Вы находитесь на странице: 1из 14

NAME:Micheal Hinds NAME: Andrew Grant Date: Monday 17th September 2012

Lab Day: Monday Week 3 Experiment 6

Experiment 8: Activation energies for an enzyme-catalyzed and acid-catalyzed hydrolysis Theory: Enzymes have the ability to bring about vast increases in the rates of reactions; in most cases, the rates of enzyme-catalyzed reactions are faster than those of uncatalyzed reactions by factors of 10 6- 10 12. Enzymes show remarkable specificity for their substrates and for formation of specific products. According to Emil Fischer theory, enzymes have the ability to distinguish between and glycosidic linkages that led him to formulate the lock and key hypothesis for enzyme specificity. According to the lock and key hypothesis, the specificity of an enzyme and its substrate comes from their geometrically complementary shapes. In an enzyme-catalyzed reaction, the enzyme and the substrate combine to form an enzyme-substrate complex. Formation of the enzymecatalyzed complex often induces a conformational change in the enzyme called an induced fit that allows it to bind the substrate more effectively.

Diagram above showing activation energy, is the amount of energy needed to get the reactants to the transition state, in which bonds are broken and new bonds are formed. Enzymes are proteins that catalyze chemical reactions. Enzymes use less activation energy and so speed up the reaction (Enzymes lowers the activation energy neede to start up a reaction).

RESULTS: TABLE 1: SHOWING ABSORBANCE READINGS TAKEN AT 290 nm FOR THE ENZYME-CATALYZED REACTION AT 303K, PERFORMED AT THREE (3) MINUTE INTERVALS Tube No. Time(mins) Time (seconds) Absorbance 0.00 Blank 0 0 0.471 1 3 180 0.929 2 6 360 1.020 3 9 540 .611 4 12 720 .810 5 15 900 1.092 6 18 1080 1.031 7

TABLE 2: SHOWING ABSORBANCE READINGS TAKEN AT 290 nm FOR THE ENZYME-CATALYZED REACTION AT 313K, PERFORMED AT THREE (3) MINUTE INTERVALS Tube No. Time(mins) Time (seconds) Absorbance 0 0 0.833 8 3 180 1.031 9 6 360 0.987 10 9 540 0.886 11 12 720 0.753 12 15 900 1.249 13 18 1080 1.119 14 TABLE 3: SHOWING THE RATE CONSTANTS OBTAINED FOR THE ENZYMECATALYZED REACTIONS CARRIED OUT AT 303K AND 313K, AND THE VALUES PLOTTED TO GET THE GRAPH THAT DESCRIBES THE ENZYME-CATALYZED REACTION Temperature (T)(K) (1/T) Rate constant (k)(s-1) ln k 0.0033 3.7667 10-4 -7.8841 303 0.0032 3.8778 10-4 -7.8551 313

TABLE 4: SHOWING ABSORBANCE READINGS TAKEN AT 290 nm FOR THE ENZYME-CATALYZED REACTION AT 338K, PERFORMED AT FIVE (5) MINUTE INTERVALS AND THE VALUES USED FOR THE PLOT OF ln(A-At) AGAINST TIME (SECONDS) Test Tube Blank 1 2 3 4 5 6 7 Time (secs) 0 300 600 900 1200 1500 1800 Absorbance 0.00 0.0771 0.78 0.871 0.976 0.827 0.66 0.86 A 1.361 1.361 1.361 1.361 1.361 1.361 1.361 1.361 A-At 1.2839 0.581 0.49 0.385 0.534 0.701 0.501 ln(A-At) 0.24990232 -0.54300452 -0.71334989 -0.95451194 -0.62735944 -0.35524739 -0.69114918

TABLE 5: SHOWING ABSORBANCE READINGS TAKEN AT 290 nm FOR THE ENZYME-CATALYZED REACTION AT 348K, PERFORMED AT THREE (3) MINUTE INTERVALS AND THE VALUES USED FOR THE PLOT OF ln(A-At) AGAINST TIME (SECONDS) Test Tube Time (secs) Absorbance A A-At ln(A-At) 8 0 1.361 0.978 -0.02224561 0.383 9 300 0.445 1.361 0.916 -0.08773891 10 600 0.525 1.361 0.836 -0.17912667 11 900 0.527 1.361 0.834 -0.18152188 12 1200 0.546 1.361 0.815 -0.20456717 13 1500 0.596 1.361 0.765 -0.26787945 14 1800 0.648 1.361 0.713 -0.33827386 TABLE 6: SHOWING THE RATE CONSTANTS OBTAINED FOR THE ACIDCATALYZED REACTIONS CARRIED OUT AT 338K AND 348K, AND THE VALUES PLOTTED TO GET THE GRAPH THAT DESCRIBES THE ACID-CATALYZED REACTION Temperature(T)(K) (1/T) Rate constant (k)(s-1) ln k 0.00296 1.8271 10-4 6.0273 x 10-5 338 -4 0.00287 2.7837 10 1.0238 x 10-4 348

CALCULATIONS: Determination of the rate constant, k, using values of the enzyme-catalyzed reaction at 303K: Graph of Absorbanceagainst time (t) (seconds) gave a straight line, where the gradient, m, is equal to the rate constant, k.

Gradient

y 2 y1 x2 x1

Taking the coordinates, (180, 0.071) as (x1, y1) and (1080, 0.410) as (x2, y2): ( 0.410 0.071) (1080 180)s k = 3.7667 10-4s-1 Determination of the rate constant, k, using values of the enzyme-catalyzed reaction at 313K: Graph of Absorbanceagainst time (t) (seconds) gave a straight line, where the gradient, m, is equal to the rate constant, k.

Gradient

y 2 y1 x2 x1

Taking the coordinates, (180, 0.482) as (x1, y1) and (1080, 0.831) as (x2, y2): (0.831 - 0.482) (1080180)s k = 3.8778 10-4s-1 Determination of the rate constant, k, using values of the acid-catalyzed reaction at 338K: Graph of Absorbanceagainst time (t) (seconds) gave a straight line, where the gradient, m, is equal to the rate constant, k.

Gradient

y 2 y1 x2 x1

Taking the coordinates, (300, 0.25386672) as (x1, y1) and (1800, -0.02020271) as (x2, y2): ((-0.02020271) 0.25386672) (1800 300)s k = 1.8271 10-4s-1

Determination of the rate constant, k, using values of the acid-catalyzed reaction at 348K: Graph of Absorbanceagainst time (t) (seconds) gave a straight line, where the gradient, m, is equal to the rate constant, k.

Gradient

y 2 y1 x2 x1

Taking the coordinates, (180, -0.08773891) as (x1, y1) and (1080, -0.33827386) as (x2, y2): ((-0.33827386) (-0.08773891)) (1080 180)s k = 2.7837 10-4s-1

For the preparation of reaction solution: 80/100 x 50 = 40mL of water 20/100 x 50 = 10 mL of 1-propanol For the enzyme catalysed reaction: Where R= gas constant= 8.314 J K-1 mol-1 Lnk2 lnk1 = (-Ea/R)((1/T2) (1/T1)) -7.8551 (-7.8841) = -Ea/8.314 ((1/(313))-(1/(303)) 0.0029 = (-Ea/8.314) (-0.0001) -Ea/8.314 = 0.0029 - 0.0001 -Ea/8.314 = -290 -Ea= -290 x 8.314 -Ea = -2411.06 Hence Ea = 2411.06 Jmol-1 (divide by 1000 to convert to kJmol-1) Ea = 2.411 kJmol-1 For the acid catalysed reaction: Lnk2 lnk1 = (-Ea/R)((1/T2) (1/T1)) 1.0238 x 10-4 6.0275 x 10-5= -Ea/8.314 ((1/(313))+(1/(303))) 4.2105 x 10-5= (-Ea/8.314) -0.00009 -Ea/8.314 = 4.2105 x 10-5 -0.00009 -Ea/8.314 = -0.4678 -Ea= -0.4678x 8.314 -Ea = -3.8893 Jmol-1 Hence Ea = 3.8893 Jmol-1

(divide by 1000 to convert to kJmol-1) Ea = 3.8893 x 10-3 kJmol-1

GRAPH 1: ABSORBANCE VERSUS TIME GRAPH FOR THE SEVEN TUBES THAT CARRIED OUT THE ENZYME-CATALYZED REACTION AT 303K
0.45 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 0 200 400 600 800 1000 1200

y = 0.0003x + 0.0357

Absorbance at 290nm

TIME (seconds)

GRAPH 2: ABSORBANCE VERSUS TIME GRAPH FOR THE SEVEN TUBES THAT CARRIED OUT THE ENZYMECATALYZED REACTION AT 313K
0.9

0.8

y = 0.0003x + 0.4364
0.7

0.6

0.5

Absorbance at 290nm

0.4

0.3

0.2

0.1

0 0 200 400 600 800 1000 1200

TIME (seconds)

GRAPH 3: ln(A-At) VERSUS TIME GRAPH FOR THE SEVEN TUBES THAT CARRIED OUT THE ACID-CATALYZED REACTION AT 338K
0.35

0.3

0.25

0.2

ln(A-At) 0.15

0.1

0.05

0 0 -0.05 200 400 600 800 1000

y = 1400 0.31551800 1200 -0.0002x +1600

2000

TIME (seconds)

GRAPH 4: ln(A-At) VERSUS TIME GRAPH FOR THE SEVEN TUBES THAT CARRIED OUT THE ACID-CATALYZED REACTION AT 348K
0 0 -0.05 200 400 600 800 1000 1200

-0.1

-0.15

ln(A-At) -0.2
-0.25

-0.3

y = -0.0003x - 0.0401
-0.35

-0.4

TIME (seconds)

GRAPH 5: PLOT OF ln k AGAINST (1/T) IN ORDER TO DETERMINE THE VALUE OF THE CONSTANTS, EA AND A
y = 1x - 1E-04 1 0 -9 -8 -7 -6 -5 -4 -3 -2 -1 -1 -2 -3 ln k -4 -5 -6 -7 -8 -9 0 1

(1/T)

GRAPH 6: PLOT OF ln k AGAINST (1/T) IN ORDER TO DETERMINE THE VALUE OF THE CONSTANTS, EA AND A
0.0035

0.003 y = 0.9544x + 4E-05 0.0025

0.002 ln k 0.0015

0.001

0.0005

0 0 0.0005 0.001 0.0015 (1/T) 0.002 0.0025 0.003 0.0035

DISCUSSION: After doing calculations, it was found that the Activation energy for the enzyme-catalyzed reaction,which was calculated to be 2.411 kJ mol-1,was much higher than that of the acid catalysed reaction, which was found to be 3.8893 x 10-3 kJmol-1.This is a deviation of the expected trend, since, according to theory, enzymes lower the activation energy of any chemical reaction. An explanation for this is that they, enzymes, providean active site, where reagents and substrates involved in a given reaction, coagulate, and thus, react. The active site allows the substrate to be in a particular orientation for the reaction to occur.This, in turn, facilitatesa smaller activation energy value to be obtained. As a result, enzymes are biological catalyst that do not influence the thermodynamics of the reaction, but changes the reaction pathways, i.e. the kinetics of the reaction by lowering the energy of transition. The anomaly aforementioned could be accounted for from the fact that the enzyme may have been denatured or it lost some activity, due to prolonged storage or being exposed to unoptimal conditions for a long period of time.

With reference to the enzyme catalysed reaction, it was seen that the rate of reaction increased with increasing temperature. Each enzyme hasan optimal temperature and hence the enzyme performed betterat its optimal activity at the higher temperature. The formation of glycosidases was optimal between pH 6.5 and 7.5, and theoptimum temperature for synthesis of glycosidaseswas between 33 and 370C. 3Hence it can be proposed that the higher of the two temperature is closer to 37C

With reference to Graphs 5 and 6, these graphs were plotted and the gradient used to determine the Activiation Energy, EA, as shown in the calculations aforementioned. Graphs 1 to 4 were plotted so that the gradient could be used to determine the rate constants for each of the reactions carried out. The constants were tabulated as shown in tables 3 and 6. The constant, k, determined using the slopes of Graphs 1 to 4, represented the rate constant. As such, the values were taken to be positive, even though a negative gradient was obtained, as seen especially with graphs 3 and 4, as only k is considered.

It was observed that with the increase in temperature the activity increased, this may be due to a higher population of particles with increased kinetic energy, thus causing increased collision and therefore an increased rate, and additionally, the heat may have contributed to vibrational energy which may have contributed to the breaking of bonds.

Generally, the trend observed was that the absorbance of the reaction contents increased, as more products was formed with increased time, as such, the absorbance increased.

One precaution taken to prevent gross experimental errors from occurring was that the enzyme was stored at low temperatures in a refrigerator and in order to prevent moisture condensation, the bottle containing the enzyme was allowed to warm up to ambient temperature, around ten to fifteen minutes before opening.

Some sources of error within the experiment were: due to parallax errors in measuring the phosphate buffer and the solutions prepared, i.e. the solutions of salicin and emulsion. Furthermore, at the start of mixing, the reactants were not properly shaken which was essential for the quenching of the samples.

References: Fryhle,Craig.T.WSolomons. Organic Chemistry 10th Edition 2011.New York: John Wiley and Sons Limited.