Investigate The Effect Of Substrate Biology Essay

Enzymes are biological accelerators which are protein molecules and effects the rate of the reaction without undergoing any alteration themselves. They catalyse all the metabolic procedures. The rate of an enzyme catalysed reaction is investigated by altering the variable parametric quantities like pH, temperature, concentration of substrate and enzyme and the procedure is called enzyme dynamicss.

The most important parametric quantity impacting the rate of an enzyme catalysed reaction is the concentration of the substrate, which is maximal at the starting of the reaction. The concentration of the substrate decreases bit by bit with clip as the concentration of the merchandise increases. This characteristic belongings is called the concentrated phenomena. The secret plan of the reaction rate ( Vi ) and substrate concentration [ S ] gives a inflated curve as shown in figure 1.

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Figure 1. The secret plan of reaction rate against concentration of substrate.At low concentration of [ S ] , the reaction gives a consecutive line and follows first order rate jurisprudence. The rate of the reaction decreases with the addition in concentration of the substrate.

At really high concentration of [ S ] the reaction becomes zero order.The enzyme i??-chymotrypsin is a crystalline compound and its belongings can be determined on a molecular footing. It acts as a accelerator in the breakage of the peptide bond and besides breaks amides to single amino acids.

[ 1 ]It besides catalyses the hydrolysis of esters and exhibits the characteristic impregnation phenomena where the concentration of the enzyme is really low and that of the substrate is really high. A passage province takes topographic point where the enzyme binds to the substrate prior to the induction of the reaction. ( Equation 1 )Where E = EnzymeS = SubstrateKm = Dissociation rate of adheringkcat = Catalytic rte invariableThe status when [ S ] O is greater than [ E ] O is given by Michaelis-Menten equation. ( Equation 1 )Where V = Reaction rateVmax = Maximum rate of the reactionThe dynamicss of i??-chymotrypsin catalysed reaction can be observed spectrophotometrically.

4-nitrophenyl ethanoate is a chromophoric substrate which on combination with the enzyme can be observed easy.[ 2 ]The hydrolysis of 4-nitrophenyl ethanoate catalysed by i??-chymotrypsin gave 4-nitro phenol was found to be biphasic.


A Gensys spectrophotometer was used in kinetic manner to enter the optical density at 410 nm every 30 seconds for 9 proceedingss. The cell compartment was thermostated at 25oC.

A buffer solution of K dihydrogen phosphate ( 0.04M, pH 7.4 ) was prepared. A enzyme solution was made by fade outing i??-chymotrypsin in the buffer ( 2 gL-1 ) . Enzyme solution ( 26 milliliter ) is assorted with buffer solution ( 30 milliliter ) in a beaker. A stock solution is prepared utilizing 4-nitro phenyl ethanoate ( 0.04 g ) in acetonitrile ( 25 milliliter ) . A survey was conducted at 5 different concentration of the substrate.

The aqueous and the organic bed is assorted together to get down the hydrolysis reaction of 4-nitrophenyl ethanoate. Each of the experiment has run and clean measured by spectrophotometer. The rate of non enzymatic hydrolysis is taken to be zero.


The consequences of optical density against clip are encapsulated in Table 1.Time ( min )Optical density 1Optical density 2Optical density 3Optical density 4Optical density 500.758-0.



















8261.8371.2461.2110.626Table 1. The table screening optical density against clipThe computation of Absorbance/17700 = optical density /i??l = 4-nitrophenolate/ M is shown in table 2Time ( min )4-nitro phenolate/ M 14-nitro phenolate/ M 24-nitro phenolate/ M 34-nitro phenolate/ M 44-nitro phenolate/ M 504.2825×10-05-4.

011 x10-063.6554 x10-05-4.8023 x10-06-5.0282 x10-060.54.

5989 x10-054.3842 x10-054.0565 x10-053.82486 x10-052.

62712 x10-0514.9266 x10-054.3842 x10-053.6554 x10-053.82486E-052.62712E-051.

55.2486 x10-054.3842 x10-054.0565 x10-053.82486 x10-052.62712 x10-0524.2825 x10-054.3842 x10-053.

6554 x10-053.82486 x10-052.62712 x10-052.54.5989 x10-054.3842 x10-054.

0565 x10-053.82486E-052.62712E-0534.9266 x10-054.3842 x10-053.6554 x10-053.82486 x10-052.62712 x10-053.

55.2486 x10-054.3842E-054.0565 x10-053.82486 x10-052.62712 x10-0544.2825 x10-054.3842 x10-053.

6554 x10-053.82486 x10-052.62712 x10-054.54.

5989 x10-054.3842 x10-054.0565 x10-053.82486 x10-052.62712 x10-0554.

9266 x10-054.3842 x10-053.6554 x10-053.82486 x10-052.62712 x10-055.

55.2486 x10-054.3842 x10-054.0565 x10-053.82486 x10-052.

62712 x10-0564.2825 x10-054.3842 x10-053.6554 x10-053.82486 x10-052.

62712 x10-056.54.5989 x10-054.3842 x10-054.

0565 x10-053.8248 x10-052.62712 x10-0574.9266E-054.3842E-053.

6554 x10-053.82486 x10-052.62712 x10-057.

55.2486E-054.3842E-054.0565 x10-053.82486 x10-052.62712 x10-0584.2825E-054.

3842E-053.6554 x10-053.82486 x10-052.62712 x10-058.54.5989E-054.3842E-054.

0565 x10-053.82486 x10-052.62712 x10-0594.9266E-054.3842E-053.

6554 x10-053.82486 x10-052.62712 x10-05Table 2. Table demoing optical density /M against clip


Factors like pH and temperature which can consequence the substrate or the enzyme can besides alter the reaction rate. The chromophores play an of import function in spectrophotometric reactions as they can be easy monitored. The rate of the enzyme catalysed reaction can be measured either by the rate of disappearing of the substrates or the rate of formation of the merchandises.

The reaction is allowed to go on by blending both the substrate and the accelerator and the rate is measured by mensurating the concentration of the merchandises formed which helps to cipher the rate of activity of the enzyme per unit clip. The concentration of the merchandises formed is measured by ciphering the optical density with the aid of Michaelis Menten equation. The affinity of the substrate to the enzyme is given by the Michaelis Menten invariable.


A survey on the consequence of substrate on the rate of hydrolysis of 4-nitrophenyl ethanoate catalysed by i??-chymotrypsin was carried out.


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