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I.

1 Structure Determination of a Polypeptide

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
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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)

Lyases EC 4 Addition (other than H2O) of Decarboxylases (removal of CO2)


groups to double bonds or Dehydratases (removal of H2O)
removal of groups to form Aldolase (cleavage of aldol)
double bonds
Isomerases EC 5 Convert the substrate into its Racemases (D-L isomers)
isomer Epimerases (sugar epimers)
Transfer of groups within cis-trans-Isomerases
molecules to form isomers Tautomerases (keto-enol groups)
Mutases (functional/positional isomers)
Ligases EC 6 Joining 2 molecules with the Carboxylase
hydrolysis of ATP Synthetase

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

The rate is measured


either by determining
how fast the substrate
is depleted OR how
fast product/s form

Pharmaceutical Biochemistry
Hand-out 2nd Shifting Gladys I. Bathan
Michaelis-Menten equation rate equation for a one-substrate enzyme catalysed reaction.

where Vo = Initial velocity


[S] = Substrate concentration
Vmax = Maximum velocity
Km = Michaelis-Menten constant

Lower Km, higher affinity of the enzyme to the substrate

Lineweaver-Burk Equation
- derived by taking the reciprocal of Michaelis-Menten equation
- more practical way of determining experimental Km and Vmax

y = mx + b

2 Types of Enzyme Inhibition


1. Reversible Inhibition
a. Competitive Inhibition

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

REGULATORY ENZYMES enzymes involved in metabolic pathways

Mode of Control (Regulation) of Regulatory Enzymes


1. Allosteric Control
Enzyme shape/conformation changes when an
allosteric effector or modulator binds to the enzyme
leading to a LESS ACTIVE or MORE ACTIVE
enzyme.

Allosteric Modulators / Allosteric Effectors


- small metabolites or cofactors
- may be inhibitory or stimulatory

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

2. Reversible Covalent Modification

Pharmaceutical Biochemistry
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3. Proteolytic Cleavage

ZYMOGEN
inactive precursor of an enzyme
- activated by cleavage of small peptide
fragment
Ex. chymotrypsinogen, trypsinogen

Some enzyme precursors are called


PROENZYMES or PROPROTEINS
Ex. proelastin, procollagen

Pharmaceutical Biochemistry
Hand-out 2nd Shifting Gladys I. Bathan