7/29/2019 Renee Lab 5

1/3

Lab 5: Enzyme Activity

1 | S h e r i f f

Rene Sheriff

Lab Assignment 5

Biology 101 Lab D2

Csanad Gurdon

22 October 2012

7/29/2019 Renee Lab 5

2/3

Lab 5: Enzyme Activity

2 | S h e r i f f

Title: Effect on Substrate Concentration Upon the Rate of Enzyme-catalyzed Reaction

Abstract:tt

Introduction:

The main subject of this lab is to observe how substrate concentration affects the rate ofthe enzyme-catalyzed reaction. In order to understand this, we must first understand what anenzyme and reaction are and how they are connected.

Reactions are classified as either exergonic or endergonic and have an energy barrier

called activation energy. Exergonic reactions occur spontaneously and the reactants possess ahigher energy than the products; there is a net loss of free energy. This type of reaction can also

be called exothermic which means the reaction provides a net output of heat. Endergonic

reactions occur nonspontaneously and the products possess a higher energy than the reactants;

there is a net gain of free energy. This type of reactions can also be called endothermic whichmeans the reaction provides a net input of heat. Activation energy is the energy required to begin

the reaction. If this set level of energy requirement is not reached, the reaction will not begin,

unless an enzyme helps initiate the chemical reaction.Most enzymes are proteins, some are RNA molecules that catalyze reactions, this helps

reduce the amount of activation energy needed for a reaction. It helps facilitate this reaction or

speeds up the reaction rate. The enzyme itself is never consumed by the reaction; it affects the

kinetics of the reaction but not the thermodynamics of it. This means that the enzyme onlylowers the activation energy for the chemical reaction; it does not govern the final concentrations

of reactants and products. If molecules have a high kinetic energy and are able to reach the

activation energy without the use of enzymes then the chemical reaction will begin and finishwith end results. The same end results or products would have been formed if the enzyme were

present. The only difference would be in the faster rate at which the chemical reaction occurred.

An enzyme can speed up chemical reactions because of its most distinct characteristics:

specificity. It is very likely that the active site of the enzyme is the shape or close to the shape ofits substrate. The substrate is the substance on which an enzyme acts. The active site is a region

in which the substrate binds to form an enzyme-substrate complex. An enzyme-substrate

complex or ES complex is a noncovalent complex that is comprised of both the enzyme and thesubstrate. At any time the substrate can dissociate itself from the enzyme, forming products,

leaving the enzyme free to create more ES complexes with other substrates.

*In order for the enzyme to work efficiently, its optimal conditions must be met.This means that each enzyme has its own most beneficial environment in which it operates the

best, operating the best means being able to create ES complexes with substrate. If the enzyme is

denatured, this means that the active site no longer has its unique original shape and cannot bind

to its substrate, thereby slowing the reaction rate and product formation within a given amount oftime (or with respect to time).

In lab 5, enzyme activity is observed with different substrate concentrations in order to

find the rate at which the enzyme catalyzes the reaction. In Exercise II, Assay II, the rate

indicates the effect of substrate concentration on enzyme activity. I predict the higher thesubstrate concentration the higher the reaction rate.

7/29/2019 Renee Lab 5

3/3

Lab 5: Enzyme Activity

3 | S h e r i f f

Materials and Methods:

The materials used for assay II of lab 5 are as follows: 7 clean spec 20 tubes, a

spectrophotometer (Spectronic 20), water (H2O), buffer (pH 8), enzyme (alkaline phosphatase),and substrate (p-Nitro-Phenyl Phosphate or pNPP).

2.0ml of 2 mM pNPP was transferred from the substrate beaker to tube 1. This is one of

the controls of assay II because it is a spec tube that only contains substrate. Then 1.0 ml is takenfrom tube 1 and transferred to tube 2, which also contains 1.0ml of water (H 20). Then 1.0ml istaken from tube 2 and transferred to tube 3, which also contains 1.0 ml of water. This continues

for tubes 4 and 5 as well. Once tube 5 becomes a mixture of 1.0ml from tube 4 and its own 1.0ml

of water, take 1.0ml of the mixture from tube 5 and discard it so that all tubes contain the samequantity of 1.0ml.

Results:

Discussion:

jknLiterature Cited:

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

0 0.5 1 1.5 2 2.5 3 3.5

Series 1

Series 2

Series 3