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UNIVERSITI MALAYSIA PERLIS
Peperiksaan Pertengahan Semester (2)
Sidang Akademik 2010/2011
Oktober 2010
ERT 106-Biochemistry
[Biokimia]
Masa : 1 jam
Please make sure that this question paper has THREE (3) printed pages including this front page
before you start the examination.
[Sila pastikan kertas soalan ini mengandungi TIGA (3) muka surat yang bercetak termasuk
muka hadapan sebelum anda memulakan peperiksaan ini.]
This question paper has FOUR (4) questions. Answer ALL questions and each question
contributes 25 marks.
[Kertas soalan ini mengandungi EMPAT (4) soalan. Jawab SEMUA soalan dan setiap soalan
memberikan 25 markah.]
Name
:.......................................................................................
Matrix No. : .....................................................................................
Question 1
a)
Denaturation is define as the loss of secondary, tertiary and quartenary stucture of protein
except for the primary stucture. Discuss on TWO (2) conditions that may contribute to
protein denaturation.
[10 marks]
 Strong acid or base – changes in pH result in protonation of some protein side group,
which alter/disrupt hydrogen bonding & salt bridge
 Organic solvents – water-soluble organic solvents
eg. Ethanol interfere with hydrophobic interaction
 Detergents – these amphiphatic molecules disrupt hydrophobic interaction causing
proteins to unfold into extended polypeptide chains
(amphiphatic = contain nonpolar and polar components)
 Reducing agents – eg. Urea, β-mercaptoethanol, will convert disulfide bridge (S-S) to
sulfhydryl group (SH). urea disrupt H bond & hydrophobic interaction
 Heavy metal ions – mercury (Hg+) and lead (Pb2+) disrupt salt bridge by forming ionic
bond with negatively charge group.
 Temperature change – as temp increase, the rate of molecular vibration increase. So
weak H bond will be disrupt and protein will unfold
 Mechanical stress – stirring & grinding actions disrupt the delicate balance of forces
that maintain protein strcuture.
eg. Foam formed when egg white is beaten vigorously contains denatured protein
b)
Distinguish the important characteristics of primary, secondary and tertiary structures of
proteins.
[15 marks]
Primary structure
 Primary structure of protein is its unique sequence of amino acids forming its
polypeptide chain
 Every polypeptides has a specific amino acid sequence
 the primary structure of a protein is starting from the amino-terminal (N) end to the
carboxyl-terminal (C) end
secondary structure
 These coiled & folded referred as secondary structure.
 2 types of secondary structure :
- α-helix
stabilized by hydrogen bond
- β-pleated sheet between carbonyl & amino groups
in the polypeptide’s backbone
tertiary structure
 Three-dimensional structure of a single protein molecules
 The α-helices and β-pleated sheets are folded into compact globule.
 Protein folding occurs as consequence of interactions between the side chains in their
primary structure
Question 2
a)
Enzymes are protein with high molecular weight.
(i)
State THREE (3) important properties of enzyme
[6 Marks]
- enzymes enhance the rate of a chemical reaction but is not permanently altered by the
reaction
- enzymes perform their work at moderate temperature
- enzymes are specific in the reactions that each one catalyzes
- each type of enzyme molecule contains a unique, intricately shaped binding surface
called active site
(ii)
Each enzyme is currently classified and named according to the type of
reaction it catalyzes. Explain any TWO (2) classes of enzyme
[8 Marks]
1 )Oxidoreductase
- Catalyze oxidation-reduction (redox) reaction
-eg dehydrogenases, oxidases, oxygenases, reductases, peroxidases & hydroxylases
2) Transferases
- catalyze reactions that involve the transfer of groups from one molecule to another
- eg. Transaminases, transcarboxylases
3) Hydrolase
- cleave bonds by adding water
- eg. Phosphatases, peptidases, esterase
4) Lyases
- catalyze reactions in which groups (eg. H20, CO2, NH3) removed to form a double
bond
- eg. Decarboxylases, hydratases, deaminases
5) Isomerases
- catalyze intramolecular rearrangements
-eg. epimerases or mutases
6) Ligases
- catalyze bond formation between two substrate molecules
- eg. Synthetase, carboxylase
(iii)
Describe by using appropriate diagram an’ induce-fit model’
[6 Marks]
 In this model, substrate does not fit precisely into a rigid active site
 Noncovalent interaction between enzyme & substrate change the active site structure,
make it fit for the substrate
Question 3
a)
The activity of enzymes can be inhibited by inhibitors such as drugs, antibiotics, poisons
and food preservatives. Discuss and compare with appropriate diagram on competitive
inhibitors and uncompetitive inhibitors
[10 Marks]
 Competitive inhibitors bind reversibly to free enzyme, not the ES complex, to form an
enzyme-inhibitor (El) complex. (2M)
 The inhibitors binds only to the enzyme-substrate complex (ES), and not the free enzyme
b)
Enzymes use the same catalytic mechanisms as nonenzymatic catalysts. Describe on
TWO (2) factors that contribute to enzyme catalysis.
[20 Marks]
(1) proximity and strain effects
 For a biochemical reaction to occur, the substrate must come into close proximity to catalytic
functional groups within the active site
 The substrate must be precisely oriented to the catalytic group
 Once substrate correctly positioned, result in a strained enzyme-substrate complex.
 This strain help to bring the enzyme-substrate complex into the transition state
 When an enzyme and substrate are in very close proximity, they behave as if they are part of the
same molecule
(2) electrostatic effects
 the strength of electrostatic interactions is related to the capacity of surrounding solvent
molecules to reduce the attractive forces between chemical groups.
 The local dielectric constant is often low
 this may influence the chemical reactivity of the substrate.
 weak electrostatic interactions in both the active site and the substrate, are believed to
contribute to the catalysis.
 An efficient binding of substrate lowers the free energy of the transition state
(3)Acid-Base catalysis
 Enzyme active sites contain side chain groups that act as proton donors or acceptors.
 These groups are referred to as general acids or general bases
 The side chain of histidine often participates in concerted acid-base catalysis because its pKa
range is close to physiological pH
 The protonated imidazole ring can serve as a general base
 Enzymes use several functional groups that behave as general bases to transfer efficiently
(4) Covalent catalysis
 In some enzymes a nucleophilic side chain group forms an unstable covalent bond with the
substrate
 The enzyme-substrate complex then forms product
 The covalent bond must, at a later stage in the reaction, be broken to regenerate the enzyme.
 This mechanism is found in enzymes such as proteases like chymotrypsin and trypsin, where an
acyl-enzyme intermediate is formed.
Question 4
The following is a diagram illustrating steps of a technique in DNA technology.
1
2
3
4
a)
Determine and discuss the steps 1 to 4.
[20 marks]
1) restriction fragment preparation
 DNA samples to be tested are treated with restriction enzymes that cut at specific
nucleotides sequences to produce a restriction fragments
2) electrophoresis
 The mixture of restriction fragments from each sample are separated by electrophoresis
according to their size
 Each sample forms a characteristic patterns of band
 The gel soaked with 0.5M NaOH to convert dsDNA to ssDNA
3) hybridization with radioactive probe
 Nitrocellulose filter is exposed to radioactively labeled probe, which bind to ssDNA with
a complementary sequence
4) Autoradiography
 Rinse away unattached probe
 Autoradiograph showing hybrid DNA fragment
b)
Name the technique and discuss ONE (1) purpose of this technique.
[5 marks]
Southern blott
 Enable researcher to detect and analyze particular DNA sequence
 Hybridization can be used to locate and/ or identify specific genes or other sequence