Reviewer

BIOCHEMISTRY LABORATORY
Exercise 4: Salivary Amylase

Enzymes Shape (exactly the same with active site)

of the substrate fits into the
 Biological catalysts for chemical reactions in all binding/active site of enzyme
living organism  “Complementary Shape” occur naturally
 Large protein molecules specialized to catalyze  Active Site = rigid
biological reactions (LOWERS activation B. Induced Fit Model (Glove and Hand Model)
energy)  The flexibility of the enzyme to conform
 binding of substrate to the enzyme is due
 INCREASES that rate of reaction (106 to 1012
to the ability of the E to adjust and be
times faster) = reusable & remain unchanged;
flexible
lowering of activation energy
 “Complementary Shape” forms after
 Are 3D polypeptide capable of adapting a binding
unique shape (ACTIVE SITE) into which a  Active Site = flexible
substrate possessing similar shape can fit
Enzyme Inhibitor
 Have extraordinary catalytic power (acts on a
 Inhibitor bonds to the E to alter or destroy the E’s
specific substrate)
activity
 Very specific; in terms of the type of reaction &
 Can be reversible or irreversible
affected by the following :  Inhibitor Types:
1. pH 1. Noncompetitive Inhibitor
2. temperature o Bonds to the enzyme but not to the active
3. substrate concentration site
 Operates at a very high speed o When this inhibitor is bound, the active
 Works in a 2-step reaction process: site changes shape and the substrate no
o E + S <-> ES longer fits
o ES <-> E + P 2. Competitive Inhibitor
o Has a shape & structure similar to
 includes a Cofactor = a metal ion/ organic
substrate, so it competes with the
molecule needed for enzyme
substrate for binding to the active site
 Reusable and remain unchanged
Enzyme Classification
Enzyme Mechanism
1. An enzyme contains an active site that binds the Class # Class Name Catalyzed Reaction
substrate 1 Oxidoreductase Transfer of electrons
 This forms an enzyme-substrate complex s (hydride ions or H
2. Once the reaction has occurred, the catalyst atoms)
released the products 2 Transferases Group transfer reactions
3. Specificity occurs as explained by the following 3 Hydrolases Hydrolysis reaction
models: (transfer of functional
A. Lock and Key Model groups to water)
 Both components (enzyme & subs) fit 4 Lyases (1) Addition of groups to
double bonds
each other to a very high degree
(2) Formation of double
bonds by removal of

pg. 1 (Dumaguit, 2020)

 groups - α-amylase - utilize starch as a substrate
5 Isomerases Transfer of groups within and produces reducing sugars as
molecules to yield products:
isomeric forms  Maltose (Disaccharide)
6 Ligases Formation of C-C, C-S, C-  Dextrin (Oligosaccharide)
O & C-N bonds by
condensation reactions
coupled to cleavage of Starch
ATP or similar cofactor
 white, tasteless, solid carbohydrate
 has linear amylose & branched amylopectin
Naming Enzymes  chemical formula: (C6H10O5)n
 converted to glucose as a source of energy for
1. Trivial organisms
 Trypsin and Pepsin  can be detected by the blue-black color when I2
2. Substrate + “ase” ending solution is added
 Sucrase  Yellow Solution = complete hydrolysis/
3. Action + “ase” ending disappearance of starch
 Dehydrogenase  Major component of starch can enzymatically
4. Combo + “ase” ending hydrolyze by α -Amyloase
 Succinic acid dehydrogenase 
 Substrate = succinic acid  Occurs in 2 Forms:
 Action = removes H+ 1. α -Amylose
 Enzyme = -ase ending o Linear with α -(1-> 4) glycosidic linkages
5. Numerical o Produces glucose and maltose
 Used more often in chemistry 2. Amylopectin
o Contains two glycosidic linkage
Amylase
- linear α - (1-> 4)
 Enzyme that breaks down starch into sugars - branched α - (1-> 6)
 Class # 3 - Hydrolases enzyme o Produces glucose, maltose and dextrin
 Found in saliva = begins the chemical reaction of
Starch Hydrolysis
digestion
 α- Amylase  Conversion of starch to dextrin, maltotriose,
o Found in starch maltose & glucose
o Also called “α - (1-> 4) - D - glucan - 4 -  Enzymatic hydrolysis of starch breaks large,
gulcanohydrolase" or “glycogenase” insoluble starch molecules into soluble (in H2O)
o Are calcium metalloenzymes completely starches (smaller polysaccharide initially formed):
unable to function in the absence of calcium 1. Amylodextrin - gives BLUE color in I2 solution
o Both salivary & pancreatic amylases belong to 2. Erythrodextrin - gives RED color in I2 solution
this type 3. Achrodexrin - gives NO COLOR in I2 solution =
o Breaks down carbohydrates yielding: salivary amylase is active
- Amylose NaCl
- Amylopectin
o Salivary Amylase  To mimic the normal physiological condition in the
- Also called “EC 3.2.1.1” body (amylase needs Cl- to be able to perform its
- Digestive enzymes = α-amylase function)
 Chloride Ion (Cl-)

pg. 2 (Dumaguit, 2020)

 o An allosteric effector (selectively binds to a  Complex formed by complexation of amylose
protein & regulates its biological activity) with an iodine polymer where triiodide (I3-)
- Allosteric Regulation - regulation of forms by reacting iodine (I2) with iodide (I-)
enzyme by binding an effector molecule
at the protein’s allosteric site 2. Determination of Achromic Point
o Acts a ligand that can increase or decrease  Time taken by S.A. to hydrolyze starch to
enzyme activity achrodextrin at optimum conditions
o α-Amylase’ binding site has positively
Factors Affecting Enzyme Activity
charged amino acid residues that assists in the
binding of negatively charged Cl- = causes 1. Enzyme & Substrate Concentration
conformational change to the enzyme  Inc. rate of rxn = Inc. S conc & constant E conc
switching it to the more active state  Inc. the # of S molecules = Inc. probability that
Addition of Iodine S will collide with E to form an E-S complex. (E
is excess)
 BLUE = Presence of starch  At a certain conc, the rate of rxn reaches the
 YELLOW = Complete hydrolysis -> disappearance of maximum level
starch
2. Inhibitors
Influence of the Substrate
 Non-competitive & competitive inhibitor
 Keeping the concentration of the enzyme constant
while increasing the concentration of substrate 3. pH & Temperature
results in an increase in the rate of reaction  Constant Factors:  Starch - substrate 
 At a certain concentration, the rate of reaction  Amylase - enzyme 
reaches a maximum level (After increasing the  NaCl - to mimic normal physiological condition
substrate levels does not increase the rate of in the body (amylase needs Cl- to be able to
reaction) perform its function)

pH

Substrate: Starch
Enzyme: Amylase
Variable: pH Buffer (4,5,6.7,8,10)
Constant: 37 C (normal body temp.)

Optimum pH
 pH = 6.7
 Activity of the enzyme is at its maximum
 Hydrolyzes the starch at the least time
 pH = 6.5
o The pH of a saliva of a normal healthy
individual is 6. 5
Basis for the Rate of Reaction of Salivary α- Amylase
 The substrate attaches itself to the enzyme via 2
1. Disappearance of Reactant (Starch) ionic bonds
 Disappearance of blue-lack color given by the o Ionic bonds are caused by the transfer of a H
starch-iodine complex ion from a -COOH group in the side chain of
AA1 residue to an -NH2 group in the side chain
of AA2

pg. 3 (Dumaguit, 2020)

  Increase in temp = increase amount of energy
available for the reactants to reach its transition
state = E-S complex
 The rate of most Enzyme reactions approx. double
for every 10°C temp rise
Lower pH  When Enzyme denatures, conformation or
 The -NH3+ group will not be affected, but the COO- structure alters irreversibly
will pick up a H ion (protonation) = inability to form  Inc in thermal agitation break down the 2° & 3°
ionic bonds -> enzyme is inactive level that rely on H-bonds, Vander Waals Forces &
ionic bonds = alters shape of AS
 Will affect the polar & non-polar intramolecular
attraction & repulsive forces and interaction
between Enzyme & Substrate
 Affect the conformation of the enzyme
 Acidic/ Basic Medium = Enzyme is denatured ->
change in conformation -> 3° structural level
 From lower pH Enzyme activity will increase until
Optimum pH then after Enzyme activity decreases
 Ionic bonds are caused by the transfer of a H ion
from a -COOH group in the side chain of AA1
residue to an -NH2 group in the side chain of AA2
 Affects the conformation of the enzyme

Temperature

Substrate: Starch
Enzyme: Amylase
Variable: Temperature (°C) - 4, RT, 37, 50, 60, 70 )
Constant: pH (6.7 – normal body condition)
Rate of Reaction: 1/ t or min-

Optimum Temperature

 37°C (Maximum amount of product is formed)

Low Temperature

 Amylase is ineffective because it is inactive

High Temperature

 Amylase is less effective due to thermal

denaturation
o Amylase becomes permanently denaturation

 Reaction rate increases proportionally with the rise

of temp
 Proportionality due to increase in kinematic energy
which increases frequency of collision of Enzyme &
Substrate
pg. 4 (Dumaguit, 2020)