Investigate The Effect Of Substrate Biology Essay

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Enzymes are biological catalysts which are protein molecules and effects the rate of the reaction without undergoing any change themselves. They catalyse all the metabolic processes. The rate of an enzyme catalysed reaction is investigated by changing the variable parameters like pH, temperature, concentration of substrate and enzyme and the process is called enzyme kinetics. The most significant parameter affecting the rate of an enzyme catalysed reaction is the concentration of the substrate, which is maximum at the starting of the reaction. The concentration of the substrate decreases gradually with time as the concentration of the product increases. This characteristic property is called the saturated phenomena. The plot of the reaction rate (Vi) and substrate concentration [S] gives a hyperbolic curve as shown in figure 1.

Figure 1. The plot of reaction rate against concentration of substrate.

At low concentration of [S], the reaction gives a straight line and follows first order rate law. The rate of the reaction decreases with the increase in concentration of the substrate. At very high concentration of [S] the reaction becomes zero order.

The enzyme -chymotrypsin is a crystalline compound and its property can be determined on a molecular basis. It acts as a catalyst in the breaking of the peptide bond and also breaks amides to individual amino acids. [1] It also catalyses the hydrolysis of esters and exhibits the characteristic saturation phenomena where the concentration of the enzyme is very low and that of the substrate is very high. A transition state takes place where the enzyme binds to the substrate prior to the initiation of the reaction. (Equation 1)

Where E = Enzyme

S = Substrate

Km = Dissociation rate of binding

kcat = Catalytic rte constant

The condition when [S]o is greater than [E]o is given by Michaelis-Menten equation. (Equation 1)

Where V = Reaction rate

Vmax = Maximum rate of the reaction

The kinetics of -chymotrypsin catalysed reaction can be observed spectrophotometrically. 4-nitrophenyl acetate is a chromophoric substrate which on combination with the enzyme can be observed easily. [2] The hydrolysis of 4-nitrophenyl acetate catalysed by -chymotrypsin gave 4-nitro phenol was found to be biphasic.

Method

A Gensys spectrophotometer was used in kinetic mode to record the absorbance at 410 nm every 30 seconds for 9 minutes. The cell compartment was thermostated at 25oC. A buffer solution of potassium dihydrogen phosphate (0.04M, pH 7.4) was prepared. A enzyme solution was made by dissolving -chymotrypsin in the buffer (2 gL-1). Enzyme solution (26 mL) is mixed with buffer solution (30 mL) in a beaker. A stock solution is prepared using 4-nitro phenyl acetate (0.04 g) in acetonitrile (25 mL). A study was conducted at 5 different concentration of the substrate. The aqueous and the organic layer is mixed together to start the hydrolysis reaction of 4-nitrophenyl acetate. Each of the experiment has run and blank measured by spectrophotometer. The rate of non enzymatic hydrolysis is taken to be zero.

Results

The results of absorbance against time are encapsulated in Table 1.

Time (min)

Absorbance 1

Absorbance 2

Absorbance 3

Absorbance 4

Absorbance 5

0

0.758

-0.071

0.647

-0.085

-0.089

0.5

0.814

0.776

0.718

o.677

0.465

1

0.872

0.836

0.774

0.750

0.525

1.5

0.929

0.898

0.822

0.807

0.561

2

0.988

0.961

0.869

0.855

0.585

2.5

1.047

1.023

0.914

0.901

0.600

3

1.106

1.086

0.959

0.945

0.611

3.5

1.165

1.149

1.001

0.987

0.618

4

1.224

1.211

1.041

1.028

0.621

4.5

1.284

1.274

1.079

1.065

0.623

5

1.344

1.335

1.114

1.098

0.625

5.5

1.403

1.398

1.145

1.127

0.626

6

1.462

1.460

1.172

1.152

0.625

6.5

1.523

1.523

1.194

1.172

0.626

7

1.583

1.586

1.213

1.188

0.627

7.5

1.644

1.649

1.227

1.199

0.627

8

1.704

1.711

1.237

1.205

0.627

8.5

1.766

1.774

1.243

1.209

0.626

9

1.826

1.837

1.246

1.211

0.626

Table 1. The table showing absorbance against time

The calculation of Absorbance/17700 = absorbance /l = 4-nitrophenolate/ M is shown in table 2

Time (min)

4-nitro phenolate/ M 1

4-nitro phenolate/ M 2

4-nitro phenolate/ M 3

4-nitro phenolate/ M 4

4-nitro phenolate/ M 5

0

4.2825x10-05

-4.011 x10-06

3.6554 x10-05

-4.8023 x10-06

-5.0282 x10-06

0.5

4.5989 x10-05

4.3842 x10-05

4.0565 x10-05

3.82486 x10-05

2.62712 x10-05

1

4.9266 x10-05

4.3842 x10-05

3.6554 x10-05

3.82486E-05

2.62712E-05

1.5

5.2486 x10-05

4.3842 x10-05

4.0565 x10-05

3.82486 x10-05

2.62712 x10-05

2

4.2825 x10-05

4.3842 x10-05

3.6554 x10-05

3.82486 x10-05

2.62712 x10-05

2.5

4.5989 x10-05

4.3842 x10-05

4.0565 x10-05

3.82486E-05

2.62712E-05

3

4.9266 x10-05

4.3842 x10-05

3.6554 x10-05

3.82486 x10-05

2.62712 x10-05

3.5

5.2486 x10-05

4.3842E-05

4.0565 x10-05

3.82486 x10-05

2.62712 x10-05

4

4.2825 x10-05

4.3842 x10-05

3.6554 x10-05

3.82486 x10-05

2.62712 x10-05

4.5

4.5989 x10-05

4.3842 x10-05

4.0565 x10-05

3.82486 x10-05

2.62712 x10-05

5

4.9266 x10-05

4.3842 x10-05

3.6554 x10-05

3.82486 x10-05

2.62712 x10-05

5.5

5.2486 x10-05

4.3842 x10-05

4.0565 x10-05

3.82486 x10-05

2.62712 x10-05

6

4.2825 x10-05

4.3842 x10-05

3.6554 x10-05

3.82486 x10-05

2.62712 x10-05

6.5

4.5989 x10-05

4.3842 x10-05

4.0565 x10-05

3.8248 x10-05

2.62712 x10-05

7

4.9266E-05

4.3842E-05

3.6554 x10-05

3.82486 x10-05

2.62712 x10-05

7.5

5.2486E-05

4.3842E-05

4.0565 x10-05

3.82486 x10-05

2.62712 x10-05

8

4.2825E-05

4.3842E-05

3.6554 x10-05

3.82486 x10-05

2.62712 x10-05

8.5

4.5989E-05

4.3842E-05

4.0565 x10-05

3.82486 x10-05

2.62712 x10-05

9

4.9266E-05

4.3842E-05

3.6554 x10-05

3.82486 x10-05

2.62712 x10-05

Table 2. Table showing absorbance /M against time

Discussion

Factors like pH and temperature which can effect the substrate or the enzyme can also change the reaction rate. The chromophores play an important role in spectrophotometric reactions as they can be easily monitored. The rate of the enzyme catalysed reaction can be measured either by the rate of disappearance of the substrates or the rate of formation of the products. The reaction is allowed to continue by mixing both the substrate and the catalyst and the rate is measured by measuring the concentration of the products formed which helps to calculate the rate of activity of the enzyme per unit time. The concentration of the products formed is measured by calculating the absorbance with the help of Michaelis Menten equation. The affinity of the substrate to the enzyme is given by the Michaelis Menten constant.

Conclusion

A study on the effect of substrate on the rate of hydrolysis of 4-nitrophenyl acetate catalysed by -chymotrypsin was carried out.