Name: Mubatsiri Makoni

Reg. Number: R136543L

Program: MBCHB

Year: 2013

Biochemistry Practical Write up: Enzymology

Date: 2 October 2013

Title: Determination of The Effect Of pH On Enzyme Activity By The Measuring The Rate

At Which Amylase Breaks Down Sugars.

Introduction: Almost all enzymes are globular proteins that catalyse reactions in living

systems. They have extraordinary specificity, which is each enzyme catalyses only one

reaction or one group of closely related reaction, and they are very effective as they may

speed up reactions by factors of 1020 (Gillespie et al, 1989).

Substrates are molecules on which an enzyme works. Substrates are converted into products

by bond-making and bond-breaking processes. The substrate interacts with side chains of the

amino acids on the enzyme and these interactions cause the making and breaking of bonds

(Wilbraham et al, 1987).

Reactions in living systems progress very slowly in the absence of an enzyme and it would

take a very long time to produce an adequate quantity of products. Enzymes are catalysts

which remain unchanged after the reaction (Rayner-Canham et al, 1983). Small quantities of

they are therefore needed in comparison to the quantity of substrate.

An enzyme is made under the direction of a gene (a segment of a DNA molecule). DNA is

transcribed to RNA which is then translated to an enzyme which can then catalyse other

reactions (Brady and Holum, 1981).

Denaturation of an enzyme is an alteration in its structure brought about by heat, chemical

changes and other factors in the enzyme’s environment. This changes its three dimensional

shape and hence the shape of the active site, so the substrate cannot bind to it anymore and

this slows down and ultimately stops the enzyme activity (Compton, 1979). Enzymes

therefore have an optimum pH which is a range in which they function most efficiently.

Outside this pH range their activity reduces and ultimately they are denatured. In this
experiment, the rate of action of the enzyme amylase was measured at different values of pH

in order to determine the effect of pH on enzyme activity.

Materials and methods: Solutions of phosphate buffer solutions were mixed in a series of

tubes to produce seven solutions with pH ranging from 5.5 to 8.0. These were added to some

starch, placed in a water bath and warmed to 37oC and saliva was added to each tube. The

saliva was the source of the enzyme salivary amylase which breaks down starch. After ten

minutes the reaction was stoped by adding 20% NaOH to each tube. 5% Methylamine

hydrochloride was added to each tube and the tubes were place in a boiling water bath for

two minutes. They were then cooled and absorbance readings at 520nm were obtained using

a spectrophotometer (SpectronicR 20 GenesysTM) and blanking with a solution similar to the

ones in each test tube but without the enzyme.

Results:

Tube pH Reaction Velocity

(colorimeter reading)
1 5.5 0.118
2 6.0 0.485
3 6.4 0.670
4 6.8 0.794
5 7.2 0.092
6 7.6 0.078
7 8.0 0.012
Table 1

Discussion: Almost all enzymes are globular proteins. Their shapes allow them to form an

active site into which the substrate can fit and this is a specific system. The overall charge of

the surface of the enzyme affects this shape and therefore enzyme activity. In this experiment

the absorbance was directly proportional to the rate of enzyme activity since the absorption

was an indirect measure of the amount of substrate formed per unit time.
The graph, graph 1, indicates an increased rate of reaction from pH 5.5 approaching pH 6.8.

This was because the pH of the solutions was nearing the optimum pH range for amylase

which is pH 5.6-6.9. When the solutions are too acidic, the overall charge on the surface of

the enzyme distorts its shape. At an extremely acidic pH, the enzyme is denatured and no

reactions can be catalysed. As the pH increases the extent of distortion is lowered until pH

6.8, which results in an increase in enzyme activity up to this point. In this range the overall

charge in the solution promotes the maintenance of an optimum and stable 3-dimensional

structure which can catalyse the reaction more efficiently. The rate of reaction then

decreased significantly with each increase in pH, from pH 6.8 onwards. This was because the

optimum pH range for the enzyme was passed and the solution was now too alkali for the

maintenance if the enzymes structure. Enzyme activity was lost until the solution became too

alkali and denaturation occurred. At an extremely alkali pH all the enzymes will be denatured

and no reactions will be catalysed.

Conclusion: Enzymes have an optimum pH which is the pH they work at best. Out of this pH

range, their rate of action is lowered until they are denatured and no longer function. The

optimum pH for alpha amylase is between 5.6 and 6.9.

Questions:

a) NaOH stopped the reaction by denaturing the enzyme alpha amylase. Adding NaOH Made

the solution very basic, changing the concentrations of charges in the solution. The enzyme

has an active site to which the substrate binds and this is changed when the protein is

denatured since some bonds holding it together would have been broken. The enzyme also

relies on charges in its active site for it to attract and bind to the substrate and this system is

disrupted by the addition of the alkali.

b) 37oC is he optimum pH for amylase activity. This temperature was chosen and used

because it would allow for a clearer pattern to be seen as the rate of the enzyme activity drop

or rise will not be a result of the temperature.

c) Tube 8 was there to give us a reference point. It was a solution in which no reaction

happened since there was no enzyme, but since every other ingredient was present it could be

used for comparison. Tube 8 was therefore used to blank the spectrophotometer.

d) I would expect the optimum pH for pepsin to be lower because it works in the stomach<

which is a very acidic environment.

e) Alkalosis would affect the effect of enzyme activity more. This is because the optimum pH

of the enzyme is 5.6-6.9 which is slightly acidic. Alkalosis will therefore mean the pH in the

body is further away from the optimum pH of the enzyme so its activity will be decreased

more.

References

1) Gillespie, R Humphreys, D Baird, N and Robinson, E. (1989). Chemistry. 2nd edition.

(Allyn and Bacon, Inc, Massachusetts, USA) pg 1099

2) Wilbraham, A Staley, D Simpson, C and Matta, M. (1987). Addison-Wesley

Chemistry. (Addison-Wesley Printing Company, Inc, California, USA) pgs 644-645

3) Rayner-Canham, G Last, A Perkins, R and Roode, M. (1983). Foundations of

Chemistry. (Addison-Wesley Publishing Company, Massachusetts, USA). pgs 248-

249

4) Brady, J and Holum, J. (1981). Fundamentals of Chemistry. (John Wiley and Sons,

New York, USA) pg 664

5) Compton, C. (1979). Inside Chemistry. (McGraw-Hill Book Company, New York,

USA) pgs 212-264