Faith Gwenzi

Biochemistry Practical
Experiment 5 Enzymology
R227908S
Introduction
Enzymes are central to every biochemical process. Acting in organized sequences, they catalyze
the hundreds of stepwise reactions that degrade nutrient molecules, conserve and transform
chemical energy, and make biological macromolecules from simple precursors. Through the
action of regulatory enzymes, metabolic pathways are highly coordinated to yield a harmonious
interplay among the many activities necessary to sustain life (Nelson and Cox, 2005).

Enzyme molecules contain a special pocket or cleft called the active site. The active site contains
amino acid side chains that create a three-dimensional surface complementary to the substrate.
The active site binds the substrate, forming an enzyme-substrate (ES) complex. ES is converted
to enzyme-product (EP), which subsequently dissociates to enzyme and product. Most enzyme-
catalyzed reactions are highly efficient, proceeding from 103 to 108 times faster than
uncatalyzed reactions. Typically, each enzyme molecule is capable of transforming100 to 1000
substrate molecules into product each second. The number of molecules of substrate converted to
product per enzyme molecule per second is called the turnover number. Enzymes are highly
specific, interacting with one or a few substrates and catalyzing only one type of chemical
reaction (Champe, 2005).

Just as the amount of substrate can limit the rate of an enzymatic reaction, so the amount of
active enzyme can also limit the rate of an enzymatic reaction. Cells are able to regulate the
number of active enzymes present at any given time. Gene expression is the first way to regulate
the amount of enzyme present, and cells are able to activate genes as particular enzymes are
needed (Mader, 2004). In order to study the effect of increasing the enzyme concentration upon
the reaction rate, the substrate must be present in an excess amount; i.e., the reaction must be
independent of the substrate concentration. Any change in the amount of product formed over a
specified period of time will be dependent upon the level of enzyme present.

Salivary α-amylase randomly cleaves the α(1-4) glycosidic linkages of amylase to yield dextrin,
maltose, or maltotriose. It adopts a double displacement mechanism with retention of anomeric
configuration. This enzyme breaks down starch (and glycogen, since the two are chemically very
similar) by addition of water between glucose units (hydrolysis). The end products of the
amylase reaction are shorter polymers of glucose along with the disaccharide maltose, while
glucoamylase liberates single glucose sugars. Amylase occurs in mammalian saliva and small
intestines and is found in the intestinal tract of most animals. It is also common in plants, where
it degrades stored starch and in some bacteria and fungi that attack plants. Glucoamylase occurs
only in fungi and selected bacteria. Both types of amylases are industrially significant in the
conversion of biomass to refined sugars and sweeteners.
When enzyme concentration is increased, the rate of reaction also increases. This parameter is of
particular interest because it has a biological correlate. In phenylketonuria patients lack
sufficient amounts of the enzyme phenylalanine hydroxylase, which catalyzes the transformation
of phenylalanine to tyrosine. As a result, large concentrations of phenylalanine, phenylpyruvate,
and phenyllactate accumulate in the plasma and urine. Phenylketonuria occurs clinically in the
first few weeks after birth, and if the infant is not placed on a special diet, severe mental
retardation and seizures will occur (Devlin,1997). Therefore the objective of this experiment is to
find out the effect of enzyme concentration on rate of reaction, using salivary amylase as the
sample enzyme.
Materials
Salivary amylase
0.06M Na2HPO4
1% starch solution in 0.01M NaCl
20% NaOH
5% Methylamine hydrochloride

Procedure
Prepare buffer by mixing 5ml of 0.06M Na2HPO4 with 5ml of 0.06M KH2PO4 to give the
optimum pH of 6.8 of salivary amylase
Prepare reaction mixtures as shown in Table 1.0
Table 1.0
Tube Buffer(ml) Starch Water(ml)
Number (ml)

1 1 3 0.8
2 1 3 0.6
3 1 3 0.4
4 1 3 0.2
5 1 3
6 1 3 1.0

Incubate tubes in a water bath at 37°C for approximately 5 minutes.

1 0.2
2 0.4
3 0.6
4 0.8
5 1.0
Stop the reaction after exactly 10 minutes by adding 5 drops of 20% NaOH and mixing well.
Add 5 drops of 20% NaOH to tube 6.
Remove tubes from water bath
Add 0.2ml of 5% Methylamine hydrochloride to each of the six tubes and immediately place the
tubes in a boiling water bath and heat for 2 minutes.
Transfer the tubes to a water bath at room temperature and leave to cool for about 3 minutes.
Read the absorbance at 520nm as before against the blank to be used(tube 6)

Results
Table 1.3 showing the concentrations of salivary amylase enzyme against the colorimetric
readings accumulated.
Tube Enzyme Reaction
Numbe concentratio velocity
r n (ml) (Colorimete
r reading)

1 0.2 0.034
2 0.4 0.129
3 0.6 0.253
4 0.8 0.410
5 1.0 0.460
DISCUSSION

Concentration of salivary amylase in the reaction mixture is directly proportional to the volume
of the diluted saliva added to each test tube, therefore this volume can be taken to represent
enzyme concentration in that tube. When substrate concentration is constant, the reaction
velocity is directly proportional to the concentration of the enzyme thus increasing the
concentration of enzyme increases reaction velocity. However, this will only have an effect up to
a certain concentration, where the enzyme concentration is no longer the limiting factor
([Link], 2007). This coincides with the results from fig 2 which clearly show that rate
of reaction increases linearly with an increase in enzyme concentration.

When substrate concentration is not rate limiting, these reactions are said to be “zero order”
because the rates are independent of substrate concentration, and are equal to some constant k.
The formation of product proceeds at a rate which is linear with time. The addition of more
substrate does not serve to increase the rate. In zero order kinetics, allowing the assay to run for
double time results in double the amount of product. Enzyme activity is generally greatest when
substrate concentration is unlimiting. As substrate is used up, the enzyme’s active sites are no
longer saturated, substrate concentration becomes rate limiting, and the reaction becomes first
order, ie the graph flattens out as there is no longer any increase in rate of reaction (Segel, 1984)

The enzyme binds to or somehow Interacts with the substrate to speed up the reaction. There are
certain sites on the substrate that the enzyme will act on. So as you increase the concentration of
enzymes it increases the number of successful collisions and so to a point the rate of reaction is
directly proportional to enzyme concentration and the higher the concentration of the enzyme the
faster the reaction. However, at some point this will ‘plateau’ – that is it will ‘level’ or reach
equilibrium where the reaction reaches its maximum rate regardless of the enzyme concentration
because all the sites on the substrate are ‘full’ and so the excess enzyme is just floating around
doing nothing because it has nothing to react with(Darnell,1986).

CONCLUSION

The rate of a biochemical reaction increases as the enzyme concentration increases up to a

a) Pre-incubation was necessary to ensure that the reagents in the test tube were brought to
370C which is the normal body temperature and optimum temperature for salivary
amylase. This was also done so that temperature would not be a limiting factor in this
reaction. If this stage was omitted, the reaction would have been very slow due to
inactivation of the enzyme by low temperatures.

b) Amylase hydrolyses starch to maltose and glucose. Glucose is used as a fuel in the cell to,
carry out essential processes that the cell requires in order to live.

c) No hydrolysis will take place because amylase is specific for starch only hence it will not
react with glucose or protein. Increasing enzyme concentration will have no effect on rate
of reaction since there is no substrate.

d) No reaction would occur since amylase does not act on maltose or protein. Increasing
enzyme concentration would have no effect on the reaction since there is no substrate

e) Amylase will act on starch producing maltose and glucose but it will not act on the
protein as it is only specific for starch. Increase in enzyme concentration will increase the
rate of reaction provided the substrate is in excess. Presence of protein may lower the rate
of reaction by disrupting interaction of the enzyme and substrate.

REFFERENCES

Champe, P. C., Harvey, R.A. and Ferrier, D.R. (2005) Lippincott illustrated reviews of
Biochemistry. 3rd Edition (Philadelphia: USA), pg 86
Darnell, J., Lodish, J. and Baltimore, D. (1986). Molecular cell biology. (Scientific American
Books, New York), pg 231
Devlin, T.M. (1997) Textbook of Biochemistry with clinical correlations. 4th Edition. (Wiley-
Liss Inc: New York), pg 96
Lieberman., Mark, A.D., Smith, C. (2007) Mark’s essentials of Biochemistry, A clinical
approach. (Lippincott Williams and Wilkins: New York,USA), pg 149
Mader, S.S. (2004) Biology. 8th Edition (The Mc-Graw Hill companies: New York,USA), pg 109
Nelson, D.L., Cox, M.M. (2005). Lehninger, Principles of Biochemistry. 4th Edition (W.H
Freeman: New York), pg 201
Segel, L.A. (1984). Modeling dynamic phenomena in molecular and cellular biology.
(Cambridge University Press, New York), pg 134