Professional Documents
Culture Documents
General Steps:
1. Purify the polypeptide
2. Reduce all the disulphide bonds
3. Determine the N- and C-terminal amino acid
4. Break into fragments by internal cleavage
5. Determine the amino acid sequence of each fragment
6. Combine all fragments to get the complete amino acid sequence of the polypeptide.
Terminal Cleavage
Method Reagent Specificity
Edman Degradation phenylisothiocyanate Forms a PTH derivative with the N-terminal
amino acid
Carboxypeptidase Carboxypeptidase Cleaves the peptide bond on the N-side of the C-
terminal amino acid
Aminopeptidase Aminopeptidase Cleaves the peptide bond on the C-side of the N-
terminal amino acid
Internal Cleavage
Method Reagent Specificity
Cyanogen Bromide CnBr Cleaves the peptide bond on the C-side of
methionine
Trypsin Trypsin Cleaves the peptide bond on the C-side of
arginine and lysine
Chymotrypsin Chymotrypsin Cleaves the peptide bond on the C-side of
tryptophan, phenylalanine and tyrosine
II. Enzymes
A. Terms
PROSTHETIC GROUP
cofactor tightly bound or covalently bonded to the
enzyme
SUBSTRATE
- molecule that binds to the enzyme and converted
to a product
ACTIVE SITE
- part of the enzyme where the substrate binds
Examples of Cofactors
Inorganic ions Coenzyme
Coenzyme Precursor
Cu2+ Flavin adenine dinucleotide (FAD) Riboflavin (vit B2)
Fe2+, Fe3+ 5-deoxyadenosylcobalamin (coenzyme B12) vit B12
Zn2+ Lipoate Pantothenic acid and other compounds
Mg2+ Coenzyme A ------
Ni2+ Biocytin Biotin
Se Nicotinamide adenine dinucleotide (NAD) Niacin
Mo Tetrahydrofolate Folate
Thiamine pyrophosphate Thiamine (vit B1)
Pyridoxal phosphate Pyridoxine (vit B6)
Pharmaceutical Biochemistry
Hand-out 2nd Shifting Gladys I. Bathan
B. Enzyme Class
Class Enzyme Class Type of Reaction Catalyzed Examples
(EC) Number
Oxidoreductases EC 1 Redox reactions Oxidases
Transfer of electrons (hydride Reductases
ions or H atoms Dehydrogenases
Transferases EC 2 Transfer of a group from one Kinases (phosphate grp)
substrate (donor) to another Transaminases (amino grp)
(acceptor)
X-Y + Z = X + Z-Y
Hydrolases EC 3 Hydrolysis (addition of H2O) Proteases (peptide bonds)
of C-C, C-O, C-N and bonds Phosphatases (Phosphoester bond)
like phosphoanhydride Glycosylases (glycosidic bond)
Nucleases (phosphosugar bond)
Esterases (ester bonds)
C. Enzyme Activity
ENZYMATIC ACTIVITY
- capability of an enzyme to catalyse a specific reaction at specific pH and temperature per unit time.
- affected by:
1. SUBSTRATE CONCENTRATION
2. pH
Optimum pH pH at which the enzyme activity is at maximum
Extreme pH can denature enzymes.
3. TEMPERATURE
Optimum temperature temperature at which the enzyme activity is at maximum
Extreme temperatures can denature enzymes.
4. ACTIVATORS/INHIBITORS
Activators molecules that can enhance/increase the enzyme activity
Inhibitors molecules that can decrease the enzyme activity
D. Enzyme Kinetics
- determines the rate of enzyme-catalyzed reaction and how it changes in response to changes in experimental
parameters.
- the rate is determined by measuring: a. the amount of product (P)formed per unit time or
b. measuring the amount of substrate (S) consumed per unit time
Pharmaceutical Biochemistry
Hand-out 2nd Shifting Gladys I. Bathan
Michaelis-Menten equation rate equation for a one-substrate enzyme catalysed reaction.
Lineweaver-Burk Equation
- derived by taking the reciprocal of Michaelis-Menten equation
- more practical way of determining experimental Km and Vmax
y = mx + b
Competive inhibitors
- bind to the enzymes active site
- compete with the substrate
- structures are similar to the substrates structure
- combine with the enzyme to form EI complex
- inhibitions lessens when [S] isincreased
Pharmaceutical Biochemistry
Hand-out 2nd Shifting Gladys I. Bathan
b. Non-competitive/Uncompetitive Inhibition
Uncompetitive inhibitors
- bind at a site other than the enzymes active site
- binds only at the ES complex
c. Mixed Inhibition
Uncompetitive inhibitors
- bind at a site other than the enzymes active site
- binds either at E or ES complex
2. Irreversible Inhibition
- inhibitor binds permanently to the enzymes active site by forming a covalent bond or a stable noncovalent
modification
Pharmaceutical Biochemistry
Hand-out 2nd Shifting Gladys I. Bathan
E. Regulatory Enzymes
ALLOSTERIC SITE
- binding site of allosteric modulators/effectors other
than the enzymes active site
ALLOSTERIC ENZYMES
- do not follow the hyperbolic Michaelis-Menten
Relationship
- exhibit sigmoidal saturation curve
FEEDBACK INHIBITION
- when the regulatory enzyme is inhibited by the end
product of a pathway
Pharmaceutical Biochemistry
Hand-out 2nd Shifting Gladys I. Bathan
3. Proteolytic Cleavage
ZYMOGEN
inactive precursor of an enzyme
- activated by cleavage of small peptide
fragment
Ex. chymotrypsinogen, trypsinogen
Pharmaceutical Biochemistry
Hand-out 2nd Shifting Gladys I. Bathan