Enzymes

The cell of the body are chemical Factories

• Many compounds are needed by the body to sustain life.
• The food that we eat are sources of reactants to synthesize these compounds
• Human activity requires large amounts of these products, so that fast reactions are desired
• The processes with which these compounds are synthesized uses catalyst to hasten the reactions
○ We need enzymes that would catalyze the reactions so that the reaction that take place is fast.
These chemical reactions propel that we are dojng, without enzyme it would take slower to move
or talk.

Catalysis is Necessary for Life

catalyst is a substance that speeds up a chemical reaction, while emerging unchanged at the end
• a catalyst is never used up, thus a very small amount can facilitate a very large number of reactions
• a catalyst never affects the equilibrium state of a reaction

Illustration of catalysis What are enzymes?

• large protein molecules that
increase the rate of chemical
reaction without themselves
undergoing any change.
• are Biological Catalysts
• are (almost) always proteins
(Ribozomes are enzymes made
of ribonucleic acid)
• are extraordinarily efficient
(109-1020)better than chemical
catalyst (glucose reaction with
O2 )
• Are remarkably specific (for
every 1 reaction, 1 enzyme
catalyst)

• Need mild physiological conditions to function (temperature, salt, pH, etc.)

• Often require a cofactor (metal ion, prosthetic group, or coenzyme)
• May be modified for regulatory purposes

Enzyme Property
• Approximately 3,000 enzymes in a single cell
• Distributed according to the body’s need
○ Protein splitting enzymes are in the blood for clotting
○ Digestive enzymes are in the stomach and pancreas
○ Even in organelles enzymes are localized

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 Stereospecificity
• Enzymes selectively recognize proper substrates over other molecules
• Enzymes produce products in very high yields - often much greater than 95%
• Specificity is controlled by structure - the unique fit of substrate with enzyme controls the selectivity for
substrate and the product yield
○ Enzymes are conscious or aware of the stereospecificity and isomeric forms

Terms Used with Enzymes

• Holoenzyme: the active species
• Apoenzyme: the protein component of a holoenzyme
• Active site: pocket or cleft in the enzyme where the reaction occurs or the specific portion of the enzyme to
which a substrate binds during reaction.
• Proenzyme: inactive form of enzymes
Why is there an Because some proteins are not in use at the moment are inactive form, but if
○ inactive form? they are needed in the body, they will be activated. One example is blood
clotting.
• Cofactor: a nonprotein portion of an enzyme that is necessary for catalytic function; examples are metallic
ions such as Zn2+ and Mg2+.
• Coenzyme: a nonprotein organic molecule, frequently a B vitamin, that acts as a cofactor.
• Substrate: the compound or compounds whose reaction an enzyme catalyzes. Or reactant bound at active
site

How does an enzyme work Catalytic Power

• Enzymes provide an alternative pathway for reaction, one
with a significantly lower activation energy and, therefore, a
faster rate.

Cofactors
• In addition to the protein part, many enzymes also have a non-protein part called a cofactor
• The protein part in such an enzyme is called an apoenzyme, and the of combination apoenzyme plus
cofactor is called a holoenzyme. Only holoenzymes have biological activity; neither cofactor nor apoenzyme
can catalyze reactions by themselves
• A cofactor can be either an inorganic ion or an organic molecule, called a coenzyme
• Many coenzymes are derived from vitamins, organic molecules that are dietary requirements

Mechanism of Enzyme Action

• Lock and key model
-That the active site has a shape where only a specific substrate could fit in. Rigid.
• Induced-fit model
• The active site becomes modified to accommodate the substrate. Adaptable.

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 Lock-and-Key Model
• by Emil Fischer
• Simplest and most quoted model
• Explains specificity by comparing the active site to a lock and the substrate to a key
• Assumes that enzymes is a rigid 3-D molecule
• Too restrictive for the dynamic structure of enzymes

“Lock & Key” Hypothesis

• “Lock & Key” postulated that substrate(s) fit a perfectly matched active site on the enzyme –

Induced Fit
• Introduced by Daniel Koshland
• According to this model the enzyme modifies the shape of the active
site to accommodate the substrate
• Both the lock-and-key model explain the phenomenon of competitive
inhibition
• Induced-fit-model explains noncompetitive inhibition

Important Features that Contribute to the Catalytic Ability of Enzymes

• Reacting groups are brought together at the active site in the proper orientation for reaction
• Some of the amino acids in the enzyme serve as catalytic groups; many enzymes have metal ions as catalysts
of the reaction Groups on the enzyme can stabilize the transition state

Factors Influencing Enzyme Activity

• Enzyme and Substrate Concentration
• Temperature
• pH

Enzyme Activity
• The effect of enzyme concentration on the rate of an enzyme-catalyzed reaction. Substrate concentration,
temperature, and pH are constant.
• The higher the enzyme and substrate concentrations, the higher the enzyme activity.
• When the enzyme concentration increases, the rate of reaction also increases proportionately.
• The effect of substrate concentration on the rate of an enzyme-catalyzed reaction. Enzyme concentration,
temperature, and pH are constant.
-At sufficiently high substrate concentrations, a saturation point is reached.
-After this point, increasing substrate concentration no longer increases the reaction rate because the
excess substrate cannot find any active site to which to bind.

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 • No matter how much substrate added, if the enzyme is this much only then the reaction tapers
down.

Enzyme Activity

• The Michaelis-Menten constant Km is defined as the substrate concentration at 1/2 the maximum velocity.
• Michaelis-Menten constants have been determined for many of the commonly used enzymes.
The size of Km tells us several things about a particular enzyme:
• A small Km indicates that the enzyme requires only a small amount of substrate to become saturated.
Hence, the maximum velocity is reached at relatively low substrate concentrations.
• A large Km indicates the need for high substrate concentrations to achieve maximum reaction velocity.
• The substrate with the lowest Km upon which the enzyme acts as a catalyst is frequently assumed to be
enzyme's natural substrate, though this is not true for all enzymes.

• The effect of temperature on the rate of an enzyme-catalyzed reaction. Substrate and enzyme
concentrations and pH are constant. We heat food to stop spoilage.

Enzyme does not work about 50 degrees celsius. Activity of

enzyme is around 38 to 40 degrees celsius. A temperature lower,
the enzyme don't work well.
• Each enzyme has an optimal temperature and pH at which it has
its greatest activity.
• Temperature affects enzyme activity because it changes the
conformation of the enzyme.
• Once the optimal temperature is reached, any further increase in
temperature, alters the enzyme conformation.
• The substrate may not fit properly onto the changed enzyme
surface, the rate of reaction actually decreases.
• Enzymes can break intermolecular forces.
• The effect of pH on the rate of an enzyme-catalyzed reaction. Substrate and enzyme concentrations and
temperature are constant. Peak is about between 6 and 7. pH lower or higher than the neutral will
deactivate or hamper.
• As the pH of its environment changes, the conformation of the protein changes similar to those of observed
in temperature changes.

Enzyme Regulation
• Feedback control
○ A process in which the formation of a product inhibits an earlier reaction in the sequence
○ The reaction product of one enzyme may control the activity of the other
• Proenzymes or zymogens
○ A protein that becomes an active enzyme after undergoing a chemical change
 Trypsin’s inactive form is called trypsinogen

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 • Allosterism
○ An enzyme regulation in which the binding of a regulator on one site on the enzyme modifies the
enzyme’s ability to bind the substrate in the active site

• Feedback control: an enzyme-regulation process where the product of a series of enzyme-

catalyzed reactions inhibits an earlier reaction in the sequence. The product, when there is enough
it will hamper the action of the enzyme that catalyzes the production of that product. When we
take things that are not natural to the body, we are hampering the natural reactions in the body.

• The inhibition may be competitive or noncompetitive.

• Proenzyme (zymogen): an inactive form of an enzyme that must have part of its polypeptide
chain hydrolyzed and removed before it becomes active.
• An example is trypsin, a digestive enzyme.
• It is synthesized and stored as trypsinogen, which has no enzyme activity.
• In digestive system, when there is no food there is also a stop in the enzymes.

The Allosteric Effect

The allosteric effect. Binding of the regulator to a site other than the active site changes the shape of the
active site.
• Allosterism: enzyme regulation based on an event occurring at a place other than the active site
but that creates a change in the active site.
○ An enzyme regulated by this mechanism is called an allosteric enzyme.
○ Allosteric enzymes often have multiple polypeptide chains.

Protein Modification
• Change in the primary structure by the addition of a functional group
covalently bonded to the apoenzyme.
• Would result to either activation or inhibition of enzyme activity
e.g.

Isoenzyme
• Isoenzyme: an enzyme that occurs in multiple forms; each catalyzes the same reaction.
• Example: lactate dehydrogenase (LDH) catalyzes the oxidation of lactate to pyruvate.
• The enzyme is a tetramer of H and M chains.
• H4 is present predominately in heart muscle.
• M4 is present predominantly in the liver and in skeletal muscle.
• H3M, H2M2, and HM3 also exist.
• H4 is allosterically inhibited by high levels of pyruvate while M4 is not.
• H4 in serum correlates with the severity of heart attack.

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How are Enzymes Named
• Names of enzymes are commonly derived from the reactants they catalyze or substrate on which
they act on.
e.g. lactate dehydrogenase-speeds up the removal of hydrogen from lactate
• The names of most enzymes end in “ase” some enzymes however were named before their actions
were even understood e.g. trypsin, pepsin….

Classification of Enzymes
Main class Some subclasses Type of reaction catalyzed
Hydrolases Lipases Hydrolysis of an ester group
Nucleases Hydrolysis of a phosphate group
Proteases Hydrolysis of an amide group
Isomerases Epimerases Isomerization of a chirality center
Ligases Carboxylases Addition of CO2
Synthetases Formation of new bond
Lyases Decarboxylases Loss of CO2
Dehydrases Loss of H2O
Oxidoreductases Dehydrogenases Introduction of double bond by removal of H2
Oxidase Oxidation
Reductases Reduction
Transferases Kinases Transfer of a phosphate group
Transminases Transfer of an amino group

1. Oxidoreductase:

2. Transferase:

3. Hydrolase:

4. Lyase:

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5. Isomerase:

6. Ligase:

Use of Enzyme in Medicine

• Enzyme assays useful in medical diagnosis.

First Lab Exam: Expt 1, 2, and 3

Experiment #1: The Living Cell
The Cell Components and Distribution of Biomolecules in a cell

The different components of the cell maybe separated by :

• homogenizing the tissue sample
• subject it to differential centrifugation.
• the homogenate is always immersed in ice bath. In this way individual functions of the cell organelles and
component biomolecules is preserved.

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The different portions of homogenate
Differential centrifugation as a separation technique is based on the fact that at a lower speed centrifugal
force, large and dense particles settle down first. At a slightly faster speed, the next dense particles settles
down and centrifugation continues, until only the homogenate is left.

Separation of cell components

• Homogenize by adding a solution of 1% sulfuric acid to help break down
tissues.
○ Sediment 1: nuclei and unbroken cells, rich in nucleic acids and
proteins; also lipids and carbohydrates.
○ Sediment 2: Mitochondria, made up of lipids, proteins, nucleic
acids, & carbohydrates
○ Sediment 3: lysosomes, components include

Positive Results for the Qualitative Analysis

• Carbohydrates: Molisch test • Lipids: Sudan IV Solution test

• Proteins: Biuret test • Nucleic acid, DNA: Dische reaction

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• Nucleic acid, DNA: Feulgen's reaction (alternative) Biuret Test (Cu++ ion in citrate solution)
• Test used to detect peptide bonds
• Cu++ ions is reduced to Cu+
• Copper ions forms a complex with the Nitrogen
and Carbon of the peptide bond in an alkaline
solution
• Pink to violet coloration is an indication of a
positive test
• Nucleic acid, RNA: Orcinol test
Molisch’s Test by: Hans Molisch
(α-naphthol)
• carbohydrates are dehydrated by H2SO4 to form
an aldehyde
• aldehydes condenses with molecules of
α-naphthol forming a furfural (violet or red ring) which
is seen in the junction of the two liquids

Sudan IV
• is a chemical that is fat soluble.
• lysochrome (fat-soluble dye) diazo dye
• When lipids are present in solution, it dissolves and stains the lipid and forms a separate reddish-orange
layer.

Dische (diphenylamine) test

• Dische reaction distinguishes DNA from RNA with its reaction to the pentose portion of the nucleotide
• The sugar portion of RNA is a ribose, while that of DNA is a deoxyribose.
• Dische reacts with the deoxyribose to form a blue –colored complex
• It won't react on ribose.
• It is highly toxic. Feulgen’s Test
• Purine base of the DNA is removed
using hydrochloric acid.
• When the aldehyde portion is
exposed schiff’s reagent is added
resulting to a magenta colored
compound

Orcinol test
• Is a tests for pentoses in RNA.
• Test reagent dehydrates the
pentoses to form furfural which
reacts with orcinol(FeCl3/H2O).
• The iron ion in the test reagent will
produce a bluish product to green
and precipitate may also form.

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Qualitative Analysis of the Cell Fractions
Cell Fraction CARB PROTEINS LIPIDS NUCLEIC
ACIDS
DNA RNA
Molisch Biuret Sudan IV Dische Feulgen’s Orcinol
T Test
solution reaction reaction Test
Sediment 1 ++++ + ++ ++++ ++++ ++++
(Nucleus, whole cell)
Sediment 2 (mitochondria) +++ ++ ++++ +++ +++ +++
Sediment 3 ++ +++ + ++ ++ ++
(lysosomes)
Supernate 3 (microsomes, + ++++ +++ + + +
soluble proteins, enzymes,
inorganic ions)
Experiment # 2 Proteins
• Proteins are made up of amino acids. To test for the amino acid component of a sample, different tests was
done to a solution of egg white

Egg White
• Albumin is the name for the clear liquid (also called the egg white) contained within an egg.
• The primary natural purpose of egg white is to protect the yolk and provide additional nutrition for the
growth of the embryo (when fertilized).
• Egg white consists primarily of about 90% water into which is dissolved 1% and 4% proteins (including
albumins, mucoproteins, and globulins).
• Unlike the yolk, which is high in lipids (fats), egg white contains almost no fat, and carbohydrate content is
less than 1%.
• Egg whites contain just over 50% of the protein in the egg.

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Test for the different amino acid in egg albumin
Test/component Amino acid tested Result
Biuret Peptide bonds -copper(II) ion forms violet-colored coordination complexes in
an alkaline solution
Xanthoproteic Tyrosine/tryptophan Yellow precipitate
Millon’s Phenol Salmon pink
group/tyrosine
Hopkins Cole Tryptophan Violet ring
Pauly’s Histidine / tyrosine Red color
Lead acetate Cysteine/methionin Gray to black
reaction e
Nitroprusside Thiols/cysteine Red coloration
Sakaguchi Arginine Wine red
Hellers Ring Aromatic amino White ring
acids

Test Components and Results

Biuret • 1% NaOH (peptide bonds are cleaved)
• Coppersulfate
• Potassium sodium tartrate

Xanthoproteic Conc. HNO3 /heat

Millon’s Mercury in HNO3

Hopkins Cole Glyoxylic acid reaction

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Test Components and Results
Pauly’s diazotized sulfanilic acid under alkaline conditions

Lead acetate Lead acetate

Nitruprusside sodium nitroprusside

Sakaguchi α-naphthol and

sodium
hypobromite

Heller’s Ring conc. HNO3

Color Reactions
Biuret Test- test for peptide bonds present in
proteins
• component 10% NaOH + 0.5% CuSO4
and potassium sodium tartrate.
• Potassium sodium tartrate is added
to chelate and thus stabilize the cupric
ions.
• The reaction of the cupric ions with the
nitrogen atoms involved in peptide bonds
leads to the displacement of the peptide
hydrogen atoms under the alkaline
conditions.

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Xanthoproteic Test (conc. HNO3)
• the test gives a positive result in those proteins with amino acids carrying aromatic groups, tyrosine and
tryptophan.
• neutralized with an alkali, turning dark yellow is due to xanthoproteic acid which is formed due to nitration of
certain amino acids.

Millon’s test ( Hg+ in HNO2 and HNO3)

• the test is for the phenolic group in tyrosine
• Result maybe reddish-brown or brown-pink precipitate

Hopkin’s-Cole Reaction/Glyoxylic Acid Reaction (glyoxylic acid and conc.H2SO4)

• Test for the presence of indole group in detecting the presence of tryptophan in proteins.

Pauly’s test (Sulfanilic acid + sodium nitrite + sodium

carbonate = diazonium component)
• is a chemical test to detect the amino acid histidine
and tyrosine.
• the basic principle in pauly's test is diazotization.
Sulfanilic acid that has diazotized will form diazonium
component. Diazonium component react with the
imidazole ring of histidine and a phenol group of
tyrosine to form dark red compound.
• Diazonium component + (imidazole ring of histidine /
phenol group of tyrosine) = dark red color solution
Lead acetate reaction
The sulfur-containing test for proteins, which is known as the lead acetate test, is used to detect the presence of
sulfur within a protein. Sulfur is commonly found in methionine, cysteine, homocysteine and taurine.

Sakaguchi Reaction (1-Naphthol and a drop of sodium hypobromite.)

• chemical test used for detecting the presence of arginine in proteins.
• The guanidine group in arginine reacts with Sakaguchi reagent to form a red-coloured complex

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Heller's test (conc. HNO3)
• is a chemical test that shows that strong acids cause the denaturation of precipitated proteins.
• Concentrated nitric acid is added to a protein solution from the side of the test tube to form two layers.
• A white ring appears between the two
• layers if the test is positive. Heller's test is commonly used to test for the presence of proteins in urine.
Protein denaturation
• Denaturation- refers to the destruction of the 3-dimensional structure of proteins
due to the disruption of the tertiary and secondary structures (H-bonds, salt/ionic
bonds, disulfide bonds, hydrophobic interaction) but not of the peptide bonds.
• Hydrolysis- break down of the primary structure of peptide bonds.
• Precipitation- coagulation or formation of insoluble solids or precipitates.
• Denaturating agents: heat, extreme pH, alcohol, heavy metals, alkaloidal agents,
uv, oxidizing and reducing agents that can destroy the tertiary and secondary
protein structures.

Results:
1. Denaturation by heat and extreme pH

Test tube no. Reagent added Observation after heating Observation after neutralization
1 HCl White ppt ppt decreased
2 NaOH White ppt ppt decreased
3 H 20 Cloudy solution
Heat and extreme pH can disrupt H-bonds, hydrophobic interaction, S-S and bonds.
2. Precipitation with alcohol
Result: formation of white ppt.
Alcohol can disrupt H- bonds.
3. Precipitation with heavy metal ions
Test tube Reagent Observation after addition of a few drops of Observation after addition of excess
No. added reagent reagent
1 1% HgCl2 White ppt ppt decreased
2 1% AgNO3 White ppt ppt decreased
3 1% White ppt ppt decreased
Pb(C2H3O2)2
4 1% CuSO4 Bluish white ppt ppt decreased
• Addition of heavy metals results to disruption of the salt bridges/ionic bonds.
• Forms insoluble metal proteinates

4. Precipitation with alkaloidal reagents

Test tube Reagent Observation after addition of a few drops Observation after addition of excess
No. added of reagent reagent
1 Satd. picric yellow ppt ppt increased
acid
2 10% tannic brown ppt ppt increased
acid
3 5% K4Fe(CN)6 yellow ppt ppt increased
4 20% TCA white ppt ppt increased

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• Disruption of salt bridges/ionic bonds
• Formation of protein salts (e. g. protein tannate, protein picrate)

Experiment #3: Enzymes and Vitamins

Enzymes
>biological catalyst that hastens the rate of biochemical reactions.
A. Digestion of starch
Starch + Saliva ----> Amylodextrin, Erythrodextrin,
Achrodextrin ----> Maltose
Starch + I2 ------> Blue colored complex
Maltose + I2 ------> absence of blue complex

Iodine - non-reducing sugar

Definition of terms
Monosacharides - reducing sugar Enzymes - are biological catalyst that catalyze the
chemical reactions necessary for growth, repair and
D. Catalase Activity maintenance of a living organism without which it
2H2O2 ---------> 2H2O + O2 could not survive
catalase
What are vitamins?
Tissue Time for filter paper to resurface (sec) Vitamins are similar to hormones in many ways that
are considered additional substance required for
1. Saliva normal functions and development of the body.
2. Tears slower
3. Fresh blood
4. Pancreas
5. Muscle faster
6. Brain
7. Potato extract
• Tissues which are physiologically active have higher catalase activity.
• Tissues which are not physiologically active have lower catalase activity.

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Biochemistry Page 16
Biochemistry Page 17
Vit. Common name Source Defficiency
K anti-hemmorrhage green leafy tissues Hemorrhage
vitamin
E anti-oxidant milk, eggs, fish, muscle meat, Muscle destrophy to paralysis
(tocopherol) cereals, veg
B2 Riboflavin Yeast, milk, liver, heart and leafy Cheilitis (mouth lesions) glossitis
vegetables (tonue inflammation)
B3 Niacin eggs, milk and fruits Pellagra or rough skin
B5 Pantothenic Liver, kidney, fish, eggs, milk and Degeneration in the adrenal cortex
lean meats and impotency
B6 Pyridoxine Yeast, egg yolk, liver, and grain dermatitis
germs
B7 Biotin Liver, kidney, fish, eggs, milk Dermatitis, muscle pains and
depression
B9 Folic Green leaves, yeast, liver, kidney Megaloblastic anemia gastrointestinal
and cauliflower disturbances
B12 Cobalamine Liver, kidney, fish, eggs, milk, Pernicious anemia
oysters, and clams

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