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Influence of pH and Concentration to enzyme Activity

December 1, 2015

Page 1

I TITLE OF EXPERIMENT :

Effect of pH and concentration enzyme by enzyme activity

II DATE OF EXPERIMENT :

November 24th, 2015

III END OF EXPERIMENT :

November 24th, 2015

IV PURPOSE :

To showing that pH and concentration of enzyme effected enzyme activity

V BASIC THEORY

“ nzym ”

With the exception of a small group of catalytic RNA molecules, all enzymes

are proteins. Their catalytic activity depends on the integrity of their native

protein conformation. If an enzyme is denatured or dissociated into its subunits,

catalytic activity is usually lost. If an enzyme is broken down into its component

amino acids, its catalytic activity is always destroyed. Thus the primary,

secondary, tertiary, and quaternary structures of protein enzymes are essential to

their catalytic activity.

Enzymes, like other proteins, have molecular weights ranging from about

12,000 to more than 1 million. Some enzymes require no chemical groups for

activity other than their amino acid residues. Others require an additional

chemical component called a cofactor—either one or more inorganic ions, such as

Fe2, Mg2, Mn2, or Zn2, or a complex organic or metalloid-organic molecule called a

coenzyme. Some enzymes require both a coenzyme and one or more metal ions for

activity. A coenzyme or metal ion that is very tightly or even covalently bound to

the enzyme protein is called a prosthetic group. A complete, catalytically active

enzyme together with its bound coenzyme and/or metal ions is called a

holoenzyme. The protein part of such an enzyme is called the apoenzyme or

apoprotein.

Coenzymes act as transient carriers of specific functional groups. Most are

derived from vitamins, organic nutrients required in small amounts in the diet. We

consider coenzymes in more detail as we encounter them in the metabolic


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pathways discussed in Part II. Finally, some enzyme proteins are modified

covalently by phosphorylation, glycosylation, and other processes. Many of these

alterations are involved in the regulation of enzyme activity.

HOW ENZYMES WORK

The enzymatic catalysis of reactions is essential to living systems. Under

biologically relevant conditions, un-catalyzed reactions tend to be slow—most

biological molecules are quite stable in the neutral-pH, mild temperature, aqueous

environment inside cells. Furthermore, many common reactions in biochemistry

entail chemical events that are unfavorable or unlikely in the cellular environment,

such as the transient formation of unstable charged intermediates or the collision

of two or more molecules in the precise orientation required for reaction.

Reactions required to digest food, send nerve signals, or contract a muscle simply

do not occur at a useful rate without catalysis.

An enzyme circumvents these problems by providing a specific environment

within which a given reaction can occur more rapidly. The distinguishing feature

of an enzyme-catalyzed reaction is that it takes place within the confines of a

pocket on the enzyme called the active site (Fig. 6–1). The molecule that is bound

in the active site and acted upon by the enzyme is called the substrate. The surface

of the active site is lined with amino acid residues with substituent groups that


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bind the substrate and catalyze its chemical transformation. Often, the active site

encloses a substrate, sequestering it completely from solution.

The enzyme substrate complex, whose existence was first proposed byCharles-Adolphe Wurtz in 1880, is central to the action of enzymes. It is also the

starting point for mathematical treatments that define the kinetic behavior of

enzyme-catalyzed reactions and for theoretical descriptions of enzyme

mechanisms.

ENZYME ACTIVITY DEPENDS ON PH

Enzymes have an optimum pH (or pH range) at which their activity is

maximal, at higher or lower pH, activity decreases. This is not surprising. Amino

acid side chains in the active site may act as weak acids and bases with critical

functions that depend on their maintaining a certain state of ionization, and

elsewhere in the protein ionized side chains may play an essential role in the

interactions that maintain protein structure. Removing a proton from a His

residue, for example, might eliminate an ionic interaction essential for stabilizing

the active conformation of the enzyme. A less common cause of pH sensitivity is

titration of a group on the substrate.

The pH range over which an enzyme undergoes changes in activity can

provide a clue to the type of amino acid residue involved. A change in activity near

pH 7.0, for example, often reflects titration of a His residue. The effects of pH must

be interpreted with some caution, however. In the closely packed environment of

a protein, the pKa of amino acid side chains can be significantly altered. For

example, a nearby positive charge can lower the pKa of a Lys residue, and a nearby

negative charge can increase it. Such effects sometimes result in a pKa that is

shifted by several pH units from its value in the free amino acid. In the enzyme

acetoacetate decarboxylase, for example, one Lys residue has a pKa of 6.6

(compared with 10.5 in free lysine) due to electrostatic effects of nearby positive

charges. Every enzyme has an optimum pH (or pH range) at which it has maximal

activity.

EXAMPLES OF ENZYMATIC REACTIONS


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Thus far we have focused on the general principles of catalysis and on

introducing some of the kinetic parameters used to describe enzyme action. We

now turn to several examples of specific enzyme reaction mechanisms. An

understanding of the complete mechanism of action of a purified enzyme requires

identification of all substrates, cofactors, products, and regulators.

Moreover, it requires a knowledge of (1) the temporal sequence in which

enzyme-bound reaction intermediates form, (2) the structure of each intermediate

and each transition state, (3) the rates of interconversion between intermediates,

(4) the structural relationship of the enzyme to each intermediate, and (5) the

energy contributed by all reacting and interacting groups to intermediatecomplexes and transition states. As yet, there is probably no enzyme for which we

have an understanding that meets all these requirements. Many decades of

research, however, have produced mechanistic information about hundreds of

enzymes, and in some cases this information is highly detailed.

We present here the mechanisms for four enzymes: chymotrypsin,

hexokinase, enolase, and lysozyme. These examples are not intended to cover all

possible classes of enzyme chemistry. They are chosen in part because they are

among the best understood enzymes, and in part because they clearly illustrate

some general principles outlined in this chapter. The discussion concentrates on

selected principles, along with some key experiments that have helped to bring

these principles into focus. We use the chymotrypsin example to review some of

the conventions used to depict enzyme mechanisms. Much mechanistic detail and

experimental evidence is necessarily omitted; no one book could completely

document the rich experimental history of these enzymes. Also absent from these

discussions is the special contribution of coenzymes to the catalytic activity of

many enzymes. The function of coenzymes is chemically varied, and we describe

each as it is encountered in Part II.


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VI TOOLS AND MATERIALS

A Tools

- Steam Bath

-

Beaker Glass

B Materials

- Saliva solution

- Starch solution 0.4 mg/mL

- Iodine solution

VII FLOW CHART

1.

Influence of pH to enzyme activity

saliva

- diluted 100x with distillate

Enzyme solution

1 mL starch with pH (1,3,5,7 and 9)

- pour into test tube- Let it for about 2 minutes

+ 10 drops enzyme solution

- Mixed it well and wait for 3minute

+ 1 mL iodine solution

+ 10 mL distillate water

Result the solution

- read the absorbance

Result the absorbance

1 mL starch solution

- pour into test tube B

(Blank)- Let it for about 2 minutes+ 10 drops distillate water

- Mixed it well and wait for 3

minute+ 1 mL iodine solution


Result the solution




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2. Influence of concentration to enzyme activity

saliva

- diluted 100, 200, 300, 400,

500 x with distillate water

Enzyme solution


- pour into test tube- Let it for about 2 minutes+ 10 drops enzyme solution

concentration 100, 200, 300,400, 500- Mixed it well and wait for 3

minute

- heated in 60 ˚C

+ 1 mL iodine solution+ 10 mL distillate water

Result the solution




- pour into test tube- Let it for about 2 minutes+ 10 drops distillated water

- Mixed it well and wait for 3minute- heated in 60 ˚C + 1 mL iodine solution


Result the solution




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VIII RESULT OF EXPERIMENT

Procedure of the ExperimentsObservation data

Reaction/Assumption ConclusionBefore After

1. Effect of pH towards enzyme activity - Starch

solution:

turbid solution

-

Distillatewater:

colorless

solution

- Iodine: yellow

solution

- Enzyme:

turbid solution

blank solution

- Starch + I2

= blackish

blue-

+ distillate

water =

blackish

blue

Test tube I U

- Starch pH 1

+ enzyme +

iodine =

blue (+++)

solution

- + distillate

water =blue (+++)

solution

Tube II U

- Starch pH 3

+ enzyme +

pH affects to the

enzyme activation

and enzyme work

effectively at pH 7

+

+ I2 ↛

+ I2 ↛

saliva

- diluted 100x with distillate

Enzyme solution

1 mL starch with pH (1,3,5,7 and 9)

- pour into test tube

- Let it for about 2 minutes

+ 10 drops enzyme solution


minute



Result the solution




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turbid

solution

- + iodine =

blue (++)

solution

- + distillate

water =

blue (++)

solution

Tube III U

- Starch pH 5

+ enzyme =

turbid

solution

- + iodine +

distillate

water =

blue (+)

solution

Tube IV U

- Starch pH 7

+ enzyme =

turbid

solution

-

+ iodine =

∆ A1 = 0.887

∆ A3 = 0.863

∆ A5 = 0.825

∆ A7 = 0.066

∆ A9 = 0.117

- Enzyme activation affected by pH. pH

optimum of amylase is 7 (Mohammad

Sadikin, 2002)


- pour into test tube B

(Blank)


+ 10 drops distillate water

- Mixed it well and wait for 3minute



Result the solution




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light brown

- + distillate

water =

colorless

Tube V U

- Starch pH 9

+ enzyme =

turbid

solution

- + iodine +

distillate

water =

light blue

2. influence of concentration to enzyme activity - starch solution

= turbid

solution

- iodine solution

= yellow

solution

- distillate

water =

colorless

- enzyme

solution =

turbid solution

Blank test tube

-

starch +

water =

colorless

-

+ I2 = deep

blue

Test tube

- Starch +

enzyme

(dilute

100x) =

colorless +

Concentration of

substrate can affect to

the enzyme

activation.

Enzyme work

effectively in

concentration 100x

diluted

+

saliva

- diluted 100, 200, 300, 400,

500 x with distillate water

Enzyme solution


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I2 =

colorless

solution

-

Starch +

enzyme

(dilute

200x) =

colorless +

I2 = light

blue

- Starch +

enzyme

(dilute

300x) =

colorless +

I2 = blue

(++)

- Starch +

enzyme

(dilute

400x) =

colorless +

I2 = blue

(+++)

-

Starch +

∆ A100 = 0.030

∆ A200 = 0.487

∆ A300 = 0.682

∆ A400 = 0.745

∆ A500 = 0.545

Greater concentration of enzyme cause

enzyme work properly (Hafiz Soewoto, 2000)

+ I2 ↛

+ I2 ↛




+ 10 drops distillated water- Mixed it well and wait for 3

minute

- heated in 60 ˚C + 1 mL iodine solution


Result the solution




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enzyme

(dilute

500x) =

colorless +

I2 = blue

(+)




+ 10 drops enzyme solutionconcentration 100, 200, 300,

400, 500


minute

- heated in 60 ̊ C + 1 mL iodine solution


Result the solution




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IX EXPLANATION

In this experiment, we do the experiment about factor which effected

enzyme amylase activity in saliva where breaking starch solution. Factor which

effected enzyme activity there’s a concentration of enzyme, pH, temperature, and

substrate concentration.

Effect of pH towards enzyme activity

First experiment, with the aim to knowing effected of pH to enzyme activity.

We used tube B for blank solution and tube U for diluted saliva. In tube B, added

with 1 mL starch solution 1% turbid solution, which the function of starch is as a

substrate. Then, let it 2 minutes to make starch work correctly and starch can

degradation perfectly. Next add with 10 drops distillate water colorless solution,

where the function of distillate as a changes of enzyme in blank solution. Then,

mixed well and wait about 3 minute for the solution mixed correctly. Add 1 mL

iodine solution yellow solution, where the function is for indicator of amylum and

for formed complex solution in starch solution. Then add again with 10 mL

distillated water colorless solution. Distillated water is for the solution not too

concentrated. After that read the absorbance in UV-Vis spectrophotometer.

In 5 tubes U for diluted saliva, we add 100x dilute saliva in each tube 10

drops. Then we added with starch solution pH 1, starch solution pH 3, starch

solution pH 5, starch solution pH 7, and starch solution pH 9, where the function of

starch is for substrate too. Then let it for 2 minute, to make the starch mix

correctly. Then add 10 drops enzyme solution, with function for knowing

enzymatic reaction from enzyme amylase and break amylum become glucose.

Then, wait for 3 minute to make the saliva broke and changes become glucose

correctly. After that, added with 1 mL iodine yellow solution with the function as

indicator color change from saliva which specific for tested existence of amylum

and for formed complex solution in starch solution. Then, added with 10 mL

distillate water to make the saliva not too concentrated. After that, read the

absorbance in UV-Vis spectrophotometer. The principle of absorbance is from

starch solution is colorless solution, so for measuring absorbance using


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spectrophotometer, starch solution must change become complex solution for

giving color in solution and can measured the absorbance.

After we tested absorbance in UV-Vis spectrophotometer, we get the data

and the color of solution like:

pH Abs

blanko 0.937

1 0.887

3 0.863

5 0.825

7 0.066

9 0.117

pH 1: Blue (+++) solution

pH 3: Blue (++) solution

pH 5: Blue (+) solution

pH 7: Colorless solution

pH 9: Light blue solution

From this experiment, we can conclude that value of pH affects to the

enzyme activation. And enzyme work effectively at pH 7, because in pH 7 is pH

optimum. So the enzyme correctly reacted with starch very fast, so the color

become colorless.

Influence of concentration to enzyme activity

Second experiments, we used saliva which has been diluted with 100x, 200x,

300x, 400x, and 500x. Next, we prepare test tube for blank solution and 5 tubes for

saliva solution.

+


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First, to make blank solution, we used 1 mL starch turbid solution where the

function of starch as substrate. Then let it for 2 minutes to make starch can

degradation perfectly. After that, add 10 drops distillate water colorless solution,

where the function as changes of saliva in blank solution. Mixed it well and wait

for 3 minute, for the solution mixed correctly. Then, heated solution in 60 ˚C in 1

minute, where the function of heated is for make the reaction work faster. Then

added with iodine yellow solution, where the function of iodine is as indicator for

determining existence of amylum. After that, add with 10mL distillate water

colorless solution, to make the solution not too concentrated. And read the

absorbance in UV-Vis spectrophotometer, and we get the absorbance.

For 5 tubes saliva solution which has been diluted in 100x, 200x, 300x, 400x,

and 500x. First tube, we add with starch turbid solution where the function of

starch as substrate. Then, let it for 2 minute is for the starch degradation perfectly.

After that, add with 10 drops enzyme turbid solution 100-500x diluted in each

tube and keep it until 3 minute. It will make the starch solution happened

hydrolysis partial. Starch solution can be hydrolysis by enzyme amylase in saliva

so become glucose. After that, heated the saliva in 60 ˚C 1 minute to made the

reaction work faster. Then, added with 1mL iodine yellow solution with function

as indicator to determining existence of amylum. Then added with 10mL distillate

water colorless solution is to make the solution not too concentrated. After that

read the absorbance in UV-Vis spectrophotometry. The principle of absorbance is

from starch solution is colorless solution, so for measuring absorbance using

spectrophotometer, starch solution must change become complex solution for

giving color in solution and can measured the absorbance. And we get for the

result:

Conc Abs

blanko 0.953

100x 0.03

200x 0.487

300x 0.682

400x 0.745

500x 0.545


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Conc. 500x: Blue (+) solution

Conc. 400x: Blue (+++) solution

Conc. 300x: Blue (++) solution

Conc. 200x: Light blue solution

Conc. 100x: Colorless solution

From this experiment, we can conclude that concentration of substrate can

affected to the enzyme activation and enzyme work affectively in concentration

100x dilute. Concentration affected of this enzyme is product formed, where

bigger concentration of enzyme as much as product which produce so we can

conclude that rate reaction inversely proportional with enzyme concentration.

X CONCLUSION

Based on our experiment we can conclude that:

1. pH affect to the enzyme activation and enzyme work affectively at pH optimum

is pH 7

2. Concentration of substrate can affected to the enzyme activation. Enzyme work

affectively in concentration 100x diluted

XI QUESTION AND ANSWERS

QUESTION

1. Create curve which showed the relationship between reaction rate

enzymatic (V= ∆A/minute) with pH

+


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2. Create curve which showed the relationship between reaction rate

enzymatic (V= ∆A/minute) with concentration/ diluted enzym

ANSWERS

1.

2.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1 3 5 7 9

B

- U

pH

Absorbance vs pH

B - U

y = -0.1288x + 0.8416

R² = 0.52460

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.80.9

1

100x 200x 300x 400x 500x

a b s o r b a n c e

concentration

absorbance vs concentration

absorbance

Linear (absorbance)


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REFFERENCESS

Anonym. 2015. Enzyme. (online). (https://en.wikipedia.org/wiki/Enzyme accesses at

November 30th , 2015)

Bresnick, S., 2004, Intisari Kimia Organik , Hipokrates, Jakarta.

Gritter, A., 1991, Biokimia 1 , PT. Gramedia, Jakarta.

Lehninger, A.L., Dasar-Dasar Biokimia , Penerbit Erlangga, Jakarta

https://en.wikipedia.org/wiki/Enzymehttps://en.wikipedia.org/wiki/Enzymehttps://en.wikipedia.org/wiki/Enzymehttps://en.wikipedia.org/wiki/Enzyme


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ATTACHMENT

Experiment 1 : Influence of pH to enzyme activity

1 ml starch solution poured

tube B(blanco)

Added 10 drops aquadest Wait for 3 minutes and added

4 drops I2 solution

Result after added I2 Added 8 ml aquadest Saliva/enzyme


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Put into disstilate tube Added aquadest until the line Enzyme dilute 100 x

1 ml starch in pH 9 1 ml starch in pH 1 1 ml starch in pH 3

1 ml starch in pH 5 1 ml starch in pH 7 Let it for 2 minutes

Added 10 drops of enzyme in Wait for 3 minutes


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each pH

Added 4 drops I2 solution in each pH Added 8 ml aquadest in each

pH

result


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Experiment 2 : Influence of concentration to enzyme activity

Enzyme in conc 100 Enzyme in conc 200 Enzyme in conc 300

Enzyme in conc 400 Enzyme in conc 500 1ml starch solution poured in

tube B(blanco conc)

1ml starch solution poured 1ml starch solution poured 1ml starch solution poured


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into tubes labelled 500 into tubes labelled 400 into tubes labelled 300

1ml starch solution poured

into tubes labelled 200

1ml starch solution poured

into tubes labelled 100

Added 10 drops aquadest to

tube B

Added 10 drops enzyme conc

500 to tube labelled 500






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Result

Heated in 60OC Added 1 ml I2 in Tube B Added 1 ml I2 in Tube labelled

500

Added 1 ml I2 in Tube labelled

400


300


200


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100

Added 8 ml aquadest to tube

B


labelled 500


labelled 400


labelled 300


labelled 200


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labelled 100

Result


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ph WL B - U

blanko 0.937

1 0.887 0.05

3 0.863 0.074

5 0.825 0.112

7 0.066 0.871

9 0.117 0.82

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1 3 5 7 9

B

- U

pH

Absorbance vs pH

B - U


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Conc. WL absorbance

blanko 0.953

100x 0.03 0.923

200x 0.487 0.466

300x 0.682 0.271

400x 0.745 0.208

500x 0.545 0.408

y = -0.1288x + 0.8416

R² = 0.52460

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

100x 200x 300x 400x 500x

a b s o r b a n c e

concentration

absorbance vs concentration

absorbance

Linear (absorbance)

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