Carbohydrates and
Nucleic Acids
General Biochemistry
Mahidol University International College
Manchuta Dangkulwanich, Ph.D.
1
By the end of this section, you should be able to…
•
Differentiate between monosaccharides and
polysaccharides in regard to structure and
function.
•
Understand the interconversion between various
conformation of hexose and pentose sugars.
•
Differentiate among the types of glycoproteins in
regard to structure and function
•
Describe the structure and components of nucleic
acids
2
Carbohydrates
•
Carbohydrates are a common source of energy for
living organisms.
•
Each gram of carbohydrates provides ~4 cal.
•
Body stores 200-300 g of glycogen in muscles
•
Liver stores 80-100 g of glycogen
3
4
Aldose and Ketose Sugars
Aldose
Ketose
•
The simplest form of carbohydrates are monosaccharides,
either aldehydes or ketones with two or more hydroxyl groups.
•
Monosaccharides are colorless, crystalline solids that are
freely soluble in water but insoluble in nonpolar solvents. Most
have a sweet taste. The backbones of common
monosaccharide molecules are unbranched carbon chains in
which all the carbon atoms are linked by single bonds.
5
Monosaccharides
Monosaccharides with four, five, six, and seven carbon
atoms in their backbones are called, respectively, tetroses,
pentoses, hexoses, and heptoses.
The most common ones are hexoses and pentoses.
6
Monosaccharides have stereo centers
•
Most of the hexoses of living organisms are Disomers. D-isomer: the most distant stereo
specific carbon from the carbonyl carbon has
the same configurations with D-glyceraldehyde.
•
Two sugars that differ only in the configuration
around one carbon atoms are called epimers.
7
•
The four- and five-carbon
ketoses are designated by
inserting “ul” into the name of
a corresponding aldose; for
example, D-ribulose is the
ketopentose corresponding to
the aldopentose D-ribose.
8
n
A molecule with n stereo centers has 2 stereoisomers
Aldohexoses have 4 stereo
centers, 24 = 16 (8 D- and 8 L-)
Most naturally occurring sugars
are the D-isomers; however, a
few L-isomer also exists, such as
L-arabinose.
D-isomer and L-isomer are mirror
images of each other.
9
Common monosaccharides with five or more C
in the backbone form cyclic structures
10
•
Isomers of monosaccharides that
differ only in their configuration
about the hemiacetal or hemiketal
(carbonyl) carbon (anomeric C)
are called anomers.
•
In aqueous solution, the 𝛼- and 𝛽anomers are converted freely via
the linear isomer by a process
called mutarotation.
The six-membered rings are called pyranose.
The five-membered rings are called furanose.
•
Haworth perspective formulas
show the stereochemistry of the
ring forms.
•
For D-pyranose sugars, the
designation α means that the
hydroxyl group attached to the
anomeric carbon (C-1) is on the
opposite side from -CH2OH group
at C-5; β means that they are on
the same side of the plane.
•
The same nomenclature applies to
the furanose ring form of fructose,
except that α and β refer to the
hydroxyl groups attached to C-2,
the anomeric carbon atom
How are the numbers assigned?
11
Fructose can form both five-membered and six-membered rings
•
Fructose forms both pyranose
and furanose rings.
•
The pyranose form
predominates in fructose free in
solution, and the furganose
forms predominates in many
fructose derivatives.
•
12
β-D-Fructopyranose, found in
honey, is one of the sweetest
chemicals known. The β-Dfructofuranose form is not as
sweet. Heating converts βfructopyranose into βfructofuranose, reducing the
sweetness of the solution.
Pyranose and furanose rings can adopt different conformations
•
Generally, substituents in the
equatorial (e) positions are less
sterically hindered by neighboring
substituents than axial (a) position.
•
Conformations with bulky
substituents in equatorial positions
are favored.
•
The boat conformation is seen only
in derivatives with bulky substituents.
Envelope conformations of β-D-ribose.
Either the C-2 or C-3 is out of plane on
the same side with C-5.
13
Sugar derivatives
•
OH-group in parent compound is replaced by another substituent
•
Aldehyde or ketone group is reduced to hydroxyl group
EXAMPLES:
Sugar alcohol, which lacks the aldehyde or ketone group.
D-ribose
In plants, and bacterial cell wall
Artificial sweetener
14
Some important hexose derivatives in biology
15
Monosaccharides are joined to alcohols
and amines through glycosidic bonds.
• D-glucose
will react with methanol in an acidcatalyzed process: the anomeric carbon atom
reacts with the hydroxyl group of methanol to
form two products, methyl α-D-glucopyranoside
and methyl β-D-glucopyranoside.
• The
anomeric carbon atom of a sugar can be
linked to the nitrogen atom of an amine to form
an N-glycosidic bond.
• These
modifications increase the biochemical
versatility of carbohydrates, enabling them to
serve as signal molecules or facilitating their
metabolism. Three common reactants are
alcohols, amines, and phosphates.
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Monosaccharides are reducing agents
The carbonyl carbon is
oxidized to carboxyl
group by mild oxidizing
agents such as Fe3+ or
Cu2+.
•
A reducing agent removes an electron from an oxidizing
agent.
•
This property is a basis for sugar testing in Fehling’s test.
•
Reduction of Cu2+ to Cu+ in alkaline solution and forms a
red precipitate of cuprous oxide (Cu2O).
•
A reducing sugar must be readily interconverted to a
form that include free aldehyde. Ketose sugars can also
isomerize to aldehyde in the presence of base.
17
Glucose oxidase and peroxidase to measure
the concentration of glucose
colorless
brown
Fehling’s test gives positive results for all monosaccharides. To measure the
concentration of glucose specifically, enzyme glucose oxidase is employed.
18
Complex carbohydrates are formed by linkage of
monosaccharides via glycosidic bonds
19
•
The reversal of this
reaction is hydrolysis in
the presence of acids.
•
When the anomeric
carbon is involved in a
glycosidic bond, that
sugar residue cannot
take the linear form and
therefore becomes a
nonreducing sugar.
•
Having a free
hemiacetal, maltose is
still a reducing sugar.
Some common disaccharides
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Lactose is a disaccharide of
galactose and glucose:
Gal(β1–>4)Glc.
•
Sucrose (table sugar) is a
disaccharide of glucose and
fructose. Nonreducing
sugars are called glycosides.
•
Note the name change,
because it is no longer a
reducing sugar.
20
21
Glycogen and starch are storage forms of glucose
•
Polysaccharides play vital roles in
energy storage and in
maintaining the structural integrity
of an organism.
•
The most common homopolymers
in animal cells is glycogen, a very
large, branched polymer of
glucose.
•
The nutritional reservoir in plants
is starch, of which there are two
forms: amylose and amylopectin.
α-1,4-Glycosidic bond
22
Cellulose, the major structural polymer of plants,
consists of linear chains of glucose units
•
Cellulose is an unbranched
polymer of glucose residues
joined by β-1,4 linkages.
•
The β configuration allows
cellulose to form very long,
straight chains.
•
Fibrils are formed by parallel
chains that interact with one
another through hydrogen bonds.
•
Are the structure of the
polysaccharides suitable for their
functions? How?
23
Carbohydrates are attached to proteins to form glycoproteins
•
50% of the proteome consists of glycoproteins.
•
Three classes of glycoproteins.
•
Glycoproteins - the protein component is the largest by weight.
They play a variety of biochemical roles, such as membrane
proteins, cell adhesion, binding of sperms to eggs.
•
Proteoglycans - the protein is linked to a particular type of
polysaccharide called a glycosaminoglycan. Carbohydrates
make up a much larger percentage. They are structural
components and lubricants.
•
Mucins or mucoproteins - like proteoglycan, they are
predominantly carbohydrate. N-Acetylgalactosamine is usually
the carbohydrate moiety bound to the protein in mucins.
24
Carbohydrates may be linked to Asp, Ser, Thr
•
In all classes of glycoproteins,
sugars are attached either to
the amide nitrogen atoms in
the side chain of Asp (Nlinkage) or to the hydroxyl
oxygen atom in the side chain
of Ser or Thr (O-linkage).
•
The process is called
glycosylation.
GlcNAc : N-acetylglucosamine
GalNAc : N-acetylgalactosamine
25
Additional sugars are attached to create various patterns
Erythropoietin has oligosaccharides
linked to three asparagine residues
and one serine residue.
26
Proteoglycans are composed of polysaccharides and proteins
•
These proteins are attached to a particular type of polysaccharide called glycosaminoglycan.
The glycosaminoglycan makes up as much as 95% of the molecule by weight.
•
Many glycosaminoglycans are made of repeating units of disaccharides containing a derivative
of an amino sugar (figure above), either glucosamine or galactosamine.
•
At least one of the two sugars in the repeating unit has a negatively charged carboxylate or
sulfate group.
•
They function as lubricants and structural components in connective tissues. They are important
components of cartilage, which function as cushion to absorb shock. (Think about running)
27
Carbohydrates as informational molecules: The sugar code
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Cells use specific oligosaccharides to encode important
information about intracellular targeting of proteins, cell-cell
interaction, tissue development, and extracellular signals.
a) Oligosaccharides components of a
variety of glycoproteins or glycolipids,
interacting with high specific and affinity
with lectins in the extracellular milieu.
b) ,c) and d) Viruses, toxins and some
bacteria bind to the cell surface
glycoproteins before entering a cell.
e) Selectins in the plasma membrane
mediates cell-cell interactions.
f) The mannose 6-phosphate receptor/
lectin of the trans Golgi complex binds to
the oligosaccharide of lysosomal enzymes,
targeting them for transfer in the lysosome.
28
Nucleotides and Nucleic Acids
•
A nucleic acid consists of four kind of bases linked to a sugarphosphate backbone.
•
The sequence of the bases is a form of linear information.
Charges in nucleic acids?
29
•
Nucleic acids are composed of nitrogenous bases, linked to ribose sugar (RNA) or
deoxyribose sugar (DNA).
•
The strand of sugars linked by phosphodiester bridges is referred to as the backbone of the
nucleic acid. The backbone is constant in DNA and RNA, but the bases vary from one
monomer to the next.
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The bases are derivatives of purine and pyrimidine
•
The bases in DNA are ___________
•
The bases in RNA are ___________
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N-9 of a purine or N-1 of a pyrimidine is attached to the C-1’ of the
sugar.
31
Nucleotides are the monomeric units of nucleic acids
•
A nucleoside is …
•
A nucleotide is…
32
What are these molecules?
33
Absorption spectra common nucleotides
Absorbance at 260 nm is used to
measure the concentration of
nucleic acids. (A = εbc)
ε - Use the average of ε values for
each nucleotide.
34
DNA and RNA
35
•
A DNA or RNA strand has
directionality. One end of the strand
has a free 5’-OH group (or a 5’-OH
group attached to the a phosphoryl
group), whereas the other end has
a 3’-OH group, and neither end is
linked to another nucleotide.
•
By convention the base sequence
is written in the 5’- to-3’ direction.
•
The DNA and RNA strands on the
left can be written as ATG and
rUrGrC, respectively.
•
Which one (DNA or RNA) is
hydrolyzed rapidly under alkaline
conditions?
DNA molecules are very long
•
A DNA molecule must comprise many nucleotides to
carry the genetic information necessary for even the
simplest organisms.
•
The DNA of a virus such as polyoma, which can
cause cancer in certain organisms, is 5100 nucleotide
(nt) in length.
•
The E.coli genome is a single DNA molecule
consisting of two strands of 4.6 million nt.
•
The human genome comprises approximately 3 billion
base pairs distributed among 24 distinct DNA
molecules.
36
Nucleic acid strands can form a double-helical structure
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DNA was first isolated by
Friedrich Miescher in 1868. It
was determined to be genetic
materials in the 1940s.
•
Chargaff base-pairing rules in
1950 - A:T and G:C are about
equal in many species.
•
Maurice Wilkins and Rosalind
Franklin obtained x-ray diffraction
photographs of DNA fibers in the
early 1950s.
•
The structure of DNA was
deduced by James Watson and
Francis Crick in 1953.
37
Watson and Crick Hydrogen bonding patterns
38
The features of the Watson-Crick
model of DNA
1. Two helical DNA strands are coiled
around a common axis, forming a
right-handed double helix. The
strand run in opposite directions.
2. The sugar-phosphate backbones
are on the outside of the helix. The
bases are inside.
3. The bases are nearly perpendicular
to the helix axis, and adjacent
bases are separated by
approximately 3.4 Å. Helical repeats
of 10.4 bases per turn.
4. The diameter of the helix is 20 Å.
39
Base stacking interactions contribute
to the stability of the double helix
Axial View of DNA. Base pairs are
stacked nearly one on top of
another in the double helix.
40
•
The hydrophobic bases cluster in
the interior of the helix away from
the surrounding water.
•
van der Waals forces between the
bases. Although they are weak
interactions, a large number of
atoms are in van der Waals contact,
and the net effect is substantial.
DNA double helix can adopt a variety of structural forms
•
Which form is the Watson-and-Crick model,
the one that presents under physiological
conditions?
•
A-DNA: a shorter stack and broader in
diameter (~26 Å). The base pairs are tilted
rather than perpendicular to the helix axis.
A-DNA appears when DNA is less
hydrated. dsRNA adopt a structure similar
to A-DNA.
•
Z-DNA: a longer stack and narrower in
diameter (~18 Å). The structure is lefthanded. The phosphate groups are
zigzagged. Some proteins can bind to ZDNA.
41
Major and minor grooves are lined by
sequence-specific hydrogen-bonding groups
•
In B-DNA, the major groove is wider (12 Å vs 6 Å) and
deeper (8.5 Å vs 7.5 Å) than the minor groove.
•
Each groove is lined by potential hydrogen-bond donor
and acceptor atoms that enable interactions with proteins.
•
The major groove displays more features that distinguish
one base pair from another than does the minor groove.
The larger size of the major groove makes it more
accessible for interactions with proteins.
42
Nucleic acid sequences can adopt unusual structures
Palindromic: the same when read from 5’ to 3’ in one
strand and 3’ to 5’ in the complementary strand.
43
Examples of unusual structures of DNA
44
Hoogsteen pairing allows the formation of triplex DNAs
45
RNA structures
46
•
A single-stranded
RNA molecule
can fold back on
itself to form a
complex
structure.
•
A program called
Mfold can be
applied to
simulate the
structure of RNA
based on the
sequence.
DNA origami
A long single stranded DNA can be designed to
fold in to various shapes! The reaction is one pot.
http://www.dna.caltech.edu/Papers/rothemund-origami-iccad05.pdf
47
48
The double helix can be reversibly melted
•
The two strands of DNA can be disrupted by heating or
adding chemicals, such as acids, bases or urea.
•
The melting temperature (Tm) of DNA is defined as the
temperature at which half of the helical structure is lost.
•
Stacked bases absorb less ultraviolet light than
unstacked bases, an effect called hypochromism.
49
Alterations in DNA structure that produce permanent
changes in the encoded genetic information.
•
•
Several nucleotide bases undergo spontaneous loss of their exocyclic amino groups,
deamination.
•
Deamination of cytosine in DNA to uracil occurs in about one of every 107 cytidine
residues in 24 hours. Uracil is readily recognized as foreign in DNA and is removed
by a repair system.
Hydrolysis of the N-𝛽-glycosyl bond between
the base and the pentose, to create a DNA
lesion called AP (apurinic, apyrimidinic) site or
abasic site. Purines are loss at the rate of 1 in
105 purines every 24 hours.
50
UV light induces the condensation of two
ethylene groups to form a cyclobutane ring.
•
This reaction occurs most frequently between adjacent thymidine
residues on the same DNA strand.
•
Ionizing radiation (x-rays and gamma rays) can cause ring opening
and fragmentation of bases as well as breaks in the covalent
backbone of the nucleic acids.
51
DNA may be damaged by reactive chemicals
•
Delaminating agents, particularly nitrous acid (HNO2) or compounds that can be
metabolized to nitrous acid or nitrites
•
Alkylating agents
•
Oxidative damage from excited-oxygen species such as hydrogen peroxide,
hydroxyl radicals and superoxide radicals.
52
Alkylating agents alter certain bases of DNA
Methylation of Guanine
•
Adenine and cytosine are methylated more often than guanine and thymine.
•
Methylation can serve as a defense mechanism that helps the cell distinguish
its DNA fro foreign DNA.
•
5’-methylcytidine in eukaryotic DNA has an important structural function.
53
WHO added processed
meats to Group 1 Carcinogenic to humans
54
DNA polymerase catalyzes
phosphodiester-bridge formation
•
Replication is directional. DNA polymerase requires a primer with a 3’-OH to
begin synthesis. Elongation of the DNA chain proceeds in the 5’ to 3’ direction.
Mg2+ ion is required.
•
DNA polymerase has proofreading activity. The error rate is minuscule at less than
10-8 per base pair.
55
Gene Expression in the Transformation of
DNA Information into Functional Molecules
http://www.nature.com/scitable/content/ne0000/ne0000/ne0000/
ne0000/14711098/U2CP3-1_SynthesisDegredati_ksm.jpg
56
•
mRNA
•
tRNA
•
rRNA
•
snRNA - participate in the
splicing of RNA exons.
•
miRNA - small (~21 nt)
noncoding RNAs that bind to
complementary mRNA
molecules and inhibit their
translation.
•
siRNA - small RNA that bind
to mRNA and facilitate its
degradation.
•
RNA is also a component of
telomerase.
All cellular RNA is synthesized by RNA polymerases
•
RNAP requires a divalent metal ion, either Mg2+ or Mn2+ for catalysis.
•
RNAP does not require a primer, the ability of RNAP to correct
mistakes is not as extensive as the of DNAP.
57
Transfer RNAs are the adaptor
molecules in protein synthesis
•
tRNAs contain an amino acid attachment
site and a template recognition site.
•
Aminoacyl-tRNA synthetase joins an
amino acid to a tRNA molecule. There is at
least one specific synthetase for each of
the 20 amino acids.
58
Amino acids are encoded by groups of
three bases starting from a fixed point
Second letter
59
•
Codons that specify
the same amino acids
are called synonyms.
•
Messenger RNA
contains start and stop
signals for protein
synthesis.
•
The first residue is
fMet, which is
encoded by the codon
AUG, establishing a
reading frame.
•
Release factor binds
to the stop codons:
UAA, UAG, and UGA.
DNA can be sequenced by Sanger’s method
•
Primer extension by
DNA polymerase in
the presence of a
ddNTP chain
terminator.
•
Label the primer by
radio active P-32.
60
DNA sequencing can be done automatically, and cheaply.
•
Typical read length is 800-1000 nt. Sanger’s sequencing costs between
3-10 USD per rxn.
•
KhonKaen University: 300 baht per rxn. Sc MU: 665 baht per rxn.
61
DNA-Based information technologies
•
DNA Cloning: The Basics
•
From Genes to Genomes
•
From Genomes to Proteomes
•
Genome Alterations and New Products of
Biotechnology
62
DNA Cloning: The Basics
•
Cutting DNA at precise locations
•
Selecting a small molecule of DNA
capable of self-replication - vectors
•
Joining two DNA fragments covalently DNA ligase >> recombinant DNAs
•
Moving recombinant DNA from the test
tube to a host cell
•
Selecting or identifying host cells that
contain recombinant DNA
63
DNA amplification by polymerase chain reaction
64
DNA can be specifically cleaved by restriction enzymes.
•
Sticky ends or Blunt ends depends on restriction
enzyme choice.
•
DNA sequence can be synthesized. Any sequence
that you desire, with some exceptions.
65
Strategy for mutagenesis
66
Gene expression profiling in DNA microarray
67
Genome alterations and new products of biotechnology
•
RED HOT genome editing technology - CRISPR/Cas9 by Doudna and Charpentier labs
•
By delivering cas9 protein and appropriate guide RNAs into a cell, the organism’s
genomes can be cut at any desire locations
68