Determination of Kinetics of Lactase Enzyme

Determinationof Kinetics of Lactase Enzyme

Determinationof Kinetics of Lactase Enzyme

Abstract

Thepractical is used to determine the enzymatic property of the lactaseunder the influence of analogue substrate ONPG. The measurements areattained at a specific absorbance of 420nm wavelength. Severalinhibitors are used in the experiment to facilitate the determinationof their effects on the enzyme activity of interest. From the plots,the following parameters were obtained, both for the enzyme and alsofor the substrate: 200mM, 100mM, 100mM and 75mM. The values wherehowever not accurate putting into consideration the possibilities oferrors due to interferences from other molecules present in thereaction mixture. In appropriate time line, the reading of theabsorbance may also have contributed to the marginal errors. Thissignificantly affected the final plotting of the graph and thuspossibility of fairly lower or higher values of the kinetic values.However, the plots were able to facilitate the final values bygenerating relatively smooth curves. It is noted that the valuesactually decrease with increasing dilutions such that a concentratesolution generates a steep plot with a short time interval requiredto attain the maximum peek as show on the Michealin plots. This givesa higher level of Km value which rapidly falls since the enzyme doesnot act as a limiting factor in this case. However, as the dilutionincreases, the levels of the values fall since the minimal enzyme isavailable to partake the conversion process of the substrate to theend product. The last dilution is so large that the time interval istoo small to accommodate the reaction process and thus giving zerovalues in most of the cases and thus insignificant to record thevalues. At this point, the enzyme concentration is too low and thusoverwhelmed by the high level of the substrate. This combined withthe fact that low lactase level decreases the rate of collision withthe substrate molecules greatly affecting the rate of the enzymeactivity and thus taking a longer time to attain the Vmax value. Inconclusion, the rate of the lactase enzyme to convert the analoguesubstrate to the end product is greatly affected by the enzymeconcentration such that an increase in dilution greatly lowers therate of the enzyme activity. A standard plot facilitates the actualquantitative amount of substrate being converted to the end product.

Thisexperiment tests the effect of enzyme concentration on the activityof lactase enzyme activity. It also tests the effect of the inhibitoron the enzyme.

Theaim of the practical was to determine the major kinetic values underthe influence of inhibitors. The practical was based on the abilityof the analogue substrate to absorb light under the effect of thespectrophotometer at varying substrate concentration. Lactase enzymehas the ability to cleave lactose into its basic products, glucoseand galactose, as shown below.

Theprocess is hydrolytic. The experiment is however usually done usingan analogue substrate for the enzyme since the end products of thereaction in a novo system are all colorless and thus hard todetermine them using a spectrophotometer. As a resultortho-nitro-phenyl-galactoside is used, and which can be cleaved bythe enzyme to generate ortho-nitrophenolate which is yellow in colourand can be determined spectrophotometrically at 420nM while the otherproduct is galactose that is colorless (Wingard et al., 2014).

Enzymekinetics is based on the ability of the substrate to bind at theactive site of an enzyme. This is usually affected by the enzyme andsubstrate concentration in case of an enzyme-substrate reaction. Thisalso affects the rate of dissociation of the product from the activesite as shown below (Wingard et al., 2014).

Thereaction can be thought to be a key-and-lock model in which thesubstrate locks to the enzyme perfectly and that there is minimalfree enzyme at the lowest substrate concentration. There is then aconformational change in the enzyme active site such that thesubstrate gets enough time to be converted to the end products(Wingard et al., 2014).

Enzymerate of reaction can be affected by inhibitors. The type of inhibitoraffects the reaction in different ways thus affecting the finalkinetic entities.

Competitiveinhibitors compete with the substrate for the active site and thusthe reaction can be reversed to normal by increasing the substrateconcentration. This usually leads to increased Km but unchanged Vmax.On the other hand, a non-competitive inhibitor acts as a permanentallosteric regulator by binding to a different part of the enzyme andnot the active site and thus the reaction is permanent and increasingthe substrate concentration does not revert the effect. In such acase, the levels of Km is unchanged while that of Vmax falls. Allthis can be determined experimentally.

Materialsand Reagents

  • o- Nitrophenolate 0.5mL, Buffer 3.5mL

  • Enzyme 0.5mL dilution stock solutions

  • Stopwatch, Spectrophotometer, test tubes, Sodium carbonate

Procedure

Volumesof 3.5mL buffer, 0,5mL substrate and 0.5mL enzyme stock were mixedwith the enzyme being added last

Thereaction was started within the required time interval and thenstopped by the addition of sodium carbonate.

Theabsorbance was the read and recorded for the different dilutionenzyme stock solutions at an interval of 2.5 seconds.

Results

Thetable content of absorbance in relation to enzyme dilutions. Thevalues were determined and recoded involving absorbance in responseto time. The table indicates the Rate of reaction in relation tosubstrate concentration for different enzyme dilutions.

1:1

1:5

1:25

1:125

1:625

0.0

0.0

0.0

0.00

0.0

604

245.6

156

130

0.0

812.2

500

391

306

0.0

1424

780

607

459

0.0

1899

1050

800

612

0.0

Fromthe plot,the kinetic values for the enzyme lactase are as follows|:

Enzyme concentration

Vmax

1/2Vmax(when the Vmax value is half)

1:1

150

75

1:5

100

50

1:25

70

35

1:125

60

30

Thekinetic values of the substrate (Vmax in mM/sec and Km in mMolar)

Substrate concentration

Vmax

Km

1:1

150

200

1:5

100

100

1:25

70

100

1:125

60

75

Discussion

Theplot generates a parabola shape. This indicates that at some pointthe enzyme or the substrate becomes the limiting factor in theprocess. The start of the plot is steep indicating that the rate ofproduct formation is high since the substrate concentration is thelimiting factor and thus the little that is available is quicklyconverted to the end product. The levels of the kinetic parametersare found to vary from one dilution enzyme value. As theconcentration of the active lactase enzyme decreases, the values alsoreduce and the plots reduce in their steepness. This is enhanced bythe fact that the enzyme becomes the limiting factor, and thus, theactive site is fully occupied taking a longer time for theconversions. The Km value being the substrate concentration when thereaction is just halfway indicates the rate of reaction. Thus, thehigher the kM value, the higher the rate of the enzyme activity(Cornish-Bowden, 2013). At maximum rate, Vmax the reaction raterapidly falls to zero. This is due to the depletion of the substrateavailable in the reaction mixture. The early this point is attainedthe faster the completion of the reaction rate. The values of theexperiment maybe relatively lower due to experimental errors causedby several factors. These may include interference from otherparticles in the reaction mixture resulting to error reading of theabsorbance value, affecting time intervals and thus giving a slightadvantage of the time of one mixture over the proceeding reactionmixtures. A Lineweaver-Burk plot provides a direct andlinear-accurate determination of the kinetic parameters(Cornish-Bowden, 2013).

Conclusion

Measurementsof enzyme kinetics facilitate the determination of its properties.This also facilitates the determination of the presence of anyinhibitors of the enzyme of interest during the study of theallosteric regulation of enzymes. The results for the enzyme obtainedfrom the practical indicate an effect of enzyme concentration on therate of the reaction. The more concentrated the enzyme, the higherthe rate was and the larger the Km values were.

References

Cornish-Bowden,A. (2013).&nbspFundamentalsof enzyme kinetics.John Wiley &amp Sons.

Wingard,L. B., Katchalski-Katzir, E., &amp Goldstein, L. (Eds.).(2014).Immobilizedenzyme principles: applied biochemistry and bioengineering (Vol.1). Elsevier.