NAME:

I.D. #:

LAB PARTNER:

DATE:

Course Code & Title of Lab: BIOL 2363 - Properties of an Enzyme: Succinate Dehydrogenase

Results:

Table 1: Calibration Curve for UreaVol. Of 0.06% NT (mL) 0.0 0.1 0.2 0.3 0.4 0.6 0.8 1.0Vol of distilled water (mL) 1.0 0.9 0.8 0.7 0.6 0.4 0.2 0.0Vol. Of 0.35% Na2S2O4 (mL) 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0Vol. Of acetone (mL) 7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0A540nm 0.003 0.086 0.193 0.289 0.391 0.543 0.755 0.921

Table 2. Time Course ReadingsTime (min) 0’ 0’ 5’ 10’ 15’ 20’ 30’ 30’Tube No. 1 2 3 4 5 6 7 8Succinate (mL) 0.0 0.9 0.9 0.9 0.9 0.9 0.9 0.0Buffer pH 7.5 (mL) 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.50.2% NT (mL) 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5Water (mL) 1.2 0.3 0.3 0.3 0.3 0.3 0.3 1.2Homogenate (mL) 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0A540nm 0.0

00.00

0.874

0.927

0.991

0.935

0.828

0.409

Table 3: Effect of Substrate Concentration on Enzyme Activity ReadingsTube No. 9 10 11 12 13 14 15 16Succinate (mL) 0.0 0.05 0.1 0.25 0.4 0.5 1.0 1.1Buffer pH 7.5 (mL) 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.50.2% NT (mL) 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5Water (mL) 1.2 1.15 1.10 0.95 0.8 0.7 0.2 0.1Homogenate (mL) 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0A540nm 0.042 0.689 0.860 0.952 1.001 0.829 0.931 0.931A540nm with value due to endogenous activity subtracted

0.0 0.647 0.818 0.910 0.959 0.787 0.889 0.889

Table 4. Effect of Inhibitor on Enzyme Activity ReadingsTube No. 17 18 19 20 21 22 23 24Succinate (mL) 0 0.05 0.1 0.25 0.4 0.5 1.0 1.1Malonate (mL) 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1Buffer pH 7.5 (mL) 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5

1

0.2% NT (mL) 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5Water (mL) 1.1 1.05 1.0 0.85 0.7 0.6 0.1 0Homogenate (mL) 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0A540nm 0.109 0.086 0.161 0.406 0.562 0.599 0.748 0.779A540nm with value due to endogenous activity subtracted

0.0 0.077 0.052 0.297 0.453 0.490 0.639 0.670

Table 5: µmoles formazan produced/min with changing substrate concentrationSubstrate Concentration (moles/L)

[S]

1/[S] v(µmoles formazan produced/min.)

1/v v (µmoles formazan produced/min) with malonate present.

1/v with malonate

0 0 0

0.000001 1000000 5.023 0.1991 0.598 1.6722

0.000002 500000 6.35 0.1575 0.404 2.4752

0.000005 200000 7.065 0.1415 2.3 0.4347

0.000008 125000 7.446 0.1343 3.519 0.2842

0.00001 100000 6.110 0.1637 3.804 0.2629

0.00002 50000 6.902 0.1449 4.960 0.2016

0.000022 45454.5 6.902 0.1449 5.202 0.1922

Calculations:

Sample calculation for a value of v in one of the tubes in this experiment

Using the Absorbance determined for tube 10:

A= cl where =1.84 x 10^4M¯¹ x cm¯¹ε ε

c= X

l=1cm

A=0.647

Therefore 0.647=1.84 x 10^4 M¯¹cm¯¹ x X x 1cm

2

X=0.647/1.84 x 10^4 M¯¹

X=3.5163 x 10^-5 M¯¹

Converting moles (M) to µmol, multiply by 1 x 10^6

Therefore X=35.163 µmol

35.163 µmol was produced in 7minutes; therefore in 1 minute 35.163/7 µmol was produced.

i.e. in 1 minute 5.023 µmol was produced.

Hence v=5.023 µmol/min

Sample Calculation for the Substrate content [S] in one of the tubes in this experiment:

Concentration of Sodium Succinate (the substrate) used = 0.02M

Calculating the Substrate content in tube 10:

In 1000mL of Sodium Succinate solution there are 0.02 mol

In 1mL of Sodium Succinate solution there are 0.02/1000 mol

In 0.05mL of Sodium Succinate solution there are 0.02/1000 x 0.05 = 0.000001mol

Sample Calculation for 1/v in one of the tubes of this experiment:

Calculating 1/v in tube 10:

V=5.023µmol/min

1/v= 1/5.023=0.1991

Sample Calculation for 1/[S] in one of the tubes of this experiment:

Calculating 1/[S] in tube 10

[S]=0.000001mol

1/[S]=1000000mol¯¹

3

Discussion:

In this experiment, the effect of increasing substrate/succinate concentration on the oxidation reaction of succinate to fumarate and the effect of the competitive inhibitor malonate on increasing concentrations of succinate on the oxidation reaction of succinate to fumarate were investigated.

The enzyme succinate dehydrogenase can be inhibited by the competitive inhibitor malonate. Competitive inhibitors bind reversibly to the active site therefore at high substrate concentrations the action of a competitive inhibitor is overcome (Hames and Hooper 2005).This can be seen in Table 5 since the concentration of formazan increased with increasing substrate concentration.

The maximum velocity (Vmax) is the velocity at which the rate of enzyme activity becomes independent of substrate concentration and is unaffected by the presence of an inhibitor (Hames and Hooper, 2005). This is shown on Graph 5.

Km is an approximation of the affinity of the enzyme for the substrate. A small KM indicates high affinity, and a substrate with a smaller KM will approach vmax more quickly (Nelson and Cox 2008). This is the case when an inhibitor is present, e.g. malonate.

Precautions taken in this lab were to ensure that all reactions were stopped at the same time to ensure that time was not a variable. Additionally care was taken in pippetting the enzyme to ensure that exact values were dispensed and there were not excess enzyme in any tube.

Additional Discussion:

1. Turnover number (also termed kcat) is defined as the maximum number of molecules of substrate that an enzyme can convert to product per catalytic site per unit of time and can be calculated as follows: kcat = Vmax/[E]T

2. The affinity of succinate dehydrogenase for succinate and for malonate can be determined from this experiment.

This can be done by measuring the Km, which is an approximation of the affinity of the enzyme for the substrate based on the rate constants within the reaction, and it is numerically equivalent to the substrate concentration at which the rate of conversion is half of vmax. A small KM indicates high affinity, and a substrate with a smaller KM will approach vmax

more quickly. Very high [S] values are required to approach vmax, which is reached only when [S] is high enough to saturate the enzyme.

4

In this lab, increasing concentrations of succinate were allowed to react with a fixed volume of enzyme for an equal period of time. For each different concentration of succinate, a value v (concentration of product formed/ min) was found. These values were then plotted into a Lineweaver-Burk plot from which Km was found. Another Lineweaver-Burk plot was also plotted for the tubes containing malonate and Km found.

3. In the presence of malonate, the Km value for succinate would increase. Km is an approximation of the affinity of the enzyme for the substrate. A high Km indicates low affinity. Malonate being a competitive inhibitor bind reversibly to the active site therefore lowering the affinity of the enzyme for the succinate substrate since it would be unable to bind to the succinate.

4. The homogenate was prepared in 0.25M sucrose solution to separate the different organelles and proteins (enzymes) in the tissue based on their density.

During the centrifugation process, tissues can be separated according to their density. The sucrose solution in the centrifuge tube is highly concentrated below and decreases in concentration upwards. This difference in concentration allows for various organelles to move towards equilibrium where their density is equal to that of the sucrose. Different organelles would have different equilibrium densities, hence making separation easier.

5. The more dense organelles would be present in the pellet, e.g. ribosomes, mitochondria, peroxisomes and lysosomes

5

References

Hames, David, and Nigel Hooper. Instant Notes Biochemistry 3/e. New York: Taylor & Francis Group, 2005.

Nelson, David L, and Michael M Cox. Lehninger Principles of Biochemistry. New York: W.H. Freeman and Company, 2008.

6