyme but no reaction occurs. The active site is now occupied and the substrate no longer can bind to the enzyme.
However competitive inhibition is reversible as long as there is sufficient amount of substrate within the reaction. Non competitive inhibitor forms a strong covalent bond with the active site therefore is irreversible. However the non competitive inhibitor may also bond to another part of the enzyme remote from the active site this is known as allosteric inhibition. Interaction at an allosteric site changes the structure of the enzyme therefore the active site changes and the substrate no longer fits. Within the experiment phosphate ions are acting as the inhibitors. They will compete with PNP to bind with phosphatase active site. Results
Effect of substrate on enzyme activity The table below shows the absorbance obtained after different concentrations of PNP. Tube no| Amount of p-nitrophenol| Absorbance| | ??M| ??| 1| 0| 0| 2| 0. 05| 0. 382| 3| 0. 1| 0. 779| 4| 0. 15| 1. 212| 5| 0. 2| 1. 468| 6| 0. 25| 1. 785| 7| 0. 3| 2. 002| 8| 0. 4| 2. 244| 9| 0. 5| 2. 451| PNP was calculated and used in the calibration curve Using tube 1 as an example to show calculation of amount of PNP:- n=C*V Amount of PNP=0. 5mM*0. 1/1000 =0. 05mmol Graph 1 above shows the absorbance against different amounts of PNP Table 2 shows the amount of PNP formed(S) which was obtained in the calibration curve.
The rate (v) was then found out using the amount of PNP formed. Using these values 1/S and 1/v were calculated which were used to plot the Lineweaver-Burk plot. Tube no| S| Abs| v| 1/S| 1/v| | ? mol| ??| ? mol/min| ??? mol| min/? mol| 2| 0. 0333| 0. 382| 0. 065077| 30. 03003| 15. 36649215| 4| 0. 05| 0. 779| 0. 132709| 20| 7. 535301669| 6| 0. 0833| 1. 212| 0. 206474| 12. 0048| 4. 843234323| 8| 0. 167| 1. 468| 0. 250085| 5. 988024| 3. 998637602| 10| 0. 333| 1. 785| 0. 304089| 3. 003003| 3. 288515406| 12| 0. 667| 2. 002| 0. 341056| 1. 49925| 2. 932067932| 14| 1. 67| 2. 244| 0. 382283| 0. 598802| 2. 615864528| 16| 3. 33| 2. 451| 0. 17547| 0. 3003| 2. 39494084| Using tube 1 as an example S= C1V1=C2V2 10mM*0. 01mL=C2*3mL C2=0. 0333 Calculation of V Using equation obtained from the calibration curve y=5. 870x assuming the following Y=Abs X=V y/5. 870=x 0. 382/5. 870=0. 065077 Calculation of 1/S in Tube 1 1/0. 0333 =30. 03003 Calculation of 1/v in Tube 1 1/0. 065077 =15. 36649215 Graph 2 Table 3: Theoretical dependence of v on S using Km and Vmax estimated from data of Table 2 S| v| mM| ? mol/min| 0. 0333| 0. 05367| 0. 05| 0. 109447| 0. 0833| 0. 170282| 0. 167| 0. 206249| 0. 333| 0. 50787| 0. 667| 0. 281275| 1. 67| 0. 315275| 3. 33| 0. 344358| These results were then used to plot the Michaelis-Menton plot graph 3 Value of Vmax 1/Vmax = 3. 496 Vmax=1/3. 496 = 0. 286 y = 0. 004x+3. 496 0 = 0. 004x+3. 496 x= -3. 496/0. 004 x= -87. 4 -1/Km = -87. 4 Km = 1/87. 4 = 0. 0114 Graph 4 The graph can be used to find more accurate value of Km. The values in table 2 were used to plot this graph. Effect of temperature Table 4 Tube no| Temp| Abs| Abs-control| v| 1/T| log(v)| | K| ??| ??| min-1| K-1| ??| 1| 278. 2| 0. 053| -0. 094| 0. 009029| 0. 003595| -2. 044| 2| ??| 0. 147| ??| ??| ??| ??| 3| 293. 2| 0. 276| -0. 288| 0. 047019| 0. 03411| -1. 328| 4| ??| 0. 564| ??| ??| ??| ??| 5| 298. 2| 2. 008| -0. 236| 0. 342078| 0. 003458| -0. 466| 6| ??| 2. 244| ??| ??| ??| ??| 7| 310. 2| 0. 985| 0. 443| 0. 167802| 0. 003224| 0. 775| 8| ??| 0. 542| ??| ??| ??| ??| 9| 323. 2| 1. 752| 1. 11| 0. 298467| 0. 003094| -0. 525| 10| ??| 0. 642| ??| ??| ??| ??| 11| 348. 2| 2. 639| -0. 25| 0. 449574| 0. 002872| -0. 347| 12| ??| 2. 889| ??| ??| ??| ??| T the graph below shows log v against 1/t (Graph 5) Calculation of Abs-(Abs-control) in tube 1 0. 053-0. 147 =-0. 094 Calculation of v in tube 1 y = 5. 870x y=Abs and x=v y/5. 870=x 0. 053/5. 870= 0. 009029 Calculation of 1/T in tube 1 = 1/278. 2 = 0. 03595 Calculation of log(v) in tube 1 = log(0. 009029) = -1. 886 The effect of ph on the reaction rate Table 5 Tube no| pH| Abs| v| | ??| ??| min-1| 2| 3| 0. 068| 0. 01158| 4| 4| 0. 305| 0. 05196| 6| 4. 5| 0. 399| 0. 06797| 8| 5| 0. 361| 0. 06150| 10| 5. 5| 0. 549| 0. 09353| 12| 6| 0. 271| 0. 04617| 14| 7| 0. 065| 0. 01107| Graph 6 Calculation of v in tube 1 Y=5. 870x X=y/5. 870 X=0. 068/5,870 =0. 01158 The effect of a competitive inhibitor (phosphate ions) Table 6 Tube no| S| Abs| v| 1/S| 1/v| | ? mol| ??| ? mol/min| ??? mol| min/? mol| 2| 0. 0333| 0. 168| 0. 02862| 30. 03003| 34. 94048| 4| 0. 05| 0. 125| 0. 021295| 20| 46. 96| 6| 0. 833| 0. 142| 0. 024191| 12. 0048| 41. 33803| 8| 0. 167| 0. 351| 0. 059796| 5. 988024| 16. 72365| 10| 0. 333| 0. 589| 0. 100341| 3. 003003| 9. 966044| 12| 0. 667| 0. 679| 0. 115673| 1. 49925| 8. 645066| 16| 1. 67| 0. 72| 0. 122658| 0. 598802| 8. 152778| Discussion The first investigation was looking at the effect of substrate concentration on the rate of reaction. From looking at graph 2 as the concentration of substrate slowly increased the rate of reaction also increased showing a proportional curve. As the concentration of substrate became higher than the concentration of the enzyme phosphatase the reaction rate reaches equilibrium.
We can see this at the end of the curve it is becoming a horizontal straight line which is Vmax the maximum rate of reaction. It shows a constant rate of reaction when all the enzyme active sites are occupied. In the Michaelis-Menten plot,should be showing two curves. one curve is the plot of the result of the experiment and the other curve should have been a theoretical plot. If both curves were displayed they should have coincide quite well which would have showed the same estimated Vmax. Therefore, would have indicated the conditions carried out of the experiment were quite good.
The Vmax was estimated to be 0. 286 and the Km was estimated to be 0. 0114 It is known as the temperature is increased the rate of a reaction also increases. Looking at my results it shows there is a increase in the rate of reaction as the temperature was increased. At the peak of the graph it indicates the optimum temperature for the experiment when the graph begins to decreases this is due to the change in secondary and tertiary structure alteration as the weak intermolecular are broken therefore changing the active site of the enzyme and the complementary substrate no longer fits.
There were some anomalous results within this part of the investigation these may have been due to human error and experimental errors. Theses may include not using the pipettes in the correct way and also measurements. Experiment 3 was looking at the effect of different values of pH ranging from pH3 to pH7. Looking at table 5 Abs column as the pH increased the value absorbance also increased. In tube 10 with pH of 5. 5 the absorbance values does not follow the trend it suddenly increases and in tube 12 pH 6 there is a large decrease from 0. 547 to 0. 271.
This suggests the enzyme has reached its optimum pH between the pH 5. 5 and pH 6. At this point phosphatase begins to denature due to the acidic conditions within the test tube the non covalent bonds of the enzyme structure break changing the shape of the enzyme therefore changing shape of its active site. This change in state means that the substrate no longer fits into the enzymes active site and a product cannot be formed therefore decreasing the rate of reaction. From the pH graph you can see there is a anomalous due to tube 10. This may have occurred due to an experiment error such as measuring error.
Using a smaller pipette would have given a more accurate measurement also an air bubble may have occurred whilst using the pipette altering the measurements in each test tube. Within the last experiment it was looking at the effect of an inhibitor phosphate ion on the rate of reaction between the enzyme phosphatase and substrate PNP. As the concentration of phosphate ions increases a higher concentration if PNP is needed to attain the reaction velocity. Looking at graph 6 there is an interception on the y-axis which indicates phosphate ions are competitive inhibitors. Even though the slope ncreases the intercept is unchanged because a competitive inhibitor does not alter the Vmax but increases Km. Question 1 The substrate was added to both the experimental and controlled tubes at the same time as the tubes will be in the same states and the conditions will remain constant and therefore allowing a fair test to occur. Question 2 When a pH of an enzyme is reached it works at its best as it is given the right conditions for the reaction to occur at an optimum rate. Once the pH increases its optimum value it begins to break non covalent bonds within the enzyme complex such as hydrogen bonds and weak van der waals forces.
These courses the 3 dimensional shape to change. The change in shape changes the shape of the enzymes unique active site therefore the complementary substrate does not fit and a product is not made. References * http://resources. edb. gov. hk/biology/english/images/health/substrate_concentration. jpg * Biochemistry sixth edition * www. google. co. uk * http://users. rcn. com/jkimball. ma. ultranet/BiologyPages/E/EnzymeKinetics. html Student Registration Number: 07002919201 2007/08 LSC-10003 Nature’s Tools: Proteins and Enzymes Enzyme Kinetics Report Word count: 2,055