Enzyme Assays and Enzyme Kinetics - Lab Report Example

?To Investigate and Measure Km and Vmax of ?-galactosidase Enzyme The main purpose of carrying out this experiment was to investigate and measure two parameters, Km and Vmax for ?-galactosidase enzyme. Furthermore the PH change effect on the enzyme activity was investigated as well. In general terms this was to introduce concepts related to enzyme kinetics. Introduction Enzymes are among several proteins generated in living cells to catalyze or accelerate the organism’s metabolic processes. Normally, enzymes are so selective and specific on the molecules they catalyze, referred to as substrate (Dixon, 1979).They normally do react specifically with a single substrate. The substrate has the capacity of binding to the enzyme at a location referred to as active site (Di Cera, 1996). This usually takes place before the starting of the catalyzed reaction. Enzymes also have the capacity to speed up chemical reactions. The speed up can be up to a million fold, but it is known that this enzymes functions only within a certain narrow pH range and temperature. If this range exceeds beyond, it can lead to enzymes losing their structure, hence denatured (Cornish, 2001). Enzymes are favorably involved in processes such as breaking down of fat molecules, starch and large protein in food during digestion into smaller molecules, the nucleotides joining into DNA strands and phosphate group addition to ADP to make ATP (Schnell, 2002).Usually the enzymes names end in the suffix -ase. Enzyme uses: It is known that enzymes enable particular industrial processes to be undertaken at normal pressures and temperatures. As a result of this, the amount of energy needed will be reduced as well as the expensive equipment required (Schnell, 2000). Also enzymes are meant for domestic use .For instance, in 'biological' detergents. Materials and Methods The experiment was carried out in pairs of two and the measurement got was compared with the average of the class measurement. The entire substrate and PH range was used as shown below. It was expected that the reaction to be set up then monitor the reaction with spectrophotometer for 5 minutes upto the point when the absorbance reading atleast 3.The readings were recorded after every 30 seconds.The was following conditions were investigated: Substrate concentrations: 4mM, 2mM, 1mM, 0.5 mM, 0.25mM, 0.125mM, 0.062mM, 0.031mM and blank pH:3.5,4.5,6.5,7.5 and 8.5 As mentioned above, at the end of the experiment, the pair measurements was compared with the class average measurements and regression line was plotted to ascertain this comparison. Each reaction had the following component Ingredient Volume Assay buffer 600µL ?-gal 10x Volume to attain 0.25U/ml concentration ONPG Volume required to attain a reaction concentration H2O To complete the volume to 1200µL Reaction volume 1200 µL Table 1: showing the reaction component. ONPG stock solution was 68mM, thus the required amount was calculated for each reaction using the equation c1v1=c2v2. Also 50U/ml concentration of ?-gal was the stock available and every reaction needed enzyme concentration of 0.25U/ml.All the ingredients were added but for the enzyme (all the appropriate cuvettes were set up, each was set at a time for every pH condition), the cuvette was used to zero the instrument before using the spectrophotometer, the enzyme mix was added quickly and the cuvette was returned back to the instrument. Then absorbance changes were monitored for 5min minutes at 420 nm, the data was recorded after every 30 seconds (the cuvette was taken out and mixed quickly prior every reading). Volume Ingredient 4mM 2mM 1mM 0.5mM 0.25mM 0.125mM 0.062mM 0.03mM Assay buffer 600?l 600?l 600?l 600?l 600?l 600?l 600?l 600?l ? –gal 10x 6?l 6?l 6?l 6?l 6?l 6?l 6?l 6?l ONPG 70.6?l 35.3?l 17.6?l 8.8?l 4.4?l 22?l 11?l 5?l H2O 523.4?l 558.7?l 576.4?l 585.2?l 589.6?l 572?l 583?l 589?l Table 2: How the quantities of every reagents used were added in the cuvette Results Figure 1, 4, 10, 13, and 16 indicates how the products were generated over time by the action of ?-galactosidase enzyme on the substrate at different substrate concentration and pH conditions. This enabled the calculation of product formed per minute. Figure 2, 5, 8, 11, 14, and 17 indicates the effect of substrate concentration change over time on the velocity or rate of reaction catalyzed by ?-galactosidase enzyme at different pH. Figure3, 6, 9, 12, 15, and 18 show linear weaver-Burk plot. This plot has the reciprocals of Vmax and Km values; hence this enabled the determination of Km and V max values at different pH condition. pH 3.5 Figure 1: A graph showing Optical density (420nm) of different substrate concentrations against time at pH 3.5 Figure 2: A graph showing velocity (change of rate of reaction with time) against substrate concentration at pH 3.5 Figure 3: A linear weaver-Burk Plot indicating Vmax (1/Vmax-Y intercept) and Km (1/Km-X intercept) at pH 3.5. It was not possible to calculate Vmax and Km because the linear line never met the dots. Also the value of R2 was too small. At pH4.5 Figure4: A graph showing Optical density (420nm) of different substrate concentrations against time at pH 4.5 Figure 5: A graph showing velocity (change of rate of reaction with time) against substrate concentration at pH 4.5 Figure 6: A linear weaver-Burk Plot indicating Vmax (1/Vmax-Y intercept) and Km (1/Km-X intercept) at pH 4.5. It was possible to calculate Vmax and Km because the linear line met the dots. Also the value of R2 was big enough. At pH 6.5 Figure 7: A graph showing Optical density (420nm) of different substrate concentrations against time at pH 6.5 Figure 8: A graph showing velocity (change of rate of reaction with time) against substrate concentration at pH 6.5 Figure 9: A linear weaver-Burk Plot indicating Vmax (1/Vmax-Y intercepts) and Km (1/Km-X intercept) at pH 4.5. It was possible to calculate Vmax and Km because the linear line met the dots. Also the value of R2 was big enough. At pH 7.5 Figure 10: A graph showing Optical density (420nm) of different substrate concentrations against time at pH 7.5 Figure 11: A graph showing velocity (change of rate of reaction with time) against substrate concentration at pH 7.5 Figure 12: A linear weaver-Burk Plot indicating Vmax (1/Vmax-Y intercept) and Km (1/Km-X intercept) at pH 7.5. It was possible to calculate Vmax and Km because the linear line met the dots. Also the value of R2 was big enough. Class average pH7.5 Figure 13: A graph showing Optical density (420nm) of different substrate concentrations against time at pH 7.5(for class average) Figure 14: A graph showing velocity (change of rate of reaction with time) against substrate concentration at pH 7.5(for class average) Figure 15: A linear weaver-Burk Plot indicating Vmax (1/Vmax-Y intercept) and Km(1/Km-X intercept) at pH 7.5. (For class average) pH 8.5 Figure 16: A graph showing Optical density (420nm) of different substrate concentrations against time at pH 8.5 Figure 17: A graph showing velocity (change of rate of reaction with time) against substrate concentration at pH 8.5 Figure 18: A linear weaver-Burk Plot indicating Vmax (1/Vmax-Y intercept) and Km (1/Km-X intercept) at pH 8.5 Discussion and Conclusion According to the results, it is clearly evident that pH do affects the Vmax and Km. It can be interpreted that extreme pH which is not the optimum for enzyme reaction increases the value of Km and lowers the Vmax value from the values at normal pH. According to literatures, the higher the Km the lower the binding affinity while the higher the enzyme affinity the higher the vmax value (Duggleby, 1995). This was clearly indicated in the result. For instance, at pH 4.5 the Vmax value was too low while the Km was very high as compared to other pH conditions. This implies that the affinity of the enzyme was greatly affected probably denatured. Hence the enzyme never had a chance to speed up the reaction. Between pH 6.5 and pH 7.5 seemed to be the optimum pH condition because it had the lowest Km and the highest vmax. Approximation and calculation of Vmax and Km can be by plotting data points reciprocals to give a Lineweaver-Burk plot or “double-reciprocal" (Duggleby, 1986). This offers a more precise way of determining Km. and Vmax (Dowd, 1965). Vmax can be approximated by the point at which the line crosses the 1/Vi = 0 axis (thus, [S] is infinite). Km is normally equal to Vmax times the line slope. This can be determined more easily from the X axis intercept. To illustrate how pH influences Km and Vmax,regression line can be drawn to determine the association. Figure 19: The association between Vmax and pH Basing on the linear regression line in figure 19, it is clearly evident that pH condition change from optimum lowers the Vmax. The association between Vmax and pH is relatively high with R2 value of 0.893 that is close to 1.The closer the R2 value is to 1 the higher the association. Figure 20: The association between Km and pH Basing on the linear regression line in figure 20, it is clearly evident that pH condition change from optimum raises the km. The association between Vmax and pH is relatively high withR2 value of 0.959 that is close to 1.The closer the R2 value is to 1 the higher the association. R2 value is too close to +1.According to literature, the correlation coefficient is normally between -1 to +1. In general terms, the closer the R2 is to -1 or +1, the better the association is between the factor on Y-axis and the factor on x-axis (Duggleby, 1995). To facilitate the products measurement of the enzyme reaction, scientists have used lactose analogs similar to lactose. It is able to bind ?-galactosidase active site, but results to an altered product (Dixon, 1972). . Choosing of Analogs can be in a way that one of the products produced is compound that is colored and should be measured easily by a spectrophotometer. ortho-nitrophenyl-beta-D-galactopyranoside, referred to as ONPG, upon cleavage generates a yellow color hence measured easily (Duggleby,2001). There was no big difference between the Vmax and Km values of the class average and the pair values at pH 7.5 .Despite the small difference, the trend on the way pH was affected is similar. The differences could have been brought because of technician errors such as taking reagent wrong measurements and inaccurate pH conditions. As a bifunctional enzyme, ?-galactosidase hydrolyses lactose to galactose together with glucose and lactose is converted to Allolactose (Juers et al, 2003). The enzyme needs Mn2+ or Mg2+ to function optimally. This includes K+ or Na+ mono-valent cation. lacZ makes an operon with lacA (a transacetylase) and lacY (that encodes lactose permease) . lac operon expression is usually repressed by LacI that do binds on the lac operator (Juers et al, 2003).In Allolactose presence, (the physiological inducer) (Juers et al, 2003) the binding of LacI to the operator is seen to be reduced hence resulting to lac operon expression as well as proteins production important for utilization of lactose (Juers et al, 2003). As a large tetrameric molecule, ?-galactosidase consists of four identical subunits. Only 2 monomers are needed to make a full active site despite the tetramer consists of four active sites. Every monomer has five domains that include three that have a ?/? barrel structure that has the capacity to interact with the loop structure from a separate monomer domain 2 to generate bigger percentage of the active site (Juers et al, 2003). Residues deletion from the amino terminus leads to inactive dimers. Hence Supplies peptides including the residues that are missing and restores the catalytic activity and the tetrameric structure. This process is referred to as ?-complementation. Thus it is fundamental in the technique of blue/white screening employed in gene cloning that is vector based (Juers et al,2003). Bibliography Cornish-Bowden, A. 2001. Detection of errors of interpretation in experiments in enzyme kinetics. Methods 24:181–190 Di Cera, E., K. P. Hopfner, and Q. D. Dang. 1996. Theory of allosteric effects in serine proteases.Biophys. J.70:174–181. Dixon, M. 1972. The graphical determination of Km and Ki.Biochem. J.129:197–202. Dixon, M., and E. C. Webb. 1979.Enzymes. New York: Academic Press. Dowd, J. E., and D. S. Riggs. 1965. A comparison of estimates of Michaelis–Menten kinetic constants from various linear transformations.J. Biol. Chem.240:863–869. Duggleby, R. G. 1986. Progress curves of reactions catalyzed by unstable enzymes. Atheoretical approach.J. Theor. Biol.123:67–80. Duggleby, R. G. 1995. Analysis of enzyme progress curves by nonlinear regresion. Methods Enzymol.249:61–90. Duggleby, R. G. 2001. Quantitative analysis of the time courses of enzyme-catalyzed reactions.Methods 24:168–174. Schnell, S., and P. K. Maini. 2000. Enzyme kinetics at high enzyme concentration. Bull. Math. Biol.62:483–499. Schnell, S., and P. K. Maini. 2002. Enzyme kinetics far from the standard quasi-steady-state and equilibrium approximations. Math. Comput. Modelling 35:137–144. Juers DH, Hakda S, Matthews BW, Huber RE (2003). "Structural basis for the altered activity of Gly794 variants of Escherichia coli beta-galactosidase." Biochemistry 42(46);13505-11. PMID: 14621996 Read More