Locker #51

Maldisa, Abdelaziz N.
Roberto, Rafey Jhay S.
Antatico, Marty Lawrence

Date Performed:
Date Submitted:

Activity No. 7
Factors Affecting Enzyme Activity
Objectives:
1. To determine how changes in pH affect enzyme activity
2. To determine how changes in temperature affect enzyme activity
3. To determine the effect of coenzymes in enzyme activity
Introduction:
Enzyme activity is affected by a lot of factors. This includes the concentration of the substrate and
the enzyme, temperature, pH and the presence of activators or inhibitors. Changes in the conformation of
enzymes as well as the available substrate needed in order to form the desired products affects enzyme
activity by slowing it down. This is because as the conformation of the enzyme changes, due to factors
such as temperatures higher than the optimum temperature, the protein portion of the enzyme (which
contains the active site) will begin to denature, which slows down enzyme activity and if it reaches boiling
temperatures, destroys the enzyme because the secondary, tertiary and quaternary structures which is held
together by weak forces such as H bonds and disulfide linkages, are disrupted , which results to loss of
catalytic activity for the enzyme. This concept will be discussed extensively in the iodine test discussion.
The increase in enzyme concentration would exponentially increase the rate of reaction. This is
because the quantity of enzymes in the body is almost always much lower than that of its corresponding
substrate, thus increasing the concentration of enzymes will lead to higher rate of reaction because more
enzymes are present to react with the substrate present. Increasing the concentration of substrate will also
lead to an increase in enzyme activity until such time as all of the enzymes are already occupied by the
corresponding substrate, which is termed as the point of saturation. Upon reaching the point of saturation,
the excess substrate would have to wait for their turn until an available active site for enzyme-substrate
complex formation is available. Thus, upon reaching the point of saturation, the rate of reaction would not
display a net gain nor a net loss but a horizontal line to indicate that all of the available enzymes are
occupied with the available substrate, thereby showing no significant change in the rate of reaction.
(SHOW GRAPHS)
Most enzymes operate best in a certain pH. Most enzymes in the human body operate in a near
neutral pH of 7.1-7.8. Some enzymes such as pepsin which is an important digestive enzyme operate best
in an acidic pH of 2.0. Any drastic changes in pH would disrupt the protein structures secondary, tertiary

and quaternary structure and as a result, denature the enzyme.

The presence of activators and inhibitors also affect the activity of the enzyme. Activators increase
enzyme action. Examples include cations which has the capacity to bind temporarily with the enzymes
active site, giving the enzyme an intense positive charge, which facilitates the binding of the substrate.
Inhibitors make enzymes less active or inactive. Competitive inhibitors are inhibitors that bind to the active
site of an enzyme, and are generally of the same structure and conformation with the supposed substrate
that shouldve bind with the enzyme. Some examples include antibiotics such as sulfa drugs, whose active
compound, sulfanilamide, acts as a competitive inhibitor of PABA, which is used to make the important folic
acid necessary for bacterial growth. With the absence of PABA, Folic acid is not synthesized in the
bacteria, and as a result the bacteria die from its deficiency of folic acid. Humans are not affected by sulfa
drug action because humans obtain their folic acid from dietary sources and do not synthesize it unlike
bacteria. Noncompetitive inhibitors meanwhile, attach themselves to other portions of the enzyme; thereby
slowing catalytic action. These include metal ions that have the capacity to bond with sulfhydryl groups
away from the active site. Irreversible inhibitors are inhibitors that form covalent bonds with the enzymes
active site, permanently prohibiting the entry of the substrate into the active site. A notable example of an
irreversible inhibitor is the drug, penicillin. Penicillin inhibits the enzyme transpeptidase which is responsible
for forming crosslinks needed to strengthen bacterial cell walls by forming a covalent bond with the serine
residue found in the enzyme alongside its reactive amide bond found in the beta-lactam ring, permanently
preventing entry of the substrate, leading to weak bacterial cell walls, which leaves the bacteria susceptible
to lysis (to break open). (PUT PENICILLIN REACTION)
Discussion
(PUT IODINE TEST TABLE)
According to Stoker (2010), the optimum temperature of the salivary amylase enzyme is 37 C,
corresponding to the normal body temperature. As seen from the data results, the iodine color at 40 C
indicates a brown solution at a time interval of 20 minutes, which corresponds to an earlier formation of
erythrodextrin (which is red in color) than the other temperatures tested upon.
(PUT STARCH REACTION WITH AMYLASE)
The results obtained from 10 C and 60 C were identical. This is because at 10 C, molecules move
more slowly due to reduced kinetic energy, and as such lesser collisions between enzyme and substrate
happens, decreasing enzyme activity. It is of note that enzyme-substrate complex formation is governed by
Brownian motion, or constant random motion. The higher kinetic energy (which corresponds to higher
temperature) leads to an increase of randomly colliding enzyme and substrate, which increases enzyme
activity in turn, the opposite scenario happens at 10 C, resulting to decreased enzyme activity as compared
to the optimum temperature of 40 C. This is evident in the table by noticing that formation of a brown
product (erythrodextrin) only happened at the time interval of 55 minutes. The olive green pigmentation

obtained at 50 minutes may have been an oversight of the group that handled the 10 C test, which they
might have misconstrued as a transition color for the solution. (Draw temp. curve with 10C)
At 60 C, well beyond the optimum temperature of 40 C, enzyme activity is also markedly
decreased from that obtained in 40 C. This is because at temperatures well beyond the optimal
temperature, the chemical bonds that hold the enzymes secondary, tertiary and quaternary structure begin
to denature, altering the enzymes conformation permanently, which leads to subsequent loss of catalytic
activity. Unlike the previous scenario which involves cold temperatures, denaturation is permanent and the
enzymes catalytic activity will not be restored unlike enzymes subjected to cold temperatures, which can
regain increased catalytic activity as the temperature goes higher due to increase of collisions. This
explains the temperature curve wherein progressively higher temperatures up to the optimum temperature
records an increased rate of enzyme reaction while the opposite happens as the temperature is further
increased after the optimum temp due to imminent denaturation of the enzymes protein portion. At room
temperature (30 C), the data obtained was theoretically similar to that obtained from the optimal
temperature of 40 C. Since the group that performed the test on room temp recorded an orange hue rather
than the brownish hue uniformly adopted by the other groups for the iodine test, we assumed that the
formation of an orange tinged solution is equivalent to that of a brown solution. This is noted at 35 minutes,
indicating a higher reaction time as compared to 40C and 60C but lower than the optimum temp at 40 C.
The iodine test, as discussed previously in the last lab report, serves as an indicator of starch (and
in this experiment, as a measure of salivary amylase activity on starch at diff. temps). This is accomplished
through the formation of a starch iodine complex with a triiodide ion and a starch coil, resulting to a blue
color change, which eventually turns into colorless when starch is finally converted into maltose. The
colorless form of the iodine test was not observed due to time constraints.
pH (INSERT TABLE)
The test for pH entails two digestive enzymes: Pepsin and pancreatin. Pepsin is a proteolytic
enzyme whose main function is to degrade food proteins into peptides and amino acids. Proteolytic
enzymes generally have an inactive precursor called a zymogen, in this case pepsinogen. This mechanism
is a form of enzymatic control since pepsin in an active state could digest the stomach lining and other
proteins in the human body. Pepsinogen is activated by hydrochloric acid during digestion, converting it into
pepsin. As it is generally found in the stomachs acidic environment, it is only logical that pepsins optimum
pH is at 2.0. Pepsin starts to become inactive at a pH of 6.5, explaining the slight cloudy solution observed
after 1 hour of digestion utilizing pepsin and sodium carbonate, which is basic, resulting into lesser
enzymatic activity and thus a lesser rate of digestion. As pepsins optimum pH is at an acidic environment,
a cloudy solution was brought about due to the disintegration of the egg white. The supernatant or the liquid
lying above the solid residue was tested for protein presence using the biuret test, which hinges on the
concept of forming chelation complexes between the cupric ion found in cupric sulfate and the peptide
linkages in the protein. Both solutions tested positive signifying protein presence in the solution brought
about by peptide liberation from the egg white during digestion.

Pancreatin is a mixture of digestive enzymes that consists of amylase, lipase and protease.
Amylase catalyzes the breakdown of starch into maltose, whereas lipase catalyzes the hydrolysis of lipids
while protease is a proteolytic enzyme that breaks down food proteins into peptides. Proteases could be
acidic, basic or neutral. As such, pancreatin obtained from hogs is generally used for patients suffering from
cystic fibrosis, celiac disease and Crohns disease due to their relative lack of these enzymes, which
compels them to have supplemental doses of the enzyme. Porcine pancreatin however, contains an acidic
protease. Subject to both acidic and basic environments, the pancreatin enzyme slightly disintegrated the
egg white, resulting to cloudy solutions. Both substances also tested positive for the biuret test, signifying
protein presence.
Benedicts Test (table)
Benedicts Test is a test conducted to detect reducing sugars which are sugars that contain an
aldehyde functional group. It also tests positive for aldehydes and alpha hydroxyketones. Sucrose or table
sugar was used as the test reagent for this test. The mixture of sucrose and water did not result to a color
change, and stayed blue after heating. This is because sucrose by itself, is not a reducing sugar despite the
fact that both of its components (glucose and fructose) are reducing sugars. The reason behind this is that
the glycosidic bond found in sucrose prevents isomerization into an aldehyde, thus making it test negative.

The other test tubes, which contained supernatant liquid, residue and supernatant liquid with
residue all tested positive to the test, albeit with varying time intervals in forming the color change. Yeast
contains the enzyme sucrase(invertase), which is extracted into the solution after being pulverized together
with sand and mixing with water. Centrifuging the test tubes led to the accumulation of the sand and yeast
residue at the bottom and the appearance of a supernatant liquid layer. The test tube containing
supernatant liquid as well as the mixture of supernatant liquid and residue produced a green solution,
indicating traces of reducing sugars whereas the test tube containing residue had a low concentration.

Sucrase hydrolyzes sucrose into glucose and fructose, both of which will test positive to Benedicts
reagent. Benedict;s solution contains CuSO4, sodium carbonate and sodium citrate. Sodium carbonate
serves to create an alkaline environment necessary for the reduction of cu (2) ions into red cu (i) ions
whereas sodium citrate complexes with cu (II) ions so that they will not deteriorate into cu(I) during storage.
CuSO4 is responsible for the color change because as the sugars are heated in an alkaline environment,
they are converted into enediols as intermediates, reducing the Cu2+ ions into the Cu+ ion, which will
inadvertently be precipitated into the red copper (I) oxide. The resultant color changes are brought about by
varying concentrations of reducing sugars in the solution.
Sucrose + H2O ---> glucose + fructose

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%20Molecules%20Lab.htm