Download amino acids and proteins

GENERAL BIOCHEMISTRY
SEMESTER TWO
1
COURSE OUTLINE
1.
A.
a)
b)
c)
d)
AMINO ACIDS AND PROTEINS
Chemistry of amino acids
Structure
Properties
Types/classification
Reaction
–
–
Carboxyl group
Amino group
B. Peptides
2
C.
a)
b)
c)
Polypeptides and proteins
Types and classification
Function
Structure and properties
I. Denaturation
II. Determination of amino acid sequence
III. Reactions of side chain R group
d)
Isolation and purification of proteins
3
2.
a)
b)
c)
ENZYMES
Naming and classification
Nature of enzyme activity
Factors affecting rate of enzyme action
I.
II.
III.
IV.
V.
VI.
VII.
d)
Substrate concentration
Enzyme concentration
Temperature
pH
Time
Inhibitors
Cofactors
Regulation of enzyme activity
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3. COFACTORS
4. NUCLEOTIDES AND NUCLEIC ACIDS
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AMINO ACIDS AND PROTEINS
• Proteins are complex organic nitrogenous
substances in the cells of plants and animals.
• They basically contain C, H, N, O and S.
• The building block units of proteins are amino
acids.
• There are about 20 different naturally
occurring amino acids and these contribute to
the structure, properties and functions of
proteins.
6
Chemistry of amino acids
• Amino acids have a general common
structure, but differences occur in
their side chain which distinguishes
them from one another.
• They contain an amino or basic group
and a carboxylic or acidic group.
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• Both the amino and carboxylic groups
are bound to the same carbon atom, αcarbon which is adjacent to the
carboxylic group.
• The α-carbon is also bonded to a
hydrogen atom and to a side chain R
group.
• The identity of a particular amino acid
therefore depends on the nature of the R
group.
8
General structure
+
NH3
H
C
R

NH2
COO
-
or
H
C

COOH
R
9
• Amino acids are often abbreviated by 3 letter
symbols or one letter symbol. The carbon
atoms in amino acids are designated α, β, γ, δ,
ε, etc. in the order in which they are attached,
starting with the C adjacent to the carboxyl
group.
• Almost all naturally occurring amino acids are
optically active with the exception of glycine
and have L-configuration at the α-carbon. Some
D-amino acids have been found in bacterial cell
walls.
10
Functions of the R-group
• The R-group may be acidic, basic or neutral,
etc and may serve the following functions.
1. They contribute to the interaction between
different parts of the protein molecule or
different proteins, e.g. hydrophobic
interactions, disulphide linkages, covalent
modification, etc.
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2. They affect stability of proteins.
3. They affect reactivity as they serve as points of
attachment of groups other than the amino acids,
e.g. sugar, sulphates, phosphates group, etc.
4. They serve as sources of identification by
hydrolytic enzymes.
5. R-groups dictate the shape of the proteins due to
their interactions, thus determining the ultimate
functions of the protein i.e. they dictate folding
into precise 3-dimensional configuration.
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Properties of amino acids
1. With the exception of glycine, amino acids have
asymmetric centres and are therefore optically active.
2. They possess charges. Neutral amino acids are
amphoteric in nature (have both acidic and basic
properties). Amino acids with acidic R groups (COO- or
COOH) are acidic while amino acids with basic R groups
(NH3+ or NH2) are basic.
3. Due to the presence of both positive and negative
charges, amino acids behave like salts and have high
melting points normally above 200oC.
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4. They are generally colourless, crystalline
solids whose solubility in water varies with the
nature of the constituent R-group. They are
quite insoluble in non-polar solvents like ether
and chloroform.
5. Some are sweet, e.g. valine, alanine, proline.
Others are bitter, e.g. arginine, isoleucine or
tasteless, e.g. leucine.
14
Classification of amino acids
Classification may depend on:
1. their reactions, i.e. neutral, acidic or basic.
2. chemical structure i.e. presence of polar and
non-polar side groups, aromatic groups, long
hydrocarbon side chain, etc.
• A general classification is based on the polar or
non-polar nature of the R-group and the
presence of acidic or basic groups in the side
chain. There are four main groups;
15
1. Amino acids with neutral hydrophobic (non-polar / apolar)
side chain.
• e.g. glycine, valine, alanine, leucine and
isoleucine. These have aliphatic side chains.
Proline has an aliphatic cyclic structure and is
actually an imino acid since the N is bonded to 2
carbon atoms.
• Phenylalanine and tryptophan have aromatic Rgroups. (Aromatic ring of tryptophan is called
indole ring).
• Methionine contains S in addition to aliphatic
group.
16
• Non polar amino acids in a protein tend to
produce a hydrophobic environment in the
protein of which they are components.
• The amino acids in this group are generally found
buried in the interior of the proteins where they can
associate with one another and remain isolated from
water.
• Amino acids in this group play an important role in
maintaining the three dimensional structure of
proteins.
17
2. Amino acids with neutral and hydrophilic (polar
uncharged) R group in their side chain.
• These amino acids have polar side chains that
are neutral at neutral pH (pH 7). They are
serine and threonine (polar OH groups
attached to aliphatic side chain). Tyrosine has
OH group attached to aromatic chain. Cysteine
has -SH polar side chain which can react with
other cysteine –SH groups to form disulfide (-SS-) bridges in proteins.
• Sometimes, glycine is put in this group because
of the H (formation of H bonds in water).
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• The polar OH group of Ser, Thr and Tyr enables
them to participate in H bonding, an important
factor in protein structure. OH groups also serve as
points for esterification with phosphate and
attachment of sugars or carbohydrates.
• Asparagine and glutamine bear highly polar amide
side chains of different sizes.
• The amino acids within this group can associate
with one another by hydrogen bonding which
helps to maintain proper three dimensional
structures of proteins.
19
3. Amino acids with acidic and hydrophilic
(polar) side chains
• This group has an additional COOH group in
the side chain. The carboxyl group can lose a
proton to form the carboxylate ion (proton
donor).
• They are negatively charged at neutral pH, e.g.
glutamic acid (glutamate) and aspartic acid
(aspartate).
• The side chain carboxyl groups frequently
bond to NH2 to form the side chain amide
groups yielding the analogous amino acids
asparagine and glutamine.
20
4. Amino acids with basic and hydrophilic (polar) side
chains
• These bear positive charges at neutral pH and
have additional amino groups in the side
chain, e.g. histidine (side chain of histidine
referred to as imidazole).
• Lysine (side chain amino group attached to
aliphatic hydrocarbon chain), and arginine
(side chain guanidine group attached to
aliphatic hydrocarbon chain).
21
• The hydrophilic or polar amino acids are
often found on the surface of protein in
association with water.
• The negatively and positively charged
amino acids within a protein can interact
with one another to form ionic bridges,
another strong force that helps keep the
protein chain folded in a particular
manner.
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Non-protein amino acids
• These are amino acids that are found in free
or ‘uncombined’ forms and are not
constituents of proteins.
• Some play important roles in metabolism and
may be intermediates in the biosynthesis of
proteogenic amino acids, e.g. L-ornithine and
L-citrulline are metabolic intermediates of the
urea cycle and hence participate in the
biosynthesis of the amino acid arginine.
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O
+
H3N CH
C O
O
-
H2C
+
H3N CH C O
H2C
CH2
CH2
CH2
CH2
+
H3N
HN
C=O
L--Ornithine
NH 2
L--Citrulline
24
-
• β-alanine occurs free in nature and is a
component of the water soluble vitamin
pantothenic acid.
• Other non protein amino acids are
homocysteine, homoserine, γ-amino butyric
acid (GABA).
• Homoserine and homocysteine are
intermediates in amino acid metabolism.
• GABA is involved in the transmission of nerve
impulses.
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H2N CH2CH2C OOH
alanine
NH2
HS CH2CH2C COOH
H
Homocysteine
H2N CH2CH2CH2C OOH
GABA
NH2
HO CH2CH2C COOH
H
Homoserine
26
• Other examples are serotonin, thyroxine and
indoleacetic acid (IAA).
I
I
CH2 COO
HO
O
CH2CH COO
+
NH3
-
-
N
H
I
I
Thyroxine
HO
Indole acetic acid (IAA)
+
CH2 CH2 NH3
N
H
Serotonin
27
Rare amino acids
• These rarely occur but have been isolated from
hydrolysate (hydrolytic products) of some
specific proteins.
• They are all derivatives of some standard amino
acids, e.g. 4-hydroxyproline and 5-hydroxylysine
are derivatives of proline and lysine respectively.
• They are abundant in fibrous proteins and
collagen. Hydroxyproline is an important
component of animal supportive and connective
tissues.
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COO
+
H3N
HO
CH CH2
H2C
C COOH
N H
H
H
+
-
CH
CH2
H2C
H C OH
CH2
+
H3N
4-hydroxyproline
5-hydroxylysine
29
Essential amino acids
• These can not be synthesized in the body of
higher animals and have to be provided in the
diet. The inability to synthesize them may be
due to the absence of
1. The corresponding α-keto acid of the amino
acid.
2. The enzyme involved in transamination i.e. a
specific transaminase.
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+
NH3
O
R
1
C COOH
+
R
2
C
COOH
+
NH3
transaminase
R
H
Acceptor keto
acid
Donor amino
acid
1
C
COOH
+
O
R
2
C COOH
H
New amino
acid
New keto
acid
• The process is called transamination. Example;
CH3
C
O
+
H2N
C COOH
O
H
H
H2N
C COOH
COOH
H
H3C
Pyruvate
Glycine
Alanine
+
H C
C OOH
31
• Since most proteins in the body contain the full
complement of amino acids, young animals fail
to grow on a diet deficient in even one essential
amino acid since without it they are unable to
synthesize adequate proteins.
• Sufficient quantities are needed to maintain the
proper nitrogen balance in the body.
32
• Prolonged deficiency leads to the disease
kwashiorkor in children
• Other deficiencies are
1. fall in plasma protein level, and
2. low haemoglobin levels in adults
33
Essential amino acids
Non-essential amino acids
Histidine
Alanine
Leucine
Glycine
Isoleucine
Asparagine
Methionine
Proline
Phenylalanine
Serine
Threonine
Tyrosine
Tryptophan
Cysteine
Valine
Glutamine
Arginine
Glutamic acid
Lysine
Aspartic acid
Ideally, lysine and arginine are semi essential amino acids
because they can be synthesized by the body but not in
adequate quantities. Proteins from cereals are poor in
lysine and those from legumes low in methionine
34
Acid base properties of amino acids
• Amino acids contain both acidic and basic groups and
react with both alkali and acids to form salt and are
thus amphoteric in nature.
• The acid group is a proton donor and the basic group
is a proton acceptor. In the crystalline or solid state,
amino acids exist as dipolar ions. In this case, the
COOH group exists as the carboxylate ion (COO-)
bearing a negative charge and the amino group (NH2)
exists as ammonium ion (NH3+), bearing a positive
charge.
• The dipolar ion is called a zwitterion.
35
• In aqueous solution, equilibrium exists between
the dipolar ion and other anionic and cationic
forms of the amino acid.
H
H2N
H
-
C COO
R
Basic solution
anionic
H+
OH-
+
H3N
H
-
C COO
R
Zwitterion
H+
OH-
+
H3N
C COOH
R
Cationic
36
• The position of the equilibrium depends on the pH of
the solution and the nature of the amino acid especially
contributed by the R-group.
• In strongly acidic solution all amino acids exist primarily
as cations and in strongly basic solutions, they exist as
anions.
• At some intermediate pH called the isoelectric point
the concentration of the zwitterion is at its maximum
and the concentration of the cation and anion are
equal. At this pH (isoelectric point), there is no net
migration of the amino acid when placed in an electric
field.
• The dissociation of an amino acid is therefore strongly
dependent on the pH value of the solution.
37
Isoelectric pH
• It is the pH at which there is no net charge on
the amino acid. It is denoted pI.
• Each amino acid has a specific pI and this has
been the basis for precipitation of amino
acids.
• At certain pH values, the amino acid may
move either to the anode or cathode in an
electric field depending in the charge on it and
the magnitude of that charge at that pH.
38
Amphoteric nature of amino acids
• The amphoteric nature of amino acids is
responsible for the buffering action of
proteins in the blood because as an acid it can
donate proton and as a base, it can accept
proton and therefore can resist small changes
in pH.
• An amino acid with a neutral R group is
somewhat more acidic than it is basic. As a
result, its isoelectric point occurs at a pH
slightly lower than a neutral solution of pH7.
39
Amphoteric nature of amino acids
40
Titration curves of amino acids
• When an amino acid is titrated, its titration
curve indicates the reaction of each ionisable
group that is capable of reacting with H ion.
The normal titration curve of an acid with a
base is as follows;
41
• Acid base reactions involve a conjugate acid
base pair made up of proton donor and proton
acceptor. (The biochemical behaviour of many
important compounds depend on their acid
base properties).
• The ability of acids or bases to readily lose or
gain protons depends on the chemical nature of
the compounds involved. Eg. the degree of
dissociation of acids in water varies from
complete dissociation for strong acids, to partial
or no dissociation for weak acids. Intermediate
values are possible.
42
Dissociation of weak acid in solution
• Typical example is dissociation of acetic acid;
• HA
A - + H+
• The strength of an acid, which is the amount
of H+ released when a given amount of acid is
dissolved in water, can be expressed
numerically. The expression is called acid
dissociation constant or Ka, and can be
written for any acid HA according to the
equation;
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1. For each acid, Ka has a fixed numerical value at a fixed
temperature.
Ka = [H+] [A-]
[HA]
solving for [H+]
[H+] = Ka[HA]
[A-]
According to the Henderson-Hasselbalch equation
pH = pKa + log [A-]
[HA]
pH = pKa +
log
[proton acceptor/conjugate base]
[proton donor/conjugate acid]
44
• The numerical value of Ka is higher when the
acid is more completely dissociated, i.e. the
larger the Ka the stronger the acid or the
smaller the pKa value the stronger the acid.
• At pH = pKa, half of the ionizable groups are
dissociated.
45
Titration of acetic acid with NaOH
• During titration, a measured amount of base is
added to a measured amount of acid thus
changing the pH of the solution.
• The amount of base required for complete
reaction with the acid is referred to as one
equivalent.
• The point in titration at which the acid is
exactly neutralized is called the equivalence
point.
• In the course of titration of acetic acid, a point
is reached when the pH = pKa of the acetic
acid.
46
• The point of the titration curve where pH=pKa is
the inflection point. This corresponds to a
solution with equal concentration of the weak
acid and its conjugate base, in this case acetic
acid and acetate respectively.
• The pH at the point of inflection is about 4.8 and
is equal to the pKa of acetic acid. Inflection point
is attained when 0.5 equivalent of base has been
added. Near the inflection point the pH changes
very slowly as more base is added.
47
• The equivalent point is reached when one
equivalent of base has been added. At this point,
practically all the acetic acid has been converted
to acetate ion.
48
Titration of acetic acid with NaOH
pH
9.2
4.8
-
* CH3COO
x
Inflection point
* CH3COOH
0
0.5
1.0
NaOH
x CH3COOH = CH3COO-
49
Titration curves of some amino acids, e.g.
alanine
• Alanine has 2 titrable groups; the carboxyl and amino
groups.
• In it’s fully protonated form it can be considered as a
dibasic acid and can therefore donate 2 protons
during its titration with a base like NaOH. At very low
pH, alanine has protonated carboxyl group
(uncharged) and a positively charged amino group.
50
Under such conditions alanine has a net positive
charge of one. As base is added, the carboxyl
group loses its proton to become a negatively
charged carboxylate ion as the pH of the
solution increases.
• At this stage, alanine has no net charge. As
more base is added with resulting increase in
pH the protonated amino group (weak acid)
loses its proton and the alanine has a negative
charge of one.
51
Titration curve of Alanine
52
Ionization of alanine
• The pKa of the two stages of ionization of alanine is wide enough to yield
two separate regions, each region showing a titratable group. The
apparent pKa of the 2 dissociation curves can be determined from the
midpoint of each stage. pKa1 is equal to 2.34 and pKa2 is 9.69. At pKa 2.34,
NH3+CHRCOOH and NH3+CHRCOO- are present in equimolar
concentrations and at pKa 9.69 H3N+CHRCOO- and H2NCHRCOO- are
present in equimolar concentrations.
53
• At pH 6.02 there is a point of inflection between the two separate
phases of the titration curve of alanine. This is the isoelectric pH
or pI and alanine bears no net charge and therefore there is no
migration in an electric field (at pH 6.02, the dominant specie is
NH3+CHRCOO-).
• Mathematically, pI is expressed as the arithmetic mean of the 2
pKa values.
pI
=
pI alanine
pKa1 + pKa2
2
=
2.34 + 9.69
2
=
6.02
54
• At pH above the pI (alkaline) amino acids exist
as H2NCHRCOO- and migrate to the anode
(positive pole). At pH below pI (acidic) amino
acids exists as H3N+CHRCOOH and will migrate
towards the cathode (negative pole).
55
Titration curve of diprotic amino acids
• A diprotic amino acid is an amino acid with an
additional titratable group in the side chain
e.g. Histidine
• Amino acids having additional NH2 or COOH
group will have corresponding pKa2 values for
them. Example, aspartate has a pKa of 2.1 for
the α-carboxyl group and a pKa of 3.9 for the
β-carboxyl group and pKa of 9.8 for the NH3+
group.
56
• In histidine, the imidazole side chain also contains
titratable group.
• At very low pH values, histidine has a net positive
charge of 2 as both the imidazole and amino
groups bear positive charges.
• As base is added, the pH increases and the
carboxyl group loses a proton to become a
carboxylate ion and histidine now has a net
positive charge of +1.
• With further addition of more base, the imidazole
charge group loses its proton. At this point
histidine has no net charge (pI of histidine).
57
• At still higher pH values the amino group loses
its proton and histidine now has a net
negative charge of -1.
58
• Like acids, amino acids have characteristic values for
Kas and pKas of their titratable groups.
• The pKas of the α-carboxyl groups are fairly low,
approximately 2; that of the amino groups are
between 9 and 10.5.
• The pKa of the side chain groups including the
additional COOH and NH2 groups depend on the
chemical nature of the group.
59
• The classification of an amino acid as acidic or
basic depends on the pKa of the side chain.
These R groups can still be titrated within a
protein but their pKas may not necessarily be
the same as the value in the free amino acid.
60
Reactions of amino acids
• The characteristic reactions of amino acids are
those of their functional groups, i.e. the COOH
and the NH2 groups and the functional group
present in the different side chain. These
reactions are important in protein chemistry
for;
1. Identification and analysis of amino acids in
protein hydrolysate (hydrolytic product).
61
2. Identification of amino acid sequence in a
protein.
3. Identification of the specific amino acid residues
of native protein that are required for their
biological activity.
4. Chemical modifications of amino acid residues
in protein molecule to produce changes in their
biological activities and other properties.
5. Chemical synthesis of polypeptides.
62
Reactions of carboxyl group
• The carboxyl groups of amino acids can react
to form salts, esters, acid chlorides and
amides
1.Esterification with alcohol and the formation
of peptide bonds
• In the presence of HCl amino acids react with
alcohol or ethanol to form esters.
63
• When the amide bond involves an α-amino
group of another amino acid instead of NH+3, a
peptide bond is formed. This is the basis for
the formation of peptides and proteins.
• Peptide bonds are linked between amino acids
in a protein-like glycosidic bonds.
64
2. Decarboxylation
• When α-amino acids are heated in the
presence of Ba(OH)2, carbon dioxide is
released and an amine is formed.
• Decarboxylation can also be achieved by the
enzyme decarboxyalases. Decarboxylaton of
amino acids is important in the body as it
yields biologically important active amines,
e.g. Tyrosine yields adrenaline, histidine yields
histamine and tryptophan yields serotonin.
65
66
Reactions of the amino group
1. Acylation
Amino groups of amino acid may be acylated by
treatment with acid anhydride or acid
chlorides in cold alkaline medium.
67
• This method is used to protect the α-amino
group in the chemical synthesis of a peptide.
Glycine readily reacts with benzoic acid to
detoxify the benzoic acid in the body.
68
2. Reaction with mild oxidizing agent e.g. ninhydrin
• This reaction is to detect and estimate amino
acids quantitatively in small amounts. The
reaction involves oxidative deamination of the
amino acid to form ammonia, carbon dioxide
and an aldehyde obtained by loss of one
carbon from the original amino acid.
• The reduced ninhydrin can react with another
mole of ninhydrin and the ammonia to
produce a complex with an intense violet blue
colour known as Ruhemann’s purple.
69
70
• All amino acids give intense purple blue colour
whereas imino acids like proline and hydroxyproline
give yellow colour.
• Arginine reacts to give a brown colour.
• Asparagine, because it has a free amide group also
produces a characteristic brown colour with
ninhydrin because of the amide group.
• The coloured complex produced form the basis for
quantitative determination of amino acids. The
absorbance of the solution after heating with
ninhydrin is proportional to the concentration of
amino acid.
71
3. Reaction with strong oxidizing agent e.g. nitrous
acid
• Nitrous acid (HNO2)reacts with amino group
to form the corresponding hydroxyacids with
the liberation of nitrogen. This reaction is
important in estimation of α-amino groups in
amino acids, peptides, polypeptides and
proteins. Proline and hydroxyproline do not
react. The ε-amino group of lysine reacts
slowly.
72
Reaction with aldehydes
• The α-amino group of amino acids react
reversibly with aldehydes to form a Schiff’s
base. These appear to be intermediates in a
number of enzymatic reactions involving
interaction of the enzyme with the amino or
carboxyl group of the substrate.
73
Reaction with cyanate
• Amino groups of amino acid react with
cyanate to yield carbamoyl derivatives and
this reaction has been used to modify the
properties of sickle cell haemoglobin to make
it more like adult haemoglobin.
74
• Some very important reactions of amino acids
involving amino group have become very useful
in determining amino acid sequence of proteins.
Such reactions include the following;
1. Reaction with Sanger’s reagent or 2,4dinitrofluorobenzene.
• The reagent is used in determining the amino
acid sequence in a protein or peptide since it
reacts with the free N-terminal end to form an
intense yellow dinitrophenyl (DNP) derivative.
75
• Reaction occurs in cold, alkaline
medium(normally HCO3-medium is used) and
releases hydrogen fluoride. The DNP
derivative formed can be hydrolysed to yield
individual amino acids and the DNP amino
acid which is resistant to hydrolysis.
• The DNP amino acid can be distinguished by
paper chromatography, thus identifying the
amino terminal of the polypeptide chain.
76
• Disadvantage – The method is not reliable
because amino acid sequence cannot be
determined since free amino acids are
released.
77
78
Reaction with Dansyl chloride (N-dimethyl aminonaphthalene5-sulphonyl chloride)
• Dansyl chloride is a more sensitive agent for
the detection and measurement of N-terminal
amino acid residues.
• It reacts with N-terminal amino acid to yield a
dansyl amino derivative which is stable.
• The dansyl group is highly fluorescent and the
intensity can be measured in a fluorimeter.
79
• A particular intensity will indicate the specific
amino acid present. The reaction can be used
to determine minute amounts of amino acid.
• Disadvantages are the same as that of
Sanger’s reagent.
80
• Dansyl’s method has an advantage over
Sanger since smaller amounts of amino acid
can be used
81
Reaction with phenylisothiocyanate (Edman’s
degradation)
• The reagent reacts with the N-terminal amino
acid of peptide or protein under mild alkaline
conditions to form the corresponding phenyl
thiocarbamoyl peptide or derivative.
• On treatment with acid, the N-terminal
residue is split off as a phenylthiohydantoin
derivative and this can be identified by paper
chromatography.
82
• The Edman reaction has been used to
determine the sequence of amino acids in
peptides and proteins.
• Advantage of the method is that the
derivative is very stable in acid and therefore
step-wise degradation can occur, thus rest of
the chain is left intact so further cycles of the
procedure can occur.
• Disadvantage - accumulation of by-products
can occur, interfering with the procedure. Can
be limited to 25 cycles (automated).
83
84
Metal complexes
• The α-amino acids form stable complexes with
metals such as Cu, Co, and Mn. When there are 2
or more peptide bonds, there is reaction with
Cu2+ in alkaline solution to form a violet blue
complex.
• This is the basis of the Biuret’s test. It is a
quantitative test for proteins as intensity of
colour determines concentration of proteins.
85
Identification of C-terminal amino acid
1. Use of lithium borohydride (LiBH4)
• LiBH4 reduces C-terminal amino acid to form the αamino alcohol. If the peptide chain is hydrolyzed, the
hydrolysate will contain an α-amino alcohol which
corresponds to the original C-terminal amino acid.
This can be identified by chromatographic methods.
86
2. Use of hydrazine (NH2NH2) also known as
hydrazinolysis or Akabori procedure
• Hydrazine is used to cleave all peptide bonds
by converting all except the C-terminal amino
acid into hydrazides. The C-terminal amino
acid appears as a free amino acid which can
be readily identified by chromatography.
• Usually, determination of N-terminal is more
sensitive and common than C-terminal.
87
Hydrazinolysis
88
PEPTIDES
• They are intermediate compounds between
amino acids and proteins.
• Peptides are named according to amino acid
content.
• Peptides with
2 amino acids - dipeptide
3 amino acids - tripeptide
4 amino acids - tetrapeptide
less than 10
- oligopeptide
greater than 10
- polypeptide.
89
• By convention, H2N is to the left and COOH to
the right or H2N is up and COOH down
(vertically). Specific name for peptides is derived
by attaching the ending –yl to the amino acid
whose carbonyl group is involved in the peptide
link.
90
• Peptides have amino group (NH2) at one end
(N-terminal) and a COOH at the other end (Cterminal).
• Peptides also have characteristic chemical
reactions based on the NH2, COOH and the Rgroups. Examples of naturally occurring
peptides are vasopressin, oxytocin,
glutathione, opioid peptides and enkephalins.
91
Hydrolysis of peptides
• This can be achieved by boiling with either
strong acid or base to yield the constituent
amino acids. The peptide bond has a partial
double bond character and is therefore rigid.
This prevents free rotation. The peptide bond
is generally a trans bond.
• Hydrolysis of peptide bonds can be achieved
by the use of enzymes.
92
Hydrolysis of peptides
.
93
POLYPEPTIDES AND PROTEINS
• Proteins are large molecular weight
compounds which may contain a single
polypeptide chain like myoglobin or 2 or more
polypeptide chains like haemoglobin (4
peptide chains).
• The various polypeptide chains in a protein
are held together by hydrophobic interactions,
covalent linkages and disulphide bonds, etc.
94
Classification of proteins
• This may be based on their composition.
There are 2 main types of this classification
1. simple
2. conjugated proteins
95
Simple proteins
• These are made up of amino acids only, eg
albumin, globulins, glutelins, collagen, keratin,
myosin.
Conjugated proteins
• These contain non protein portions and on
hydrolysis yield amino acids and other organic
or inorganic components.
• The non protein portions are called the
prosthetic group and they are classified
according to the nature of the non-protein
portion.
96
Examples of conjugated proteins
• Nucleoproteins - nucleic acids + protein portion
• Glycoproteins - carbohydrates + protein portion
• Lipoproteins -
lipids + protein portion
• Haemoglobin - haem + protein portion
97
• Another mode of classification is with regard to the
shape or structure of the protein. On this basis,
proteins are divided into two main groups;
1. Fibrous proteins
• These are water insoluble long thread-like molecules.
They are highly resistant to digestion by proteolytic
enzymes.
98
• They consist of several coiled peptide chains which
are highly linked.
• They are physically tough and have structural and
protective functions.
• E.g. collagen of muscles, tendons, keratin of hair,
proteins of silk, nails, connective tissues and bone
elastin.
99
2. Globular proteins
• These are soluble in aqueous system, eg
enzymes, food proteins like albumin and
casein of milk.
Fibrous proteins
Globular proteins
These contain higher amounts of regular
secondary structure
Have variable molecular weight
Long, cylindrical, rod-like shapes
Spherical in shape
Have no water solubility
Have high water solubility
They play structural rather than dynamic
role
Play functional role like catalyst,
transporters, controller protein, regulation
of metabolic pathways and gene
expression
100
Properties of proteins
1. They contain L-amino acids in peptide
linkages.
2. They form colloids in solution
3. They are generally tasteless, but their
hydrolysate may be bitter or sweet.
4. They are colourless, but heating turns them
brown and continuous heating results in
charring, giving off the odour of burning hair.
101
5. Each type of protein is characterized by;
a. specific chemical composition
b. specific amino acid sequence
c. specific molecular weight
102
Functions of proteins
Proteins play important role in all biological
processes and their functions are exemplified
in;
1. Enzymatic catalysis – enzymes catalyze
chemical reactions in biological systems, thus
proteins play a unique role in determining the
pattern of chemical transformations in
biological systems.
103
2. Transport – specific proteins transport small
molecules and ions, e.g. haemoglobin carries oxygen
in erythrocytes or red blood cells, and myoglobin
carries oxygen in the muscles.
3. Coordinated motion (movement) – proteins are
major components of muscle and muscle contraction
is accomplished by the steady motion of two kinds of
protein filaments, actin and myosin.
104
4. Mechanical support – collagen, a fibrous protein
is found in bone and skin and this gives the high
tensile strength of the skin.
• Others are fibrous (silk protein) for mechanical
strength in silk, elastin/rubber-like protein found
in elastin fibers present in several tissues in the
body eg blood vessels and skin.
105
5. Immune protection or defence (antibodies) – these
are highly specific proteins that recognise and attack
foreign substances or organisms such as viruses and
bacteria and nullify their effect in the body.
6. Generation and transmission of nerve impulses
(hormones) – certain proteins help in the
transmission of nerve impulses. The response of
nerve cells to specific stimuli is mediated by specific
proteins.
106
7. Growth control and differentiation (genetical) –
controlled sequential expression of genetic
information is essential for the orderly growth
and differentiation of cells. These processes are
mediated by proteins.
107
Organisation of amino acids in protein (protein
structure)
• Amino acids present in proteins have several
functional groups and therefore contribute to
structure, properties and functions of a
particular protein.
• Protein structure may be considered under 4
levels of organisation.
• These are the primary, secondary, tertiary and
quaternary structures. Differences arise due to
the types of bond in these structures.
108
The primary structure
• This refers to the order in which the amino acids
are covalently linked together in a protein, ie
the amino acid sequence.
• It indicates the number and types of amino
acids and in which fashion or manner they are
linked.
Eg. H2N-ala-cys-pro-met-leu-ala-ala-glu-gly-COO109
The secondary structure
• This determines the coiling of the polypeptide chain into
a helical structure. This structure arises from the folding
and twisting of the polypeptide chain into a coil or spiral
form as a result of H bonding.
• In many proteins, the H bonding produces a regular
coiled arrangement called α-helix. H bonding in
secondary structure usually involves amino acids that
are quite close in the polypeptide chain.
110
• Although H bonding is weak, they are so numerous
that they are able to stabilize the molecule.
• Another type of secondary structure is the β-pleated
sheet which may be parallel or anti parallel.
• α-helix is predominant in fibrous proteins like myosin
and α-keratin of hair, wool and nails. β-pleated sheet
is common in silk protein.
111
Factors that affect α-helix
1. Proline creates a bend in the backbone of its cyclic
structure, it cannot fit into the α-helix.
2. Localized factors involving the side chains include strong
electrostatic repulsion due to the proximity of several
charged groups of the same sign, eg positively charged
group of lysine or arginine or negatively charged groups
of glutamate or aspartate.
3. Steric repulsion or crowding due to the proximity of
several bulky side chains.
112
• In the α-helix conformation, all side chains lie
outside the helix as there is not enough space
for them in the interior.
• α-helix is stabilized by intramolecular H
bonding (between –C=O-and --H-N group ie
between carbonyl O and amide H of the same
molecule).
113
• The H-bonds in β-sheet are perpendicular to
the polypeptide backbone. β-pleated sheet is
stabilized by intermolecular forces ie C=O
from one molecule and the NH of another
molecule.
• Unlike the α-helix, β-sheets have two or more
peptide chains or β-strands.
114
The tertiary structure
• This involves coiling or folding of the helical
structure into a three dimensional structure of
the biologically active native conformation.
• There are other associated forces between the
amino acid residues relatively far apart in the
chain. These include H bonding, disulphide
linkages, ionic bonding, ester bonding and
hydrophobic interactions or Van der Waals
forces.
115
• Reactivity between the R-groups of amino
acids contributes to stability of the proteins.
• The folding occurs such that maximum
numbers of polar (hydrophilic) group are on
the exterior of the molecule exposed to the
environment and the maximum number of
non-polar (hydrophobic) groups are within the
interior.
116
• The tertiary structure determines the
structure of the protein and dictates the
catalytic properties of biologically active
protein.
1. Hydrophobic interactions (Van der Waals
forces) occur between R groups of non-polar
amino acids. It is the most important noncovalent force that causes proteins to fold into
their native structure.
117
2. H bonds between the polar R groups of the
polar amino acids. It can also occur between
the OH groups of serine and threonine and
tyrosine and amino groups and carbonyl
oxygen of asparagine or glutamine and ring N
of histidine.
3. Ionic bonds (electrostatic interaction)-this
occurs between the R-groups of positively
charged and negatively charged amino acids,
e.g. lysine and glutamate.
118
• 4. Covalent bond – this occurs between the
sulphydryl containing amino acids (cysteine)
i.e. the disulphide link.
• All these bonds are in addition to the H bonds
and peptide bond of the helical structure.
119
The quaternary structure
• This is found in proteins with more than one
polypeptide chain.
• The individual chains are arranged in relation
to each other in such a way as to produce a
simple 3-dimensional structure of the overall
protein molecule.
• Each polypeptide chain in such a protein is
called sub-unit or monomer and the assembly
is called oligomer.
120
• The sub units are linked together primarily by
non covalent forces.
• The primary structure is very vital in
determining the 3-dimensional structure or
shape, i.e. the primary structure specifies or
dictates the 3-dimensional shape of the
protein.
121
• Thus the critical determinant of the biological
function of a protein is its conformation which
is defined as its 3-dimensional arrangement of
the atoms within the molecule.
• Non-covalent forces cause a polypeptide to
fold into a unique conformation and then
stabilize the native structure against
denaturation.
122
Importance of the primary structure
• The amino acid sequence (primary structure)
of a protein determines its three dimensional
structure which in turn determines its
properties and functions, eg in enzymes, the
3-dimensional structure serves to place the
crucial amino acids that are directly involved
in catalyzing reactions close to each other.
123
• Alteration in the amino acid sequence can affect
the function of the protein.
• In some cases, large changes may occur without
affecting function of a particular protein.
• In other cases, a change in only a single amino acid
residue can effect a profound alteration in the
properties of the protein.
124
Primary structure and species variation
• Studies on cytochrome C from about 40 different
organisms show that there isn’t much difference in the
primary structure. The cytochrome C from these
organisms may differ in the positions of one or a few
amino acids in the chain.
• Though amino acids in certain positions may be
different, the proteins perform the same function
irrespective of the organism.
125
• Scientists concluded that differences in amino acid
sequence have given rise to variation in the different
species. Such a change is called a conservative
change, eg in man and in monkey position 1 is
different and in man and horse, position 12 is
different.
126
Primary structure and genetic defect
• This is exemplified in the haemoglobin associated
with sickle cell anaemia. In this genetic disease,
red blood cells are unable to bind oxygen
efficiently. The red cells also assume a
characteristic sickled shape hence the name of
the disease.
127
• The sickle cells tend to become trapped in
small blood vessels cutting off circulation and
thus causing organ damage.
• A change in one amino acid residue in the
sequence or the primary structure causes
these drastic consequences.
128
• Haemoglobin is made up of 4 subunits
consisting of two α chains and two β chains
bound together. In HbS, the α–chains are intact
but the β–chains are affected. In one of these β–
chains, glutamic acid (acidic) is replaced with
valine (hydrophobic) in position 6.
• HbA
H2N-Val-His-Leu-Thr-Pro-Glu-Glu-Lys—
• HbS
H2N-Val-His-Leu-Thr-Pro-Val-Glu-Lys—
129
• The highly polar side chain of glutamate containing an
ionisable carboxyl group is replaced by a non polar one,
the isopropyl group of valine.
• In the 3-dimensional structure of haemoglobin, this
residue is on the outside of the molecule.
• One molecule of HbS can become involved in
hydrophobic interractions with other haemoglobin
molecules because of the presence of non polar residue.
130
• Such an interaction does not occur in HbA with a
polar residue in the same position. As a result groups
of molecules of HbS aggregate with each other.
• These aggregates distort the shape of the blood cells
resulting in the disease.
• Such a change is non conservative because it alters
the function and property of the molecule.
131
Primary structure and functional differentiation
• This is also a conservative replacement but
does not result in species variation but
variation in function.
• Replacement of one amino acid in the chain
gives rise to differences in function. eg
oxytocin and vasopressin.
132
Vasopressin
1 2 3 4 5 6 7 8 9
Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-Gly
S
S
Oxytocin
1 2 3 4 5 6 7 8 9
Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly
S
S
133
Protein denaturation
• In the native state, a globular protein is a highly
ordered conformation in which the biological
activity is manifested.
• The non covalent interactions responsible for
maintaining the 3-dimensional structure are weak
and can easily be disrupted leading to unfolding
of the protein.
134
• The process of unfolding is called denaturation
and this leads to loss of activity.
• Under proper conditions, the 3-dimenional
structure can be restored or recovered in
some cases (renaturation).
135
Denaturation arises from the following;
1. Changes in peptide structure due to unfolding
2. Destruction of ionic and other bonds discussed in
protein structure.
• Generally, denaturation results in changes in solubility
at the isoelectric pH where normally, insolubility is high.
• The consequences of denaturation is coagulation, ie
when protein is thrown out of solution.
136
• Water is also very vital because in its presence
proteins are easily denatured.
• Dry proteins are less susceptible to heat
denaturation or coagulation than hydrated ones or
those in solution.
• Denaturated or coagulated proteins have little
tendency to associate with water.
137
AGENTS OF DENATURATION
•
•
•
•
•
AGENTS OF DENATURATION
There are three main agents, these are
Physical
Chemical
Biological
138
PHYSICAL
PHYSICAL – eg heat, pressure, freezing, shaking
and foaming, e.g beating of eggs,
autoclaving (sterilizing).
• In sterilizing surgical instruments, heat and
pressure are employed in the use of
autoclave. The heat and pressure denature
the bacterial protein (cell wall) thus killing the
bacteria.
139
CHEMICAL
• Extreme of pH leads to changes in the charge
on the protein. Each protein has a
characteristic charge because of the R groups
of amino acids and specific amino acid
composition.
• These positively and negatively charged Rgroups on the surface interact with ions and
water molecules keeping the proteins in
solution within the cytoplasm.
140
• When the charges on the protein are neutralised
the net charge on the protein is zero and it
becomes isoelectric.
• Once this happens the proteins no longer have
means of interacting with the surrounding water
molecules and cannot remain in solution.
• Under such conditions the protein molecules
aggregate with each other and coagulation
occurs.
141
• When a base is added to a protein with +2
overall charge, some of the protonated amino
groups lose their protons and the protein
becomes isoelectric.
• (illustrate)
142
• If a protein has 2 excess negative charges, on the
addition of acid, some of the carboxyl groups
become protonated and the proteins become
isoelectric.
• E.g. When milk is stored in the refrigerator for a
long time, the bacteria in the milk begin to grow.
These use milk sugar lactose as a source of energy
during fermentation and produce lactic acid as a
by product.
143
• As the bacteria population increase, lactic acid
concentration correspondingly increases
decreasing the pH of the milk.
• The additional acid results in the protonation of
the exposed carboxylate groups on the surface of
the dissolved milk proteins. They become
isoelectric and coagulate into a solid curd.
144
• Again if pH of the blood becomes too acidic or basic,
blood proteins like albumin (carriers) fibrinogen
(involved in blood clotting) and
immunoglobulin(protection from disease) will
become isoelectric, denature and can’t carry out
required functions.
• This eventually results in death as enzymes become
denatured and oxygen cannot be transported by
haemoglobin.
145
• Also at extreme pH values strong intramolecular
electrostatic repulsion caused by high net charge
results in swelling and unfolding of the protein.
• The degree of unfolding is greater at extreme
alkaline pH than extreme acid pH values.
• Denaturation under extreme alkaline conditions
is due to ionization of partially buried carboxyl,
phenolic and sulfhydryl groups which causes
unfolding of the polypeptide chain as they
attempt to expose themselves to aqueous
environment.
146
ORGANIC SOLVENT
e.g detergents like sodium dodecyl sulphate
• Organic solvents that are miscible with water
e.g alcohol and acetone denature proteins.
• Urea forms H-bonds with the proteins that are
stronger than those within the protein.
• Urea and detergents disrupt hydrophobic
interactions.
• Mercaptoethanol reduces disulfide bonds to
sulfhydryl groups thus disrupting three
dimensional structure of proteins.
147
Application of denaturation using organic
solvents.
• Ethyl alcohol and isopropyl alcohol are good
germicides because they denature bacteria
proteins thus killing them.
Treatment of Burns
• A complex organic compound, tannic acid is
incorporated in burn ointment. On application
to the skin the tannic acid causes a protective
layer of denatured protein to form that
prevents water loss from the burnt area.
148
CHARACTERISTICS OF DENATURED PROTEINS
1. Unfolding of or uncoiling of peptide or
polypeptide chains
2. Increased viscosity of denatured proteins in
urea solution. The increase is due to the
formation of more elongated and fibrous
structures
3. Chemical characteristics differ from those of
the original proteins .
4. Isoelectric pH may be altered
149
5.Denatured proteins have less capacity to
interact with water than with native protein
6.They are difficult to crystallize
7.Denaturation of enzyme proteins lead to their
inactivation
8.Digestibility of protein by proteolytic enzymes
may be altered. E.g. native haemoglobin is not
digested by trypsin whereas denatured ones are
easily digested.
150
DETERMINATION OF PRIMARY STUCTURE OF
PROTEINS
In determining the primary structure of proteins
the following questions must be addressed.
1.What is the amino acid composition of the
protein?
2.Which amino acids occur at the N- terminal and
C- terminal ends?
3.What is the exact order or sequence of amino
acids in the protein?
151
Determination of amino acid composition
• The protein is digested by refluxing with 6N HCl
for 12 to 36 hours (Usually 24hrs) at 100 to
110oC. This hydrolyses the protein to yield a
mixture of amino acids.
Separation and identification of the amino
acids in the hydrolysate
• Ion exchange chromatography can be used. This
has been automated to produce amino acid
analyser.
152
Principle behind amino acids separation
• The technique is based on separation by
virtue of differences in sign (positive or
negative or neutral) and magnitude (which is
more positive or negative) of charge on the
amino acids.
• The acidic solution of pH 3 is passed through a
long column packed with cation resin.
153
• As the hydrolysate travels (percolates) through
the resin the amino acids are exchanged for
sodium ion.
• At this pH most of the amino acids are cations
since their pI are usually above three but the
magnitude of the charges differ.
• The positively charged amino acids are adsorbed
by the resin because of the attractive forces
between the negatively charged sulphonate
groups and the positively charged amino acids.
154
• Those with larger positive charges (i.e. most basic
eg lysine and histidine) will have more affinity for
the negatively charged resin particles and will be
held more strongly.
• Those with least positive charges at pH 3 e.g. acidic
as aspartate and glutamate will be bound more
loosely.
• All other amino acids will have intermediate
affinity. Amino acids will therefore move down the
column at different rates and be separated if the
column is eluted with buffered solution.
155
• At a given pH the amino acids will ultimately
separate and be collected into small vials
treated with ninhydrin and the absorbance of
the solution measured at wavelength 570 nm.
• The absorbance is recorded as a function of
volume of effluent (solution that comes out)
and represented on a graph. The area under the
curve or peaks corresponds to the relative
amount of the amino acid.
(Illustrate)
156
Identification of C-terminal and the Nterminal ends of proteins
• This can be achieved in several ways by
terminal residue analysis and partial
hydrolysis.
• These involve both chemical and enzymatic
methods for identifying the amino acid at the
ends of the molecule.
157
Identification of the N-terminal end
1. Use of Dansyl chloride
2. Use of phenylisothiocyanate
3. Use of Sanger’s reagent
4. Use of aminopeptidases (enzymatic)
158
•
•
These are enzymes that cleave the N-terminal
amino acid of proteins or peptides. (These
together with carboxypeptidase, are called
exopeptidases as they attack only peptide bonds
at the end of the polypeptide chain.)
The disadvantage is that they continue to cleave
off other amino acids in the sequence. If
conditions are not controlled, unreliable results
are obtained.
159
Identification of the C-terminal
1. Treatment with hydrazine (Akabori procedure)
2. Use of LiBH4
3. Use of carboxypeptidases: these cleave
polypeptides from the C-terminal end and
present similar problems as aminopeptidases
160
Separation of peptide chains
• Determination of N-terminal and C-terminal
residues can also indicate whether a given
protein has a single polypeptide or more.
• If the protein contains more than one
polypeptide chain, the individual chains which
make up the complete protein must be
separated before the sequence is determined.
161
• The disulphide bond of cysteine which joins 2
parts of the same chain or two different
chains can be cleaved by either oxidation or
reduction.
• If the disulphide is reduced, the resulting
sulphydryls are alkylated to prevent
spontaneous re-oxidation to the disulphide
form. (Illustrate)
162
Determination of amino acid sequence of the
isolated polypeptide
• After identifying the N and C-terminal ends, the
next step is to determine the amino acid
sequence of the polypeptide.
• This is achieved by cleaving the parent
polypeptide into a number of smaller fragments
and characterizing each of them.
• Points of overlap are identified and the peptide
pieced together to determine the amino acid
sequence of the original polypeptide.
163
• Determination of points of overlap can be
carried out by partial or selective hydrolysis of
the polypeptide chain.
• Agents for such hydrolysis may be chemical or
enzymatic.
164
Chemical
1. Use of dilute acids. These hydrolyse peptide
bonds but bonds between certain pairs of
amino acids are more susceptible to acid
hydrolysis than others.
2. Cyanogen bromide (CNBr). This cleaves
peptide bonds whose carbonyl functional
group is donated by methionine. After
hydrolysis, the methionine is converted to
homoserine lactone.
165
Use of proteases
Method
Peptide bonds cleaved
Trypsin (from digestive tract of animals)
Hydrolysis of peptide bonds whose carbonyl C
is provided by basic amino acids like arginine
and lysine
Chymotrypsin (from digestive tract of
animals)
This hydrolyses peptide bonds whose carbonyl
group is donated by aromatic amino acids like
phenylalanine, tyrosine, tryptophan or by
amino acids with large apolar (non polar) side
chain like leucine. These may yield fragments
that overlap those produced by trypsin.
Thermolysin (bacterial enzyme)
Hydrolyses peptide bonds whose carbonyl
group is donated by non polar amino acids eg
leucine, isoleucine and valine in addition to
those cleaved by chymotrypsin.
Papain (pawpaw)
Carbonyl group donated by lysine and leucine
Pepsin
Same as chymotrypsin
166
Ordering of peptide fragments
• A polypeptide can be broken into a number of
small fragments that can be easily sequenced.
• However this information is not sufficient to
obtain the full structure of the original
polypeptide or protein since it won’t give the
order of the fragments in the original chain.
167
• This problem is overcome by obtaining a new
set of fragments that overlap the sequences
determined in the first step.
• Combine information of overlapping peptide
to get complete sequence.
(Illustrate)
168
Steps involved in the determination of amino
acid sequence of a protein
1. If the protein contains more than one
polypeptide chain, the individual chains are first
separated and purified.
2. All disulphide groups are reduced and the
resulting sulphydryl groups are alkylated.
3. Subject each polypeptide chain to total
hydrolysis and determine its amino acid
composition.
4. Identify N and C-terminal residues of another
sample of the polypeptide chain.
169
5.Cleave intact polypeptide chain into a series of
smaller peptides by enzymatic or chemical
hydrolysis.
6.Separate peptide fragments in 5 and determine
amino acid composition and sequence each.
7.Partially hydrolyse another sample of the original
polypeptide by a second procedure to fragment
the chain at points other than those cleaved by
the first partial hydrolysis. Separate the
fragments and determine amino acid
composition and sequence as in 5 and 6.
170
8.By comparing the amino acid sequence of the
two sets of peptide fragments particularly
where there is overlapping, the peptide
fragment can be placed in the proper order to
yield the complete amino acid sequence.
9.The position of disulphide bonds and amide
groups in the original polypeptide chains are
determined.
171
Importance of determination of the amino acid
sequence i.e. primary structure
1. It helps in the elucidation of the molecular basis of the
protein’s biological activity (i.e. whether it is an enzyme,
hormone etc)
2. It helps in deducing the 3-dimensional structure of the
protein
3. Alterations in the amino acid can produce abnormal
function and disease, e.g. sickle cell anaemia.
172
4. Amino acid sequence of a protein reveals much
about its evolutionary activity – proteins resemble
one another in their amino acid sequence only if
they have common ancestor. Consequently
molecular events in evolution can be traced from
amino acid sequence.
173
Roles of specific amino acid in proteins
• The uncharged polar amino acid residues in a
protein are the sites for H-bonding leading to
potential cross linking of chains.
• Charged polar groups are susceptible to pH
changes and may markedly affect the activity of
functional proteins.
174
These amino acid include:
• Cysteine
• This forms cross linkages with other cysteine
sulphydryl groups in the same or different
polypeptide chain by oxidation to form a
covalent disulphide bond.
• The reduced cysteine serves as a site of
attachment for substrate in a number of
enzymes.
(Illustrate)
175
• Histidine
This contains a lone pair of electrons in the ring
N and may serve as a potential metal ligand
site in the iron (Fe) containing proteins, eg
haemoglobin and cytochrome C.
• Lysine
The ε amino group of lysine forms a Schiff’s
base with substrate at active sites of enzymes.
It is intimately involved in binding with
pyridoxal phosphate, lipoic acid and biotin.
176
• Serine
It has a primary alcoholic group and may serve
as a nucleophile in a number of proteolytic
enzymes. Together with histidine it serves as
component of active site of chymotrypsin.
• Proline
Due to its relatively rigid ring it forces a bend
in a polypeptide chain and disrupts α-helicity.
177
Reactions of side chain R-groups (colour reactions
of proteins)
Amino acids show qualitative colour reactions typical of
certain functional groups present in the side chain, eg
thiol group, phenol group, indole group, etc.
1. Reaction due to the presence of thiol group (SH)
Proteins containing cysteine or cystine are heated with
strong alkali. H2S is formed and this can be detected by
forming insoluble brown to black PbS on addition of lead
acetate.
178
2. Presence of indole ring
Indole ring can be detected by the Hopkin’s Cole
reaction. In the presence of concentrated H2SO4,
the indole ring of tryptophan forms coloured
(violet) condensation products with glyoxylic acid.
Proteins that do not contain tryptophan do not
give a positive test.
179
3. Folin’s reaction uses Folin’s reagent (sodium
1,2-naphthoquinone-4-sulphonate)
The reagent gives a deep red colour with
amino acids in the presence of alkaline.
180
4. Detection of aromatic group – phenylalanine,
tyrosine, tryptophan
The xanthoproteic test is used in this. The phenyl
group is nitrated by concentrated HNO3 to form
white, then yellow nitro substitution product or
precipitate on heating. Salts of the derivatives are
orange.
181
5. Presence of arginine or guanidine group
Arginine, the only amino acid containing a
guanidine group reacts with α-naphthol and
an oxidizing agent such as bromine water or
sodium hypochlorite to give a red colour. The
reaction is called Sakaguchi reaction.
182
6. Test for phenolic group (specific for tyrosine)
Millon’s reagent is used.
• This reacts with phenolic groups to give a faint
pink colour which changes to red on heating.
• Millon’s reagent is a solution of mercurous and
mercuric nitrate containing HNO3.
183
• When added to a protein solution, a white
precipitate is first formed which turns to red
on heating.
• Reaction is dependent on the formation of a
coloured Hg compound with the OH phenol
group.
184
7. Biuret’s test
This is a popular quantitative test for proteins just like
ninhydrin.
• This involves peptides with 2 or more peptide bonds.
• Protein solutions are made strongly alkaline with Na or K
hydroxides and very dilute CuSO4 is added.
• A pinkish to purple colour develops.
185
The colour depends on the complexity of the protein.
• Proteins give purple colour; peptones give a pink colour
and peptides very light pink colour.
• Gelatin gives an almost blue colour.
• In the Biuret’s test, there is coordination of cupric ions
with the unshared electron pairs of peptide N and the
O of H2O to form a coloured coordination complex.
• MgSO4 interferes with the reaction as precipitate of
Mg(OH)2 is formed.
186
Evidence supporting peptide bonds in proteins
These are obtained from both chemical and
enzymatic degradation as well as physical
measurement.
1. Intact proteins show little free amino N
(hydrolysis produces a large amount of N2).
2. Proteins have absorption band in the far UV
and IR regions that are similar to genuine
peptides.
3. X-ray diffraction analysis confirms the presence
of peptide bonds.
187
4. Enzymes that can hydrolyse proteins also
hydrolyse synthetic peptides.
5. Most proteins and peptides give the same
colour reaction with Biuret’s test.
6. Several polypeptide hormones have been
isolated in pure forms and have been
synthesized from the constituent amino
acids.
188
Determination of protein size
Lots of methods have been employed to
determine size of proteins. They include;
1. Determination of mini molecular weight which
can be computed from the quantity of
constituents.
2. Use of osmotic pressure – there are lots of
difficulties in using osmotic pressure in
calculation.
189
Difficulties in using osmotic pressure
These include:
i. Equilibrium is attained slowly and hence
reading must be taken within a long range of
time. Due to this there may be bacteria
contamination and decomposition depending
on environmental conditions.
190
ii. The gas law equation is valid only at low
solvent concentration and osmotic pressure
determination makes use of the gas law
equation. Therefore unless solution is diluted
to infinite dilution values may be wrong.
191
3. Sedimentation
• This is the most important method for
determining the shape, size and molecular weight
of proteins and in this determination, ultra
centrifugation is made use of.
• The principle underlying this is the rate at which a
particle is driven down a centrifuge tube under
the action of centrifugal force which depends on;
192
i. initial force applied when centrifuging
ii. size, shape and density of the particle being
measured
iii. density and viscosity of solvent system in
which the molecular weight is being
determined.
193
Isolation and purification of a protein (same as
isolation and purification of amino acid)
• Amino acids and proteins can be separated
from each other and from other kinds of
molecules on the basis of such characteristics
as size, solubility, charge and specific binding
affinity.
• In purifying proteins, various separation
methods are employed and their efficiency is
evaluated by assaying for distinctive
properties of the protein of interest.
194
Separation Based on Size
1. Dialysis and ultracentrifugation
• Proteins can be separated from smaller
molecules by dialysis through a semi permeable
membrane.
• The membrane retains protein molecules and
allows small solute molecules and water to pass
through.
• In ultra centrifugation, pressure or centrifugal
force is used to filter the aqueous medium and
small solute molecules through a semi
permeable membrane.
195
2.Gel filtration chromatography (molecular
exclusion/sieve chromatography)
• This is also a separation procedure based on
size.
• It is a form of column chromatography in which
the stationary phase consists of cross linked gel
particles which are hydrated.
196
• The gel particles are usually in bead form and
there are two types of polymers. One includes
carbohydrate polymer such as dextran and
agarose and the other type is polyacrylamide.
• The cross linking produces pores in the
material.
197
• The sample is applied to the top of the column.
• Smaller molecules enter the pores and appear in
solution within the beads and in between them,
but larger ones cannot.
• As a result, smaller molecules are delayed in their
progress down the column.
198
•
As the sample is eluted by the mobile phase,
the larger molecules are eluted first followed
by the smaller ones.
Advantages are;
1. It is a convenient way to separate molecules
based on their size.
2. It can be used to estimate molecular weight
by comparison with standard samples.
199
Separation based on charge
• Ion exchange chromatography is used.
• There are anion exchangers and cation
exchangers.
• If the pH is below the pI, cation exchangers
are used.
• For pH above the pI, anion exchangers are
used.
200
Electrophoresis
• The underlying principle in this procedure is
electrostatic attraction.
• In addition, size and shape of the molecule can also
influence separation.
• Electrophoresis depends on different rates of
migration of particles of different charges in an
electric field.
201
• The charged molecule moves through a liquid
that conducts an electric current.
• Inert substances like paper and gel (SDS
polyacrylamide gel electrophoresis) are used as
support for the conducting liquid.
• The sample to be separated is applied to a strip
of paper moistened with the conducting
solution, usually a buffer.
• The ends of the paper strip are placed in
reservoirs of buffer solution.
202
• A positive electrode is placed at one end of the
reservoir and a negative electrode at the other
end.
• A high voltage is then applied.
• Proteins with a net positive charge will migrate
towards the negative electrode; those with high
positive charge will move faster than those with
lower positive charges.
203
• Proteins with net negative charge will move to the
positive electrode; those with higher negative
charge will move faster than those with lower
charge.
• A protein with no net charge will not migrate in an
electric field.
• The net charge of each protein depends on pH.
• The net charge on a protein or amino acid
influences the rate of migration in an electric field.
204
•
The principle is that, the velocity of
migration (v) of the protein in an electric
field depends on;
1. the strength of the electric field (E),
2. the net electric charge on the protein (z) and
3. the functional resistance (f) which is a
function of size and shape of the protein.
205
• Hence;
V =
Ez
f
• At the isoelectric pH, there is no net charge on
the protein. Therefore electrophoretic mobility
(v) is zero.
• Molecules of the same charge but different
molecular mass move at different rates in an
electric field. Bulky ones will move at a slower
rate than non bulky ones.
206
Separation based on polarity
Example paper chromatography
• This technique is based on the principle that
polar organic molecules will dissolve more easily
in water than in a non polar organic solvent.
• The mobile phase which carries the sample to
be separated along with it is less polar than
water and flows over the stationary phase which
is polar.
207
• Mobile phase is frequently a mixture of
solvents like N-butyl alcohol and water or Nbutyl alcohol, butyric acid and water.
• In paper chromatography, the stationary
phase water, is adsorbed on the cellulose fibre
of the paper which serves as the inert
support.
208
• The various components in the sample
interact with the stationary phase to
different extents based on their polarity.
• The more polar components of the sample
are carried along more slowly by the mobile
phase than the less polar ones which
interact less strongly with the stationary
phase.
209
• The various components of the sample can be
characterized by the
-distance travelled from the origin (site of
application of the sample) compared with
-distance travelled by the solvent front.
210
• The ratio of these 2 distances is called the Rf and
its numerical value can be used in identifying
proteins or amino acids by comparison with
standards.
• Rf =
Distance travelled by substance
Distance travelled by solvent front
• (Illustrate)
211
Affinity chromatography
• This makes use of the binding properties of many
proteins.
• The column is made up of a polymer which is covalently
linked to a substrate which binds specifically to the
desired protein.
• The other proteins in the sample don’t bind to the
column and can easily be eluted with buffer while the
bound protein remains in the column.
212
• The bound protein is then eluted out by
adding high concentration of the substrate in
soluble form.
• The proteins therefore bind to the substrate in
the mobile phase and is eluted or recovered
from the column.
• Method has advantage of producing pure
proteins.
213
Separation based on solubility differences
• The solubility of proteins can be affected by
the pH of the system.
• A protein is least soluble at its pI and since
different proteins have different pIs, they can
often be separated from each other by
isoelectric precipitation.
214
Solubility and salting-out of proteins
• Every protein has its own characteristic solubility
curve at fixed
-pH,
-temperature and
-concentration of solutes.
• If conditions are controlled, the amount of protein
that dissolves to form a saturated solution is not
really dependent on the other solute particles
present.
215
• Its solubility depends on the polar hydrophilic
groups and the non polar hydrophobic groups.
• Solubility is highly influenced by pH due to the
amphoteric nature of proteins.
• Solubility increases with rise or fall in pH, e.g.
isoelectric precipitation (i.e. a protein is only
insoluble at its pI).
216
The effect of pH and salt concentration on
the solubility of β-lactoglobulin at 25oC
• β-lactoglobulin is a milk protein with pI about 5.3.
• Above or below this pH all the molecules have
either negative or positive charges and repel one
another so the protein is very soluble at either
acidic or alkaline pH.
217
• At the pI, there is no net charge though
molecules still bear positive and negative
charges.
• However ionic interactions, Van der Waals
forces etc make the molecules clump together
and precipitate.
• Therefore solubility is minimal at pI.
(Illustrate)
218
• When ionic strength is increased solubility
increases even at pI.
• This effect of putting proteins in solution by
increasing salt concentration is called ‘salting-in’.
• At very high salt concentration, much of the
water that will solvate proteins is used for the
hydration shells of the numerous salt ions.
219
• At such high salt concentration then, solubility
of proteins again decreases and the effect is
called ‘salting-out’.
• Divalent and trivalent ions are much effective
than univalent ions for salting out.
• Commonly used salts are ammonium sulphate,
magnesium salts or phosphates and sodium
sulphates.
220
• Principle behind salting out is that high
concentration of salts may remove water of
hydration from the protein molecules thus reducing
their solubilties.
• Thus anything that reduces the activity of water
reduces the solubility of the proteins.
221
• In summary, low concentration of neutral salts
increases the solubility of proteins in water
(salting in) by stabilizing the surface charged
groups.
• At high concentration, salt ions compete with
proteins for water molecules and protein
precipitation occurs (salting out).
222
Effect of organic solvents
• Addition of solvents like acetone or alcohol to
a solution of protein displaces some of the
water molecules associated with the protein
thus decreasing the water concentration
present in solution resulting in precipitation of
proteins.
• This must be done at low temperature to
avoid denaturation of proteins.
223
Effect of temperature on solubility of
proteins
• Most globular proteins increase in solubility
with increasing temperature within a limited
range of about 0-40oC.
• Above this range, most proteins become
unstable and begin to denature.
224
Precipitation by heavy metals
• At pH 7, blood proteins are usually negatively
charged. The presence of positively charged metal
ions neutralizes this charge and the protein comes
out of solution (precipitates).
• Precipitation by heavy metals is therefore most
effective at neutral to slightly alkaline pH values.
225
• The solution must not be too alkaline as metal
hydroxides will be precipitated.
• Hg2+, Pb2+ may disrupt salt/ion bridges by
forming ionic bonds with negatively charged
groups.
• Heavy metals also bind to sulphydryl groups
and denature proteins.
226
Criterion for purity
• A pure protein should have specific well-defined
characteristics peculiar to the protein alone.
• There are various ways for determining if an
isolated protein is pure.
1. Constant solubility
• The solubility of a protein in a given solvent in
relation to excess of protein added to the solvent is
determined.
227
• With pure substances as more and more of
the substance (protein) is added to a given
amount of solvent, all of the substance
dissolves until a sharp saturated limit is
reached at which no more dissolution occurs
as excess substance is added.
228
• The solubility curve is an ascending straight line
until saturation is reached where there is a sharp
break and the remainder of the curve is a
horizontal line parallel to the solute axis.
• If the solute is a mixture of substances with
different solubilities, there is more than one
break in the curve.
(Illustrate).
229
2. Homogeneity in size (determined by
centrifugation).
• If protein particles sediment at the same rate
then the protein may be but not necessarily
homogenous because both the desired protein
and its contaminant may sediment at the same
rate.
• 3. Homogeneity in charge
Done by chromatography.
230
ENZYMES
• The numerous variety of biochemical
reactions that occur in living organisms are
nearly all mediated by a series of biological
catalysts known as enzymes.
• These accelerate biological reactions without
they themselves taking part in the process.
231
• Enzymes are made up of whole proteins
or conjugated proteins.
• They can function both in vivo and in
vitro under appropriate conditions.
• Although enzyme catalysis obeys the
laws of thermodynamics, enzymes differ
from ordinary chemical catalysts in
several important respects;
232
1. High reaction rates
•
The rate of enzymatic catalysed reactions are typical
factors of 106 to 1012 greater than those of the
corresponding uncatalysed reactions.
•
They are at least several orders of magnitude
greater than those of the corresponding chemically
catalysed reactions.
233
2. Milder reaction conditions
• Enzyme catalysed reactions occur under
relatively mild conditions. Temperatures
below 100oC, atmospheric pressure and nearly
neutral pH values.
• In contrast efficient chemical catalysis often
requires high temperature and pressures as
well as extremes of pH.
234
3. Greater reaction specificity
• Enzymes have a greater degree of
specificity with respect to both the
identities of their substrates and products
than do chemical catalysts i.e.
• Enzyme catalysed reactions rarely have side
products.
235
4. Capacity for regulation
• The catalytic activities of many enzymes vary
in response to concentration of substances
other than their substrates.
• The mechanisms of these regulatory
processes include allosteric control, covalent
modification of enzymes and variation in the
amount of enzymes synthesized.
236
• Many enzymes are simple proteins but others
are conjugated, having a non protein group
more or less closely associated with the
protein apoenzyme.
• The whole complex is termed the holo
enzyme.
• The non protein portion of the enzyme may
be firmly or loosely bound.
237
• It can be regarded as an integral part of the
structure and is called a prosthetic group.
• The loosely bound ones can be regarded as
separate entities and are referred to as
coenzymes.
• Many coenzymes usually have relatively low
molecular weight and can be dialysed off from
the apoenzyme whereas the true prosthetic
groups remain attached.
238
Some features of enzymes
1. They work best at extremely low
concentration and normally completely out
of proportion with the change they catalyse.
2. They are highly specific both in the reaction
catalysed and in their choice of substrate due
to their protein nature.
3. They speed up the rate of reaction by
lowering the activation energy.
239
Naming and classification of enzymes
• Throughout the years enzymes have been
named by appending the suffix ‘ase’ to the
name of the enzyme substrate or to a phrase
describing the enzyme’s catalytic action. Eg
urease
• Currently, enzymes are classified and named
according to the nature of the chemical
reactions they catalyse.
• Under this classification, there are six major
groups of enzymes.
240
1. Oxidoreductases
• These catalyse oxidation/reduction
reactions and may add or remove
electrons, oxygen or hydrogen.
• There are three groups of enzymes under
this and the nature of electron acceptor
determines which type.
i. Oxidases – these use oxygen as Hydrogen
acceptor. Eg tyrosinase.
241
ii. Dehydrogenases - these use some other
substrates as H acceptor.
They usually involve the removal of 2 electrons
from the substrate, eg lactate dehydrogenase,
malate dehydrogenase, alcohol
dehydrogenase. (Carriers are NAD, FAD).
242
ii. hydrperoxidase – these use hydrogen peroxide
as substrate, eg catalase and peroxidase.
• Oxidoreductases usually catalyse reactions like;
• Dehydrogenase
• Eg. Oxidation of ethanol to acetaldehyde by
alcohol dehydrogenase.
• Oxidases
• Peroxidase
243
2. Transferases
• These catalyse the transfer of a group from one
organic molecule to the other and they are
important for biological synthesis, eg methyl
group transferase, acyl group transferases.
• Phosphotransferases (kinases) responsible for
transfer of energy from one system to another
in the form of high energy phosphate bonds,
• Amino transferase (transaminases) responsible
for transferring amino group.
244
3. Hydrolases
• These catalyse the cleavage of bonds by the
addition of water, e.g. those acting on ester
bonds or polysaccharide linkages (specific, that
is either α or β), peptide linkages. Examples are
esterases, amylases, peptidases, phosphatases,
lipases, etc.
• This usually involves the hydrolytic cleavage of
C-O, C-N, O-P and C-S bonds.
245
4. Lyases
• These remove groups non-hydrolytically
leaving a double bond or in reverse add
groups to double bonds. Eg decarboxylases,
aldolase, dehydratase, citrate synthase, etc.
• They usually add or remove water, ammonia
or carbon dioxide.
• They catalyse cleavage of C-C, C-S and certain
C-N bonds.
246
5. Isomerases
• These bring about a redistribution of atoms
within a molecule and catalyze the conversion
of one isomer into another. E.g. epimerases,
mutases and racemases.
• Epimerase changes glucose to galactose.
• The two can later be joined to form the milk
sugar lactose.
247
6. Ligases
• These link together 2 molecules always at the
expense of high energy compound usually ATP
or they catalyse the formation of bonds
between C and O, C and S or C and N coupled
to the hydrolysis of high energy phosphate
compound.
• Examples are enzymes which unite amino
acids to their specific tRNA’s, the enzymes
leading to the formation of acyl CoA by uniting
the free acid and CoA.
248
• As more enzymes were isolated, and an
increasing insight was obtained into the
mechanism of enzyme actions, it became clear
that the existing system of naming enzymes
was inadequate as some of the names could
be misleading.
• For this reason, the Enzyme Commission of
the International Union of Biochemistry in
1964 devised a scheme of classification which
was more specific than the existing one.
249
• The Enzyme Commission allocated a unique 4
part number and a systematic name to each
enzyme.
• All enzymes belong to one or another of the six
main groups.
• So the four part number starts with one of the
numbers from 1 to 6.
• Each of these 6 groups is then sub-divided
according to the nature of the linkage being
attacked or the group being transferred.
250
• These sub groups are further divided and
individual enzymes then designated by a 4th
number. Example;
• EC 3.4.21.5 - The first 3 numbers (3.4.21) define
major class, subclass and sub-subclass respectively.
• The last is a serial number in the sub-subclass
indicating the order in which each enzyme is added
to the list which is continually growing.
251
• Another example is acetylcholinesterase, an
enzyme which splits acetylcholine into the
base choline and acetic acid.
• It is therefore a hydrolase (group 3), which
are themselves of different kinds.
• Some hydrolyse ester linkages, some
glycosidic linkages and others peptide
linkages.
252
• Acetylcholine esterase is in the sub group 3.1
which contains enzymes that act on ester
linkages only.
• There are several types of ester linkages;
carboxylic acid esters, phosphate esters, etc.
• The enzyme acetylcholinesterase splits the
carboxylic ester linkage of acetylcholine and so
it is assigned to the sub subgroup 1 ie. 3.1.1.
253
• The designation for acetylcholine esterase is
3.1.1.7 and its systematic name is acetylcholine
acetylhydrolase.
• Another example is triosephosphate isomerase
(EC 5.3.1.1).
• It is an isomerase and in the 3rd subclass.
• It is in the first sub-subclass and is the first entry
in this sub-subclass
254
NATURE OF ENZYME ACTIVITY
• When an enzyme is introduced into a system, it first
combines with the substrate to form an enzyme
substrate complex which then breaks down to yield
the product and free enzyme.
• The enzyme then combines with more substrate to
continue the reaction.
k1
• S + E
SE
k3
P + E
k2
255
• The rate at which product is formed depends
on k3 and the rate at which substrate is
reproduced depends on k2.
• The substrate is bound to a specific region
called the binding or active site.
• Most enzymes are highly selective in their
binding of substrate.
256
• As early as 1894 the German biochemist Emil
Fischer proposed a lock and key hypothesis for
enzyme action.
• The enzyme accommodates the specific
substrate as a lock does its specific key.
• Thus the specificity of an enzyme for its
substrate arises from their geometrically
complementary shapes.
(Illustrate)
257
• However studies reveal that active sites are not
necessarily rigid and that the shape of the
active site is modified by the binding of the
substrate i.e.,
• The active site has a shape complementary to
that of the substrate only after the substrate is
bound.
258
• This is called the INDUCED FIT HYPOTHESISproposed by D. Koshland in 1958.
• It is the dominant model for enzyme catalysis
as it better explains catalysis itself.
• Thus the active site is induced to take up a
configuration approximating the transition
state.
(Illustrate)
259
BINDING AND ACTIVE SITES
Enzymes usually have
(1) binding sites and
(2) catalytic sites
• These 2 sites are critical for the catalytic activity
of the enzyme.
• The site which holds the enzyme and substrate
together is the binding site.
260
• The catalytic site contains the groupings that
are directly involved in catalysis and conversion
of substrate to product.
• The binding and catalytic sites are usually near
one another and frequently overlapping.
• Together, they are referred to as active site.
(Binding site + catalytic site = active site)
261
Enzymes are much larger than their catalytic sites for the
following reasons:
1. The folded structure is required to define and stabilize
the catalytic site.
2. The enzyme must contain specific binding sites for the
substrate, ie the binding between enzyme and substrate
should be such that atoms participating in the bond to
be made or broken are oriented with respect to catalytic
groups.
262
3. Many enzymes contain additional surface features
essential for function.
-Only a few of the 20 amino acid side chains
participate directly in catalysis.
-In general, these include polar side chain of serine,
tyrosine, glutamate, histidine and cysteine.
263
Some features of the active site
1. The active site takes up a relatively small part of
the total volume of the enzyme.
2. The active site is a three dimensional entity.
-It is not a point, a line or a plane.
-Groups come from different parts of the linear
amino acid sequence to form the active site.
264
3. Substrates are bound to enzyme by relatively
weak forces.
4. Active sites are clefts or crevices.
5. The specificity of binding depends on the
precisely defined arrangement of atoms in an
active site.
265
Enzyme specificity
• Each biological reaction has a specific enzyme to
catalyze it based on the lock and key hypothesis
or the induced fit hypothesis.
• Specificity is due to the chemical nature of the
groups involved in the reaction or active site, i.e.
the 3-dimensional characteristics of these groups
like size, shape and location.
• It is possible to subdivide enzymes into groups
according to the degree of specificity.
266
1. Absolute specificity
• Enzymes in this group are specific for a given
substrate and will not even attack closely
related molecules.
• E.g. glucokinase catalyzes the phosphorylation
of glucose only and not the other structurally
related sugars like fructose, mannose,
galactose, etc.
267
2. Relative group specificity
• These enzymes catalyze reactions involving a series
of structurally related substrates which have in
common one identical group but differ in some other
way.
• They act on one class of bonds at widely different
rates;
Eg α-glucosidase hydrolyzes several α-glucosides.
268
• The enzyme maltase is an example of αglucosidase which splits maltose into 2 glucose
units.
• In addition, maltase will attack a range of αglucosides at different rates indicating that the
affinity of the enzyme for each substrate is
different.
269
3. Stereospecificity
• Enzymes in this group are specific for the D or L
forms of the same substrate.
• The stereo specificity of enzymes arises because
by nature, enzymes have an inherent chirality:
• E.g. lactic acid dehydrogenase of animal muscle
only oxidizes L-lactic acid.
270
• Similarly it is usual for only one pair of the
geometric isomers to be acted on or formed by
an enzyme action.
• E.g. Succinate dehydrogenase converts
fumarate to succinate but has no action on the
corresponding cis-isomer maleic acid.
271
How enzymes accelerate reactions
• The function of enzymes is to hasten attainment
of the equilibrium state.
• Without them, many cellular reactions would
occur too slowly to support life.
• For a reaction to occur, the reacting molecules
should possess a certain minimal energy Ea i.e.
activation energy.
272
• When molecules of potential reactants possessing
less than this minimal energy are brought together,
they fail to react.
• Enzymes probably affect the rate of biochemical
reactions both by enabling more molecules to
overcome the energy barrier of the reaction and by
increasing the probability of correct orientation at
the moment of collision.
273
• Enzymes enable reactions to by-pass high
activation energy barrier by re-routing
reactions.
• These have their own activation energies even
though both the substances disappearing
(substrates/reactants) and those being formed
(products) are the same as when no enzyme is
present.
274
• This is usually achieved by reactant molecules
forming an intermediate complex with the
enzyme so that they are suitably placed and in
an appropriate state of electron activation for
the reaction to occur.
• Thus enzymes accelerate reactions by
decreasing the activation energy. ΔG = Ea
(Illustrate)
275
Profile of Enzyme Catalyzed Reaction
Free energy
Progress of Reaction
276
Factors affecting rate of enzyme action
•
•
•
•
•
•
•
These are :
Substrate concentration
Enzyme concentration
Temperature
pH
Time
Inhibitors
Activators
277
Substrate concentration
• At a fixed enzyme concentration, the rate of
catalysis (v) varies with the substrate concentration
in a manner indicated in the graph (Illustrate –
Figure 1).
• At low substrate concentration the initial velocity
(v) of an enzyme catalyzed reaction is proportional
to the substrate concentration.
278
Fig.1 Effect of Substrate Concentration on
Enzyme Catalyzed Reaction
Reaction
Velocity (v)
[S]
279
• As the substrate concentration increases the initial
velocity increases less so there is no more
proportionality.
• With a further increase in substrate concentration
the reaction rate becomes independent of the
substrate concentration and assumes a constant
rate as a result of enzyme being saturated with the
substrate.
280
• Michaelis and Menten (1913) suggested this
explanation for the behaviour of enzymes.
• At low substrate concentration the enzyme is not
saturated with the substrate, therefore the reaction
is not proceeding at maximum velocity.
• Maximum velocity is observed when the enzyme
becomes fully saturated with the substrate.
• The saturation effect is believed to reflect the fact
that all the enzyme’s binding sites are occupied with
substrate.
281
• They also assumed that the rate of substrate
decomposition is proportional to the E-S
complex.
• Reaction velocity at high substrate
concentration is termed maximum velocity or
Vmax and the substrate concentration at which
the velocity is half Vmax is called the Michaelis
constant (Km).
• Km indicates the affinity of the enzyme towards
the substrate - a high affinity means a small Km
value and vice versa.
282
The Michaelis –Menten(MM) theory
• The critical feature in the MM theory is that the
enzyme first forms a complex with the substrate
to form an ES complex which is the necessary
intermediate in catalysis.
• This ES complex has 2 possible fates: It can
dissociate in the reverse direction to yield free
enzyme and substrate or it can proceed to yield a
product (P) and free enzyme.
• It is assumed that none of the products reverts to
the initial substrate. This proposal is represented
by the equation 1.
283
E + S
k1
k2
ES
k3
P + E
(1)
• All the k’s are velocity constants for the various
reactions.
• Considering the first part of the equation, the
rate of formation of ES will be dependent on
the enzyme concentration and the substrate
concentration, thus:
• Vf = k1([E] - [ES]) [S]
-
(2)
284
• Where:
[E] - [ES]
[S]
[E]
[ES]
is concentration of free enzyme
is substrate concentration
is enzyme concentration
concentration of ES complex
• The rate of reaction is proportional to
concentration of free enzyme and substrate
concentration.
285
• Considering the backward reaction, or the
breakdown of ES.
• The rate of breakdown of ES is dependent on k2
and k3, thus:
• Vb = k2 [ES] + k3 [ES]
= (k2 + k3) [ES]
(3)
At equilibrium, the rate of formation of ES
is equal to the rate of breakdown of ES: ie
286
• Vf = Vb or Eqn (2) = Eqn (3)
• k1([E] - [ES]) [S] = (k2 + k3) [ES]
• Rearranging;
[E][S] – [ES][S] = (k2 + k3) [ES]
k1
287
• [E][S] – [ES][S] = (k2 + k3)
k1
[ES]
Now, (k2 + k3) can be defined by a new
k1
Constant, Km called the Michaelis const.
The equation becomes;
[E][S] – [S] = (k2 + k3) = Km
[ES]
k1
288
• [E][S] = Km + [S]
[ES]
• [ES] = [E][S]
Km + [S]
(4)
289
• The Km is a very useful parameter normally
used to characterize enzymes.
• When an enzyme can catalyze 2 or more
substrates, the Km value for each substrate
gives an insight into the affinity of the enzyme
for that particular substrate
290
• It has dimensions of mol/l and is usually in the
range of 10-1 to 10-6 (i.e. there can be ranges
outside this).
• Measurement of the free enzyme
concentration and the concentration of the ES
complex is in most cases difficult.
• Therefore a new method has been developed
to find the relationship between ES complex
and the Km.
291
• This involves quantities that can readily be
determined by experiment. Considering the
second part of equation 1, ie product
formation,
• ES
k3
P+E
292
• The velocity or rate of the reaction will be
represented by
V = k3 [ES]
The rate of formation of the enzyme and product
is dependent on [ES].
In the presence of a large excess of substrate,
almost all the enzyme becomes bound to the
substrate to form ES and at this level, the
enzyme becomes saturated with the substrate.
293
• At this stage, there is hardly any free enzyme
and therefore, [ES] = [E].
• The enzyme is now working at maximum
velocity (Vmax) at this level, so V = k3[E].
• So V at this stage of the reaction = Vmax.
• Vmax = k3[ES] = k3[E]
294
• But from Eqn (4); [ES] = [E][S]
Km + [S]
V = k3[E][S]
Km + [S]
(5)
At this velocity, the rate of breakdown
of ES complex is maximum and it can
be deduced that Vmax = k3[E].
295
• Substituting the value for Vmax into Eqn 5;
• V = Vmax[S]
Km + [S]
• Km = Vmax – 1 [S]
V
• V = [S]
Vmax Km + [S]
(6)
(7)
(8)
296
• At half Vmax, Km is numerically equal to [S] and
this explains why Km has the same dimension as
[S].
• Equations 5 and 6 are sometimes referred to as
Michaelis-Menten equation.
• From Eqn (6), at high velocity, V = Vmax/2
297
• Vmax/2 = Vmax[S]
Km + [S]
1/2 = [S]
Km + [S]
Km + [S] = 2[S]
Km = [S]
298
• This explains why Km has the same dimension
as S.
• It can therefore be concluded that at half
Vmax,
Km = [S].
299
• Thus Km is numerically equal to [S] when the
reaction is proceeding at half its maximum
rate.
• If the enzyme is has a small Km value, it
achieves maximum catalytic efficiency at low
concentration.
300
Graphical representation of Km
The double reciprocal plot or Lineweaver–Burk
plot
Practically, Km and Vmax are difficult to measure
directly.
• Therefore it is easier to set up a series of
experiments at the same [E] but different [S] and
measure the initial velocity.
301
• Equation 6 is then rearranged to obtain a
linear graph from which Km and Vmax can be
obtained.
• This is called the double reciprocal plot or the
Lineweaver-Burk plot.
302
• From Eqn. (6)
V = Vmax [S]
Km + [S]
1/v = Km + [S]
Vmax [S]
1/v = Km + [S]
Vmax [S] Vmax [S]
= Km
+ 1
Vmax [S] Vmax
y = mx + c
303
• Lineweaver-Burk plot
304
• This is equivalent to the equation of a straight
line, where y = mx + c.
• When x = 0, y = c and for Lineweaver Burk plot,
when 1/[S] = 0, the intercept on the 1/V axis is
numerically equal to 1/Vmax.
• It is thus apparent from the graph that Km and
Vmax can be readily estimated in simple cases
by measuring the velocity of the reaction at
several levels of [S].
305
The advantages of the Line weaver Burk plot
1. It allows for the easy evaluation of the critical
constants.
2. It allows discrimination between different
kinds of enzyme inhibition and regulation,
thus giving valuable information on enzyme
inhibition.
3. It is useful in the analysis of kinetic data from
enzymes requiring more than one substrate
306
The disadvantages of the Line
weaver-Burk equation
1.
Most experimental measurements involve
relatively high [S] and values are crowded
onto the left side of the graph.
2. A long extrapolation is often required to
determine Km with corresponding
uncertainty in the result.
3. For small values in [S] small errors in Vo
(initial velocity) leads to large errors in 1/v
and hence to large errors in Km and Vmax.
307
• Several other types of plot each with its
advantages and disadvantages have been
formulated for the determination of Km and
Vmax, Eg.
The Eadie-Hofstee plot
• It is possible to arrange the Lineweaver Burk plot
of equation 9 to obtain;
• V =
VmaxKmV
[S]
This represents a plot of V versus V/[S]
308
Eadie-Hofstee plot
309
• Advantages of Eadie-Hofstee plot are:
1.This yields Vmax and Km in a simple way and
magnifies departure from linearity which may
not be apparent in the double reciprocal plot.
2.The entire range of possible [S] from 0 to
infinity can be fitted on a single plot.
3.The points are nearly evenly distributed.
310
Significance of the Km
1. Km is the [S] at which the reaction velocity is
half maximal (i.e. ½ Vmax). Therefore if an
enzyme has a small Km value, it achieves
maximal catalytic efficiency at low [S].
2. The magnitude of Km varies widely with the
identity of the enzyme and the nature of the
substrate.
3. Km is also a function of other variables like
temperature, pH and ionic strength.
311
Effect of enzyme concentration
The rate of enzyme catalyzed reactions is
proportional to the enzyme concentration.
Rate of reaction
[E]
312
Effect of time.
• The effect of pH, temperature, etc depends on
time. If there is a temperature change
between 10oC, the rate of reaction is doubled.
313
Effect of pH
• As enzymes are proteins their catalytic efficiency
will be greatly affected by the pH of the
surroundings.
• pH changes may affect the amino and carboxylic
groups and hence the ionic nature and
conformation of the active site.
• In addition changes in pH can denature the
enzyme protein thus affecting the activity of the
enzyme.
314
Alteration in pH therefore;
• Affects the affinity of an enzyme for its
substrate (i.e. either reduce or enhance its
affinity).
• Affects stability of the enzyme.
• Most enzymes have optimum activity near
neutrality or acid medium.
315
• There are a few which have optimum activity
beyond pH 7.
• The pH effect varies with temperature, [S] and
time.
• Some enzymes have broad pH activity while
others have a narrow pH activity.
316
Profile of pH effect on enzyme activity
317
Effect of temperature
• The rate of enzyme catalyzed reactions increases
as the temperature is raised within the
temperature range in which the enzyme is stable
and retains maximum activity.
• Enzyme catalyzed reactions have an optimum
temperature at which the reaction is most rapid
and beyond this temperature, there is inactivation
since enzymes are denatured by heat.
318
• It shows a similar profile to that of pH.
• The optimal temperature is around the
temperature of the organism in which
they occur but usually there is
enhancement between 25oC and 60oC.
319
Effect of enzyme inhibitors
• As enzymes are proteins, they may be
inactivated by numerous chemical agents
which denature, coagulate and precipitate
proteins.
• Such substances alter the activity of an
enzyme by combining with it in a way that
influences the binding of the substrate.
• These substances are known as inhibitors and
they lower the rate of enzyme reaction.
320
• Many inhibitors bear a structural resemblance to
the enzyme substrate but either do not react or
react very slowly compared to the actual
substrate.
• The binding of inhibitors may be reversible or
irreversible.
• In the former the inhibitor competes with the
substrate for the enzyme’s active site as it bears a
similar conformation as normal substrate or bears
a strong structural resemblance.
321
• In irreversible inhibition, there is no
structural relationship.
• There are 2 main types of enzyme
inhibition, namely;
–competitive
–non competitive
322
Competitive inhibition
• A competitive inhibitor is one that directly
competes with the normal substrate for an
enzyme’s active site or binding site.
• The active sites are therefore not available for the
normal substrate.
• Such an inhibitor usually resembles the substrate
structurally thus binding to the enzyme’s active
site.
323
E +I
EI
• The inhibitor does not damage the enzyme but
only forms a loose complex with it.
• Since the EI complex formation is a reversible
reaction it follows that increasing the substrate
concentration overcomes the inhibition.
324
Competitive inhibition cont’d
• The overall rate of inhibition is governed by the
affinities of the inhibitor molecules and substrate
molecules for the enzyme active site and by the
concentration of the reactants.
• The presence of the inhibitor increases the apparent
Km of the enzyme for the substrate.
• However, Vmax is unaltered indicating that the
inhibitor does not interfere with the breakdown of the
ES complex.
325
• Example:
succinate dehydrogenase, a TCA cycle enzyme
that functions to convert succinate to fumarate is
competitively inhibited by malonate which
structurally resembles succinate but can not be
dehydrogenated.
326
Competitive inhibition cont’d
• In competitive inhibition affinity decreases,
efficiency remains the same, 1/Km decreases
as Km increases and 1/Vmax remains
unaltered.
• Competitive inhibition can be recognized by
the effect of the inhibitor on the relation
between the rate of reaction and substrate
concentration.
327
LWB plot for competitive inhibition
328
Non competitive inhibition
• In this kind of inhibition there is no
competition between the substrate and the
inhibitor, i.e. the inhibitor can either bind the
free enzyme or the ES complex.
• There is little or no structural resemblance
between the substrate and the inhibitor
molecules.
329
• The inhibitor therefore binds to a site other
than the active site.
• As a result, the ES complex is formed more
slowly or once formed it breaks up to yield the
product less rapidly.
330
Non competitive inhibition cont’d
• This effect can not be reversed by increasing
[S] and there is no relationship between the
[S] and the degree of inhibition hence Vmax is
lowered but Km is unaltered.
• In non competitive inhibition, the affinity is
the same, efficiency decreases, 1/Km remains
the same as [S] has no effect on inhibitory
action.
331
LWB plot for Non-competitive inhibition
332
Non competitive inhibition cont’d
• Reagents that cause this type of inhibition are
those that bind irreversibly to some functional
group of the enzyme that may be essential for
maintaining the catalytically 3-dimensional
configuration of the enzyme molecule.
333
• Enzymes containing SH (sulphydryl group) are
inhibited by heavy metal ions like silver,
mercury, etc.
• Iodoacetate unite with SH group which form
part of the active site of many
dehydrogenases.
334
Non competitive inhibition cont’d
• In addition, metallic ions essential for the
activity of many enzymes form more or less
stable complexes with various inhibitors,
• E.g. fluoride forms a complex with Mg2+ ion
and inactivates enolase and CO complexes with
Fe in haem groups and inactivates
mitochondrial cytochrome oxidase.
• Other examples of irreversible inhibitors are
snake venoms and nerve gases.
335
Regulation of enzyme activity
• There are many ways an organism regulates its
enzyme activities so that
-it can coordinate its numerous metabolic
processes,
-respond to changes in its environment and
-grow and differentiate in an orderly manner.
336
Inactive enzyme
• Some enzymes are synthesized in the inactive
form and are activated at a physiologically
appropriate time and place.
• These inactive enzymes are called zymogens.
• Example trypsin is synthesized in the pancreas
but not needed at that location.
337
• It is synthesized in an inactive form and
transported to the site of action, the small
intestines.
• Digestive enzymes exemplify this kind of
regulation
338
Covalent modification
• In this kind of regulation, there is a covalent
insertion of a small group in the enzyme.
• Eg enzymes that catalyze the degradation and
synthesis of glycogen, ie glycogen phosphorylase
and glycogen synthase.
339
• In such enzymes, a phosphoryl group is
attached to a specific serine group on the
enzyme.
• Phosphorylation may activate or inactivate the
enzyme.
• The modification can be reversed by hydrolysis
catalyzed by specific enzymes called
phosphoprotein phosphatases.
340
Feedback inhibition
• In many multi enzyme systems the enzyme
that catalyzes the first step is often inhibited
by the ultimate product.
E.g. the production of cholesterol is often
regulated by a feedback mechanism.
341
Allosteric regulation
• Allosteric enzymes are multi subunit proteins
with multiple active sites.
• These usually occupy key regulatory positions in
a pathway and frequently catalyze the committed
step early in a pathway.
342
• They are regulated by molecules called
effectors (modifiers or modulators) and bind
non covalently at a site other than the active
site.
• The presence of an allosteric effector can alter
the affinity of an enzyme for its substrate or
modify the maximal catalytic activity of the
enzyme or both.
343
Allosteric regulation cont’d
• The binding of an effector will either inhibit or
activate the enzyme.
• Effectors that inhibit enzymes are termed
negative effectors or negative modulators and
those that increase enzyme activity are positive
effectors or modulators.
344
• Allosteric enzymes do not give a classical
hyperbolic Michaelis Menten curve.
• The curve is rather sigmoidal when a plot of Vo
versus [S] is made.
345
Enzyme induction
• This is another way of classifying enzymes and
considers the conditions under which the enzyme is
present in the cell.
• There are 2 main types; Constitutive and Inducible
enzymes.
346
The constitutive enzyme
• The constitutive enzymes are formed at constant
rate and in constant quantities,
• ie they are present in the cell at constant levels
whether there is substrate or not.
• E.g. are enzymes of the glycolytic pathway.
347
The inducible enzymes
• These are called adaptive enzymes and are
always present in trace amounts but their
concentration varies in proportion to their
substrate.
348
Induction and repression of enzyme
synthesis
• In this mode of regulation, enzyme activity is not
affected but the amount of the enzyme present
is regulated by altering its rate of synthesis.
• The increased (induction) or decreased
(repression) synthesis of the enzyme leads to an
alteration in the number of active sites available
rather than influencing the efficiency of the
existing enzyme molecules.
349
• E.g. enzymes needed at only one stage of
development or under selected physiological
conditions.
• Enzymes involved in the synthesis of prolactin.
350
Uses of Enzymes in Medicine
• Analysis of blood serum for unusually high
levels of certain enzymes provides valuable
information on a patient’s condition.
• Such analysis is used to diagnose heart attack,
liver disease and pancreatitis.
• Elevated blood serum concentrations of the
enzymes amylase and lipase are indications of
pancreatitis (inflammation of the pancreas.
351
• Liver diseases such as cirrhosis and hepatitis
results in elevated levels of one of the
isoenzymes of lactate dehydrogenase and
elevated levels of:
-alanine aminotransferase/serum glutamatepyruvate transaminase (ALT/GPT) and
-aspartate aminotransferase/serum glutamateoxaloacetate transaminase (AST/SGOT) in
blood serum.
352
COFACTORS
• Many enzymes require certain additional
substances to perform their catalytic activities.
• These are called cofactors and they play an
indispensable role in various enzyme catalysed
reactions.
353
•
I.
II.
III.
There are three main types;
Coenzymes
Prosthetic groups
Metal activators
354
Coenzymes
• These normally act as acceptors or donors of a
functional group or of an atom that is
removed from the substrate.
• A coenzyme is not firmly bound to one
particular enzyme and therefore can interact
with different enzymes.
• They are normally stable towards heat and
have relatively smaller molecular weight.
355
Prosthetic group
• These are non protein organic molecules firmly
attached to the enzyme and therefore not easily
dialysed off the enzyme.
• In enzymatic reactions in which the enzyme operate
in partnership with a prosthetic group, the molecule
act as an acceptor of one of the cleavage part
usually a very small portion of the substrate
molecule.
• The remaining portion of the substrate leaves the
apoenzyme while the part attached to the
prosthetic group may also be liberated or passed on
along a chain of enzymes for catalytic conversion.
356
• Both the prosthetic group and apoenzyme are regenerated
in the process.
• There is no sharp distinction between coenzyme and
prosthetic groups in either case the organic cofactor or part
of it is sometimes not synthesized by animal species and
hence must be provided in the diet. Such compounds are
called vitamins.
• Several of the water soluble vitamins are important
structural components of certain coenzymes and they
include thiamine (B1), riboflavin (B2), niacin, pyridoxine
(B6), panthothenic acid, folic acid, biotin, etc. Some of the
coenzymes are involved in redox reactions and provide
energy for the organism. Others serve as group transfer
agents in metabolic processes.
357
Coenzymes involved in redox reactions
Pyridine nucleotide coenzymes
Nicotinamide Adenine dinucleotide (NAD+)
• This is obtained form the vitamin niacin and is involved in
redox reactions. The biochemically active form is the amide
nicotinamide.
• Structure – it consists of 3 parts; nicotinamide ring, an
amide ring and a 2 sugar phosphate group linked together.
• The adenine sugar-phosphate group in the molecule is
structurally related to nucleosides. The nicotinamide ring
contains the side at which oxidation reduction reactions
occurs. Nicotinic acid can be synthesized from tryptophan
but in small quantities.
358
Sources and deficiency symptoms
for Nicotinamide adenine dinucleotide
• Niacin is found in refined and enriched grains
and cereals, milk and lean meet especially
liver.
• Deficiencies include pellagra, a disease
involving the skin, gastro-intestinal tract and
CNS. Symptoms progress through 3
dimensions and these are dermatitis,
diarrhoea, dementia (loss of memory) and if
untreated, death.
359
Biochemical function of Nicotinamide
adenine dinucleotide
• The nicotinamide nucleotides are coenzymes
for the dehydrogenases.
• The enzymes are specific for both substrate
and coenzyme, ie NAD+ or NADP+.
• The dehydrogenases that require NAD+ and
NADP+ catalyse the oxidation of a variety of
substrate including primary and secondary
alcohols, aldehydes, etc. it can exist in oxidised
and reduced forms.
360
• The NAD linked enzymes often take part in
catabolic pathways. In the process NAD+ is
reduced to NADH and NADP+ to NADPH.
• The enzymes which have NADP+ as cofactor
catalysed steps in anabolic pathways. Eg
reducing power for the synthesis of long chain
fatty acids. The reduction of carbon dioxide in
synthesis also requires NADPH.
361
Mechanism and mode of action
• The action of the nicotinamide nucleotides involve the
removal of the equivalent of 2 H atoms from the substrate
in the form of 2 protons together with 2 electrons. The 2
electrons are taken up by NAD+ and NADP+ together with
one proton (in the form of hydride ion H-) to give the
reduced coenzymes NADH and NADPH.
• The hydrogen is transferred with its electron pair as a
hydride ion to position 4 para to the N.
• The other proton from the substrate is released in solution.
When the pyridine ring becomes reduced, the N loses its
positive charge and the ring looses its aromatic nature.
Such a change alters the light absorption.
362
• Both NAD+ and NADP+ have absorption spectra which
are different for the oxidized and reduced forms. The
absorption occurs in the UV region of the spectrum
within the range 230 – 400nm.
• The oxidized form shows maximum absorption at
approximately 260 nm while the reduced form shows a
rather broad band at approximately 340nm. The
difference offers an excellent method for following the
time courses of some pyridine nucleotide linked
reactions.
• The 260 nm peak is caused by the absorption of the
adenine ring while the 340 nm peak is due ti the
pyridine ring which is responsible for electron
transport.
363
• There is little change in the 260 nm because the
adenine ring structure is the same in both oxidised and
reduced forms of the molecule.
• In reactions involving NAD+ the reduced form NADH
passes the 2 electrons from the substrate to the
cytochrome chain or electron transport chain in the
mitochondria thus providing the energy for
synthesizing ATP from ADP.
• The NADH is thus reoxidized to NAD+ for further
oxidation of the substrate AH2. E.g. occur in glycolysis,
TCA cycle and fatty acid oxidation.
• Two main ways of generating NADPH from NADP+ is the
light reaction of photosynthesis and Pentose Phosphate
Pathway
364
Flavin adenine dinucleotide (FAD) and
Flavin mononucleotide (FMN)
• These are derivatives of vitamin B2 or riboflavin.
• They function as tightly bound prosthetic groups
in a class of enzymes called flavoproteins which
catalyse a wide variety of redox reactions.
• FAD is involved in catalytic reactions of
dehydrogenases, oxidases and hydroxylases.
365
Biocehemical function
• The enzymes that use FAD as prosthetic groups
catalyse fewer redox reactions and exhibit more
H acceptor specificity than the pyridine
nucleotide coenzymes.
• The isoallozine ring of the flavin coenzyme
accepts and releases 2 H atoms which tends to
transfer H in one direction from organic substrate
to molecular oxygen as the ultimate H acceptor
in biological oxidation chain.
366
Mechanism and mode of action
• The reduction of the flavine coenzymes occurs in 2
separate staps each involving the addition of a
single electron and a proton.
• The intermediate formed after the addition of the
electron with its proton is a partially reduced
compound called semiquinone.
• The semiquinone is stabilized by the existence of
other resonance hydrides in the presence of metals
like Fe and Molybdenum in the flavoprotein. The
unpaired electrons presenting the metals thus
stabilizing them. When these metals are removed
catalytic activity is impaired
367
• There are three separately distingyisehd states of flavin
coenzymes; the yellow oxidized form, the red or blue
one electron form and the colourless two electron
reduced form. FAD is electron acceptor for reactions
like;
• FAD is a proostethic group for amino acid oxidases which
are flavoproteins. Flavoproteins can accept hydride ions
from NADH together with the proton from solution or a
pair of hydrogen atoms from a wide variest of organic
metabolites such as amino acids, thioesters of fatty
acids, etc. several of these reactions involve the removal
of the H atom from adjacent C atom to form double
bonds, eg succinate dehydroganse which is a
flavoprotein catalyses the conversion of succinate to
368
fumarate.
• The flavoprotein in the reduced state may react
directly with molecular oxygen to yield
hydrogen peroxide.
• The flavoprotein may also react with oxygen to
provide water instead. This occurs when the
substrate is undergoing hydroxylation so that
the substrate consumes one atom of oxygen
while the other oxygen is reduced to water.
369
Thymine pyrophosphate (TPP)
• This is a coenzyme derived from thyamine or Vitamine
B1. it is also known as cocarboxylase. It serves as a
coenzyme in enzymatic reactions associated with the
non-oxidative decarboxylation of α-keto acids, eg
pyruvate and α-ketogluterate
• Sources include pork, whole grains and legumes. Other
layers of seeds are particularly rich of thyamine, whole
wheat bread is also a good source but white bread is low
in thymine.
• Deficiency results in beriberi which is characterized by
dry skin. Others are irritability, disorderly thincking and
progressive paralysis.
370
Mechanism and mode of action
• The active portion of the molecule is the thiazole
ring and the C between N and S of the thiazole
ring is highly reactive. This is because the proton
on this C can easily be dissociated off leaving a
carbanion which is highly nucleophilic. An
example of such a reaction involving TPP is the
pyruvate dehydrogenase complex reaction which
catalyses the conversion of pyruvate to
acetaldehyde with the release of carbon dioxide.
371
• In the pyruvate dehydrogenase complex
reaction, the carbanion of the thiazole ring
attacks the carbonyl C of the pyruvate. Carbon
dioxide split off leaving a 2C fragment. An
activated aldehyde covalently bonded to the
coenzyme. A shift of electrons releases
acetaldehyde, regenerating the carbanion.
372
Pyridoxal phosphate (PALP)
• This is a coenzyme derived from vitamin B6 which
exist in three different forms; pyridoxal,
pyridoxamine and pyridoxine.
• The phosphorylated derivative of pyridoxal and
pyridoxamine give rise to the active metabolic
coenzyme.
• Pyridoxine occurs primarily in plants whereas
pyridoxal and pyridoxamine are found in foods
obtained from animals.
• Sources are wheat, corn, egg yolk, lover and muscle
meat, etc.
373
Biochemical function
• It is involved in the transfer of amino group from
one molecule to another, an important step in
the biosynthesis of amino acids.
• Pyridoxal phosphate is loosely bound to the
epsilon amino group of a lysyl residue on the
apoenzyme by a shiffs base.
• It is also involved in other reactions of amino acid
metabolism, eg decarboxylation and
racemisation.
374
Mechanism and mode of action
• Transamination
• In such reactions the amino group of an amino acid
is reversibly transferred to the α-C atom of an αketo acid. The coenzyme acts as an intermediate
amino group carrier on the enzymes active site
from the donor α- amino acid to NH2 group
acceptor, the keto acid.
• The amino group of the incoming amino acid is
transferred to the enzymr bound pyridocal
phosphate. The resulting pyridoxine phosphate
then donates its amino group to the α-keto acid
while the coenzyme reverts to its pyridoxal form.
375
•
•
•
•
•
In transamination reactions, pyridoxal phosphate
First forms a shiff,s base with the amino acid. The aldehyde
group condenses with the α-amino group of the amino acid
with the elimination of water.
There is rearrangement followed by hydrolysis which
removes product 1 (the α-keto acid of the first substrate).
Another keto acid (substrate 3) then forms a shiff’s base
with the pyridoxamine phosphate.
There is rearrangement followed by hydrolysis which yields
a second product, an amino acid and regenerate pyridoxal
phosphate.
The net reaction is that an amino acid (substrate) reacts
with an α-keto acid (substrate 2) to form an α-keto acid
(product 1) and an amino acid (product 2)
376
Coenzyme A (CoASH)
• Derived from the water soluble B-vitamin
pathothenic acid which is also a component of
acyl carrier protein (ACP).
• Animals can not synthesize the panthothenic acid
so it must be provided in the diet after which the
rest of the molecule can be synthesized to form
the coenzyme.
• It is a complex structure consisting of several small
components covalently linked together.
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It contains 3 major protions;
• a 3` 5` ADP molecule, a derivative of
adenosine with phosphate group esterified to
the sugar;
• a central portion containing the panthothenic
acid moiety;
• a thioethyamine portion called β-mercapto
ethylamine which is the active portion of the
molecule involved in activation reaction.
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