In summary, nucleic acids are indispensable for the continuity of life, as

they govern the inheritance, expression, and regulation of genetic

information in organisms.

Their roles extend beyond genetics to include vital cellular processes and
the transfer of energy within biological systems.
 b-N-Glycosidic Bond
• In nucleotides the pentose ring is attached to the nucleobase via
N-glycosidic bond
• The bond is formed to the anomeric carbon of the sugar in b
configuration
• The bond is formed:
– to position N9 in purines
– to position N1 in pyrimidines
• This bond is quite stable toward hydrolysis, especially in
pyrimidines
• Bond cleavage is catalyzed by acid
 Conformation around N-Glycosidic Bond

• Relatively free rotation can occur around the N-glycosidic bond

in free nucleotides
• The torsion angle about the N-glycosidic bond (N-C1') is
denoted by the symbol c
• The sequence of atoms chosen to define this angle is O4'-C1'-
N9-C4 for purine, and O4'-C1'-N1-C2 for pyrimidine derivatives
• Angle near 0 corresponds to syn conformation
• Angle near 180 corresponds to anti conformation
• Anti conformation is found in normal B-DNA
 Nucleotides and Nucleosides
• Nucleotide =
– Nitrogeneous base
– Pentose
– Phosphate

• Nucleoside =
– Nitrogeneous base
– Pentose

• Nucleobase =
– Nitrogeneous base
 b-N-Glycosidic Bond
• In nucleotides the pentose ring is attached to the nucleobase via
N-glycosidic bond
• The bond is formed to the anomeric carbon of the sugar in b
configuration
• The bond is formed:
– to position N9 in purines
– to position N1 in pyrimidines
• This bond is quite stable toward hydrolysis, especially in
pyrimidines
• Bond cleavage is catalyzed by acid
 Conformation around N-Glycosidic Bond

• Relatively free rotation can occur around the N-glycosidic bond

in free nucleotides
• The torsion angle about the N-glycosidic bond (N-C1') is
denoted by the symbol c
• The sequence of atoms chosen to define this angle is O4'-C1'-
N9-C4 for purine, and O4'-C1'-N1-C2 for pyrimidine derivatives
• Angle near 0 corresponds to syn conformation
• Angle near 180 corresponds to anti conformation
• Anti conformation is found in normal B-DNA
The sequence of atoms chosen to define this angle is O4'-C1'-N9-C4 for purine, and O4'-C1'-N1-C2
for pyrimidine derivatives
 Pentose in Nucleotides
• -D-ribofuranose in RNA
• -2’-deoxy-D-ribofuranose in DNA

• Different puckered conformations of the sugar ring

are possible
Phosphate Group
• Negatively charged at neutral pH
• Typically attached to 5’ position
• Nucleic acids are built using 5’-triphosphates
• ATP, GTP, TTP, CTP
• Nucleic acids contain one phosphate moiety per
nucleotide
• May be attached to other positions
 Other Nucleotides:
Monophosphate Group in Different Positions
Polynucleotides
• Covalent bonds formed via phosphodiester linkages
• negatively charged backbone
• DNA backbone is fairly stable
• DNA from mammoths?
• Hydrolysis accelerated by enzymes (DNAse)
• RNA backbone is unstable
• In water, RNA lasts for a few years
• In cells, mRNA is degraded in few hours
• Linear polymers
• No branching or cross-links
• Directionality
• 5’ end is different from 3’ end
• We read the sequence from 5’ to 3’
Nomenclature
 Nomenclature: Deoxyribonucleotides
You need to know structures, names, and symbols (both
two-letter (dA) and four-letter (dAMP) codes)
 Nomenclature: Ribonucleotides
You need to know structures, names, and symbols (both
one-letter and three-letter codes)
 Discovery of DNA
Structure
• One of the most important discoveries in biology

• Why is this important?

“This structure has novel features which are of considerable
biological interest”
―Watson and Crick, Nature, 1953

• Good illustration of science in action

•Missteps in the path to a discovery
•Value of knowledge
•Value of collaboration
•Cost of sharing your data too early
Covalent Structure of DNA (1868–
1935)
OH

HO P

O
O
• Friedrich Miescher isolates
Thymine C5H7O
“nuclein” from cell nuclei
O

HO P O
• Hydrolysis of nuclein
Structure of DNA: O

1929 Adenine C5H7O • phosphate

(Levene & London) O • pentose
• and a nucleobase
OH

HO P O

H O • Chemical analysis
H
H H H
OH • phosphodiester linkages
Thymine
O
CH2O P O • pentose is ribofuranoside
H O
Structure of DNA:
1935 H
H H H
OH
(Levene & Tipson) Adenine CH2O P O
O O
 Road to the Double Helix
• Watson and Crick • Franklin and Wilkins
– Missing layer means – “Cross” means helix
alternating pattern – “Diamonds” mean
(major & minor groove) that the phosphate-
– Hydrogen bonding: sugar backbone
A pairs with T is outside
– Calculated helical
G pairs with C
parameters
Double helix fits the data!

Watson, Crick, and Wilkins shared

1962 Nobel Prize
Franklin died in 1958
Watson-Crick Model of B-DNA
 Hydrogen-Bonding
Interactions
• Two bases can hydrogen bond to form a base pair
• For monomers, large number of base pairs is possible
• In polynucleotide, only few possibilities exist
• Watson-Crick base pairs predominate in double-
stranded DNA
• A pairs with T
• C pairs with G
• Purine pairs with pyrimidine
 Complementarity of DNA Strands

• Two chains differ in sequence

(sequence is read from 5’ to 3’)
• Two chains are complementary
• Two chains run antiparallel
AT and GC Base Pairs
Other Forms of DNA
DNA Denaturation
• Covalent bonds remain intact
• Genetic code remains intact
• Hydrogen bonds are broken
• Two strands separate
• Base stacking is lost
• UV absorbance increases

Denaturation can be induced by high temperature, or

change in pH

Denaturation may be reversible: annealing

Thermal DNA Denaturation (Melting)

• DNA exists as double helix at normal temperatures

• Two DNA strands dissociate at elevated temperatures
• Two strands re-anneal when temperature is lowered
• The reversible thermal denaturation and annealing
form basis for the polymerase chain reaction
• DNA denaturation is commonly monitored by UV
spectrophotometry at 260 nm
Factors Affecting DNA
Denaturation
• The midpoint of melting (Tm) depends on base
composition
• High CG increases Tm

• Tm depends on DNA length

• Longer DNA has higher Tm
• Important for short DNA

• Tm depends on pH and ionic strength

• High salt increases Tm
 Denaturation of large DNA molecules
is not uniform
AT rich regions melt at a lower temperature than GC-rich regions
Two near-complementary
DNA strands can hybridize
• Detection of a specific DNA molecule in complex mixture
- radioactive detection
- fluorescent DNA chips
• Amplification of specific DNA
- polymerase chain reaction
- site-directed mutagenesis
• Evolutionary relationships
• Antisense therapy
Messenger RNA:
Code Carrier for the Sequence of
Proteins
• Is synthesized using DNA template
• Contains ribose instead of deoxyribose
• Contains uracil instead of thymine
• One mRNA may code for more than one protein
• Together with transfer RNA (tRNA) transfers genetic
information from DNA to proteins
The 5’-cap of mRNA
Translation

• mRNA gets
translated by
Ribosomes
Bacterial ribosomes
SSU rRNA
Amino Acids, Peptides, and Proteins

Fig. 3-6. Absorption of ultraviolet light by aromatic amino acids.

 Introduction to Proteins
Proteins mediate nearly every process that takes place
inside a cell. They are the most abundant biological
macromolecules in cells. All proteins, regardless of
organism, are composed of the same set of 20 amino
acids that are incorporated into them during translation.
Due to the nearly limitless variety in the sequences of
amino acids in proteins, nearly all imaginable functions
can be encoded in proteins (Fig. 3-1).
The Zwitterion at
Neutral pH
 Common Features of Amino Acids

• The genetic code specifies 20 "standard" amino

acids, which are polymerized into proteins by
ribosomal translation.
• All amino acids contain alpha carbon, of which four
different substituent groups are typically attached
(Fig. 3-2).
• These groups are the alpha-amino group, the alpha-
carboxyl group, hydrogen, and the variable R group
(side-chain).
• In glycine, the R group consists of another hydrogen
atom.
• In the other 19 amino acids, the alpha carbon is at the
chiral center.
• There are two possible
configurations for these four
substituents--the "D" and "L"
stereoisomers, which are
mirror images of each other
(enantiomers) (Fig. 3-3).
• The standard amino acids have
the L-configuration.
Mirror images, not superimposable
 D,L Classification System for Amino
Acid Configurations
The D, L system is used to specify the absolute configuration of
substituent groups about chiral carbons in amino acids and
monosaccharides. In this system, amino acids are specified as D or L
based on comparison of their configurations to the reference compounds,
D- and L-glyceraldehyde (Fig. 3-4). Note that not all amino acids actually
are levorotatory. Thus D and L designate only the configurations of the
substituent groups and not the optical properties of the amino acid.
• Amino acids are classified based
on the characteristics of their R
groups.
• The chemical properties of the
standard 20 amino acids are
summarized in Table 3-1 (next two
slides).
 Classification of Amino Acids by R
• Group
Amino acids are collected into different categories based
on similarities in the properties of their R groups.
• One such classification scheme (Fig. 3-5) relies heavily on
the polarity of the R groups.
• Note that some amino acids, such as glycine, histidine, and
cysteine, do not perfectly fit into one group.
• Thus, their assignments to a particular group are
subjective rather than based on absolute characteristics.
• The textbook divides amino acids into five categories
based on the properties of their R groups: nonpolar,
aliphatic, aromatic, polar, uncharged, positively charged,
and negatively charged (next slides).
Numbering of Carbons in Amino Acids
The conventions for labeling the carbon atoms in amino
acids is illustrated using lysine in the figure. The 
carbon is always carbon-2 of the amino acid. The -
carboxyl group is always carbon-1
The amino acids in this group lack polar
functional groups in their side chains.
Due to their hydrophobicity, their R groups often
cluster together within proteins' interiors,
stabilizing protein structure via hydrophobic
interactions.
The preferences of several of these amino acids
for regions of protein secondary structure are
discussed in next classes.
• The R groups of the polar, uncharged amino acids all
contain polar functional groups that can hydrogen
bond with water.
• Serine and threonine contain hydroxyl groups.
• Aasparagine and glutamine contain amide groups.
• Cysteine contains a sulfhydryl group, albeit whose
polarity is quite weak.
• Asparagine and glutamine are the amide forms of the
two negatively charged amino acids, aspartate and
glutamate.
• The sulfhydryl group of the cysteine side chain is a
weak acid (pKa = 8.2).
• The cysteine side chain, therefore, is mostly uncharged
at neutral pH.
 Cysteine and Disulfide Bonds

• The thiol groups of two cysteine residues are readily oxidized to form
a covalently linked dimeric amino acid known as cystine.
• In cystine, the two cysteines are joined by a disulfide bond.
• The disulfide-linked cystine residue is strongly hydrophobic.
• In proteins, disulfide bonds form covalent links between different
parts of a polypeptide chain, or between two different polypeptide
chains.
• Overall, the side chains of the aromatic amino acids
phenylalanine, tyrosine, and tryptophan are very
hydrophobic.

• The R group of tyrosine also contains a polar hydroxyl

group that can participate in H-bonding interactions.

• The R groups of tyrosine, and particularly tryptophan,

absorb ultraviolet light at a maximum wavelength of
280 nm.

• Light absorption by these amino acids is exploited in

detecting and quantifying proteins in the laboratory
using the technique of absorbance spectrometry.
 Positively Charged Amino Acids

The most hydrophilic R groups are those that are either positively or negatively charged.
The side-chains of lysine and arginine are fully positively charged at neutral pH.
In lysine, a primary amino group is attached to the  carbon of the side-chain.
In arginine, the guanidinium group of the side-chain is positively charged.
The histidine R group contains an aromatic imidazole group that is partially positively charged at
neutral pH
Histidine residues function in many enzyme-catalyzed reactions as proton donors and/or acceptors.
 Negatively Charged Amino Acids
The R groups of aspartate and glutamate contain carboxyl groups that are fully
negatively charged at neutral pH (pKRs of 3.65 and 4.25, respectively).
In aspartate, the carboxyl group is attached to the ß carbon of the amino acid
backbone.
In glutamate, the carboxyl group is attached to the  carbon.
Protein Structure
1. hydrophobic residues are largely buried in the protein interior,
away from water.
2. the number of hydrogen bonds and ionic interactions within the
protein is maximized, thus reducing the number of hydrogen-
bonding and ionic groups that are not paired with a suitable
partner.
3. The Peptide Bond Is Rigid and Planar.
• The term secondary structure refers to any chosen segment of a
polypeptide chain and describes the local spatial arrangement of its
main-chain atoms without regard to the positioning of its side chains or its
relationship to other segments.

• Regular secondary structure occurs when each dihedral angle remains the
same or nearly the same throughout the segment. There are a few types of
secondary structure that are particularly stable and occur widely in proteins.
The most prominent are the Alpha helix and Beta conformations.

• Where a regular pattern is not found, the secondary structure is sometimes

referred to as undefined or as a random coil.
 Right-handed alpha helices are the most common secondary structure
in protein

• The three-dimensional structure of myoglobin and other

proteins showed that the right-handed alpha helix is the
common form. Extended left-handed helices are theoretically
less stable and have not been observed in proteins.

• Generally, about one-fourth of all amino acid residues in

proteins are found in helices.
Why does the alpha helix form more readily than many other
possible conformations?
The answer lies in part in its optimal use of internal hydrogen bonds. The
structure is stabilized by a hydrogen bond between the hydrogen atom attached
to the electronegative nitrogen atom of a peptide linkage and the
electronegative carbonyl oxygen atom of the fourth amino acid on the amino-
terminal side of that peptide bond.
Every peptide bond (except those close to each end of the helix) participates in
such hydrogen bonding within the helix. Three to four hydrogen bonds hold each
successive turn of the helix to adjacent turns, conferring significant stability on
the overall structure.
• At the ends of an alpha-helical segment, three or four amide
carbonyl or amino groups cannot participate in this helical pattern of
hydrogen bonding. These may be exposed to the surrounding
solvent, where they hydrogen bond with water, or other parts of the
protein may cap the helix to provide the needed hydrogen bonding
partners.
• Alpha helix can form in polypeptides consisting of either L- or D-
amino acids. However, all residues must be of one stereoisomeric
series; a D-amino acid will disrupt a regular structure consisting of
L-amino acids and vice versa. The most stable form of an alpha
helix consisting of D-amino acids is left-handed.
• Each amino acid residue in a polypeptide has an intrinsic
propensity to form an alpha helix.
• Alanine shows the greatest tendency to form alpha helices
in most experimental model systems.
• The position of an amino acid residue relative to its
neighbors is also important.
• Interactions between amino acid side chains can stabilize or
destabilize the alpha-helical structure.
The position of an amino acid residue relative to its
neighbors is also important.

• For example, if a polypeptide chain has a long block of Glu

residues, this segment of the chain will not form a helix at pH 7.0.
The negatively charged carboxyl groups of adjacent Glu
residues repel each other so strongly that they prevent Alpha
helix formation at pH 7. For the same reason, if there are many
adjacent Lys and/or Arg residues with positively charged R
groups at pH 7.0, they also repel each other and prevent alpha
helix formation.

• The bulk and shape of Asn, Ser, Thr, and Cys residues can also
destabilize an alpha helix if they are close together in the chain.
 Beta conformation

• Discovered by Pauling and Corey

In 1951.
• More extended conformation.
• Structure is again defined by
set of dihedral angles
In the beta conformation, the backbone of the
polypeptide chain is extended into a zigzag rather than
helical structure.
A beta-sheet is the arrangement of several segments side by
side, all of which are in the beta conformation.

The zigzag structure of the individual polypeptide segments

gives rise to a pleated appearance of the overall sheet.

The R groups of adjacent amino acids protrude from the

zigzag structure in opposite directions, creating the
alternating pattern seen in the side view.
The anti-parallel beta-sheet is more extended than parallel beta-
sheet
The parallel beta-sheet is more compacted than anti parallel
beta-sheet
• Hydrogen bonds form between adjacent segments of the polypeptide
chain within the sheet.
• The individual segments that form a beta-sheet are usually nearby on
the polypeptide chain but can also be quite distant from each other in
the linear sequence of the polypeptide
Collagen
• Ribbon model: Easy to see helix, strand, overall globular structure,
domain.
• Ball and Stick: Easy to see the connection between atoms.
• Space filled: Show the Vander wall radius
• Collagen can have many types.
• Typically, they contain about 35 percent glycine, 11 % alanine, 21 percent
Proline, and 4-hydroxyproline.
• The food product gelatin is derived from collagen.
• As collagen is extremely low in many essential amino acids, it has significantly
less nutritious value.
• The unusual amino acid content of collagen is related to structural constraints
unique to the collagen helix.
• Collagen's amino acid sequence is generally a repeating
tripeptide unit, Gly–X—Y, where X is often Pro and Y is
often 4-Hyp.
• Only Gly residues can be accommodated at the very tight junctions
between the individual alpha chains.
• The Pro and 4-Hyp residues permit the sharp twisting of the collagen
helix.
• Glycine, because of its small size, is required at the tight junction
where the three chains are in contact.
• The tight wrapping of the chains in the collagen triple helix provides tensile strength greater
than that of a steel wire of equal cross-section.
• Collagen fibrils are supramolecular assemblies consisting of triple-helical collagen
molecules.
• Associated in a variety of ways to provide different degrees of tensile strength.
• The chains of collagen molecules and the collagen molecules of fibrils are cross-linked by
unusual types of covalent bonds involving Lys, HyLys (5-hydroxylysine), or His residues
that are present at a few of the X and Y positions.
• These links create uncommon amino acid residues such as dehydrohydroxylysinonorleucine.
• The increasingly rigid and brittle character of aging connective tissue results from
accumulated covalent crosslinks in collagen fibrils.
• Vitamin C is required for, among other things, the hydroxylation of proline and lysine in
collagen.
Enzyme
 What are enzymes?
• Enzymes are catalysts
• Increase reaction rates without being used up
 History of Enzymes
• Louis Pasteur concluded that fermentation of sugar into alcohol by
yeast is catalyzed by “ferments.” He postulated that these
ferments were inseparable from the structure of living yeast cells;
this view, called vitalism, prevailed for decades.
• Then in 1897 Eduard Buchner discovered that yeast extracts
could ferment sugar to alcohol, proving that fermentation was
promoted by molecules that continued to function when removed
from cells.
• Frederick W. Kühne later gave the name enzymes (from the
Greek enzymos, “leavened”) to the molecules detected by
Buchner.
• The isolation and crystallization of urease by James Sumner in 1926
was a breakthrough in early enzyme studies. Sumner found that
urease crystals consisted entirely of protein, and he postulated
that all enzymes are proteins.
 Intro. to Enzymes
All living organisms must be able to self-replicate and catalyze
chemical reactions efficiently and selectively. Enzymes (from
the Greek enzymos, “leavened”) are the chemical catalysts of
biological systems. Enzymes have extraordinary catalytic
power, often far greater than that of synthetic or inorganic
catalysts. They have a high degree of specificity for their
substrates and they accelerate chemical reactions
tremendously. They function in aqueous solutions under very
mild conditions of temperature and pH, unlike many catalysts
used in organic chemistry. Enzymes are central to every
biochemical process. They catalyze the hundreds of stepwise
reactions of metabolism, conserve and transform chemical
energy, and make biological macromolecules from simple
precursors. In many diseases, the activity of one or more
enzymes is abnormal. Many drugs act via binding to enzymes.
 Cofactors: Inorganic ions/complex
organic/metalloorganic

With the exception of a small group of

catalytically active RNA molecules, all enzymes
are proteins. Their catalytic activity depends on
the integrity of their native protein
conformation. Some enzymes require no
chemical groups for activity other than their
amino acid residues. Others require an
additional chemical component called a cofactor.
Cofactors can be 1.inorganic ions, or 2. complex
organic, or 3. metalloorganic molecules called
Many enzymes have been named by adding the suffix “-ase” to
the name of their substrate or a word or phrase describing
their activity.
Biochemists by international agreement have adopted a
system for naming and classifying enzymes based on the type
of reaction catalyzed.
Each enzyme is assigned a four-part classification number and
a systematic name, which identifies the reaction it catalyzes.
For example, the enzyme known commonly as hexokinase is
formally ATP: glucose phosphotransferase.
Its Enzyme Commission number is 2.7.1.1, in which the first
number (2) denotes the class name (transferase); the second
number (7), denotes the subclass (phosphotransferase); the
third number (1), a phosphotransferase with a hydroxyl group
as acceptor; and the fourth number (1), D-glucose as the
phosphoryl group acceptor.
 ACTIVE SITE

Under biologically relevant conditions, uncatalyzed reactions

tend to be slow because most biological molecules are quite
stable in the neutral-pH, mild-temperature, aqueous
environment inside cells.
Enzymes greatly increase the rates of biological reactions by
providing a specific environment within which a reaction can
occur more rapidly.
Enzyme-catalyzed reactions take place within the confines of a
pocket on the enzyme called the active site.
The reactant molecule is referred to as the substrate.
The surface of the active site is lined with amino acid residues
with substituent groups that bind to the substrate and catalyze
its chemical transformation. Often, the active site encloses the
substrate, sequestering it from the solution.
Enzyme-Substrate
Complex
• The active site is a three-dimensional cleft, or crevice.
• The active site takes up a small part of the total volume of an enzyme.
• Active sites are unique microenvironments.
• Substrates are bound to enzymes by multiple weak attractions.
• The specificity of binding depends on the precisely defined
arrangement of atoms in an active site.
Enzymes Decrease activation energy ΔG‡
How to Lower Activation energy

Enzymes organize reactive groups into close

proximity and proper orientation
Rate Enhancement by Enzymes
 What is Induced Fit
The binding of a protein and ligand is often coupled to a
conformational change in the protein that makes the binding site
more complementary to the ligand, permitting tighter binding. The
structural adaptation that occurs between protein and ligand is called
induced fit.
 The Binding Energy Between
Enzyme and Substrate is important
for catalysis
• Free energy is released by the formation of a large number of
weak interactions between a complementary enzyme and its
substrate.
• The free energy released on binding is called the binding
energy.
• The energy released by the interactions between the enzyme
and the substrate can be thought of as lowering the activation
energy.
 Definitions
• Substrate: Species that is acted upon by enzyme
• Prosthetic group: Coenzyme or cofactor tightly
(permanently) bound to enzyme
• Holoenzyme: Enzyme + prosthetic group
• Apoenzyme (apoprotein): Enzyme – prosthetic group
• Active Site: Pocket within enzyme in which reaction
occurs
• Ground State: Starting point for forward/backward
reactions; lowest ENERGY
• Transition State: Top of energy “hill”; forward and reverse
reactions are equally likely
• Nicotinamide adenine dinucleotide (NAD+) is a coenzyme for redox
reactions such as glycolysis, β-oxidation, and oxidative phosphorylation.
• It stores energy in its reduced form (NADH), which can power ATP
production.
• Nicotinamide adenine dinucleotides, NAD and NADP, are indispensable
cofactors involved in several redox reactions in all forms of cellular life.
• Conrad Elvehjem discovered that the disease pellagra (characterized by
dermatitis, diarrhea, and dementia) is caused by a dietary deficiency of
niacin and results in low NAD+ and NADP+ levels.
• It can also have non-redox functions.
• DNA repair, chromatin silencing, transcriptional regulation, metabolic
switching, calcium mobilization, and lifespan regulation. Rather than
being based on redox events, these actions are based on NAD+
consumption.
 Nicotinamide

Niacinamide or nicotinamide is a form of vitamin B3.

 Enzyme-Substrate Complex
• Enzymes act by binding substrates
• The noncovalent enzyme substrate complex is known as the
Michaelis complex
• Description of chemical interactions
• Development of kinetic equations

kcat [ E ][ S ] Vmax [ S ]
v v
Km  S
K m  [S ]
• The enzyme-substrate complex, whose existence was
first proposed by Charles-Adolphe Wurtz in 1880, and
extended by JBS Haldane is central to the action of
enzymes.

• It is also the starting point for mathematical treatments

defining the kinetic behavior of enzyme-catalyzed
reactions and theoretical descriptions of enzyme
mechanisms.
Enzyme-Substrate
Complex
Enzymes Decrease activation energy ΔG‡
 Some basic about kinetics
• Overall rate of reaction is determined by the RATE LIMITING STEP
(slowest step)
• G° involves equilibrium, but.
• Activation energy involves reaction rate.
• Rate equation (rate law):
– 1st order rate: (V) = k[S], where V and S are velocity and substrate concentration,
respectively
– 2nd order rate: V = k[S1][S2]
What is enzyme kinetics?
• Kinetics is the study of the rate at which compounds
react
• Rate of enzymatic reaction is affected by:
• enzyme
• substrate
• effectors
• temperature
Why study enzyme
kinetics?
• Quantitative description of biocatalysis
• Determine the order of binding of substrates
• Elucidate acid-base catalysis
• Understand catalytic mechanism
• Find effective inhibitors
• Understand regulation of activity
 How to Do Kinetic Measurements
Experiment:
1)Mix enzyme + substrate
2)Record rate of substrate disappearance/product formation as a function of time (the
velocity of reaction)
3)Plot initial velocity versus substrate concentration.
4)Change substrate concentration and repeat
Effect of Substrate Concentration
 Vmax [ S ]
v
Km  S
Effect of Substrate
Concentration
• Ideal
Vmax [ S ]
rate: v
Km  S

• Deviations due to:

– limitation of measurements
– substrate inhibition
– substrate prep contains inhibitors
– enzyme prep contains inhibitors
Initial rate / initial velocity designated V0, rectangular
hyperbola, Steady State Kinetics, Pre-Steady State.
Effect of Substrate Concentration
Effect of Substrate Concentration
V = Vmax [S] /Km+[S]

½ Vmax= Vmax [S]/Km +[S]

½= [S]/km+[S]

Km +[S]= 2[S]
Km= S
However, studying the effects of substrate concentration is complicated by the fact that
[S] changes during an in vitro reaction as the substrate is converted to the product.
Effect of Substrate Concentration
 Vmax [ S ]
v
Km  S
The reaction quickly achieves a steady state in which [ES] (and
the concentrations of any other intermediates) remain
approximately constant over time. The concept of a steady state
was introduced by G. E. Briggs and Haldane in 1925

The measured V0 generally reflects the steady state, even though

V0 is limited to the early part of the reaction. Analysis of these
initial rates is referred to as steady-state kinetics.
Because Vmax is approached asymptotically (see Figure 8.11), it is impossible to obtain a
definitive value from a Michaelis–Menten curve. Because KM is the concentration of
substrate at Vmax/2, it is likewise impossible
to determine an accurate value of KM. However, Vmax can be accurately determined if
the Michaelis–Menten equation is transformed into one that gives a straight-line plot.
The constant
kcat
is a first-order rate constant and
hence has units of reciprocal time.
When k2 is rate-limiting, k2 k1 and Km reduces to
k1/k1, which is defined as the dissociation constant,
Kd, of the ES complex.
It is equivalent to the number of substrate molecules converted
to product in a given unit of time on a single enzyme molecule
when the the enzyme is saturated with substrate.

Enzyme activity is measured in units which indicate the rate of

reaction catalysed by that enzyme expressed as micromoles of
substrate transformed (or product formed) per minute. An
enzyme unit is the amount of enzyme that will catalyse the
transformation of 1 μmol of substrate/min under specified
conditions of pH and temperature. The specific activity of an
enzyme is expressed as the number of units per milligram of
protein.
Enzyme-Substrate
Complex
• A competitive inhibitor competes with the substrate for the active site
of an enzyme. While the inhibitor (I) occupies the active site, it
prevents binding of the substrate to the enzyme. Many competitive
inhibitors are structurally similar to the substrate and combine with
the enzyme to form an EI complex, but without leading to catalysis.
• Nine amino acids—histidine, isoleucine, leucine, lysine, methionine,
phenylalanine, threonine, tryptophan, and valine—are not
synthesized by mammals and are therefore dietarily essential or
indispensable nutrients. These are commonly called the essential
amino acids.
• Beta-alanine is a non-proteogenic amino acid that is produced
endogenously in the liver. In addition, humans acquire beta-alanine
through the consumption of foods such as poultry and meat. By itself,
the ergogenic properties of beta-alanine are limited; however, beta-
alanine has been identified as the rate-limiting precursor to carnosine
synthesis [1, 2], and has been consistently shown to increase levels of
carnosine in human skeletal muscle
• Four weeks of beta-alanine supplementation (4–6 g daily)
significantly augments muscle carnosine concentrations, thereby
acting as an intracellular pH buffer; 2) Beta-alanine supplementation
currently appears to be safe in healthy populations at recommended
doses; 3) The only reported side effect is paraesthesia (tingling), but
studies indicate this can be attenuated by using divided lower doses
(1.6 g) or using a sustained-release formula; 4)
• d-Alanine (d-Ala) is an unusual endogenous amino acid present in
invertebrates and vertebrates.
• we focus on d-alanine (d-Ala), a lesser studied d-AA (compared to d-
Ser and d-Asp) in animals
• d-Ala is an enigmatic AA, with much of the information on its source,
function, and impact on disease still unknown. This is somewhat
surprising given the early discovery of the importance of d-Ala in the
formation of bacterial cell walls.
• Selenocysteine, also known as the 21st amino acid, is unique among
the proteinogenic amino acids. It is the only amino acid containing an
essential dietary micronutrient (selenium) as a constitutive
component, the only amino acid encoded by a UGA codon and the
only one synthesized on its tRNA in all domains of life.
• Selenocysteine, the 21st amino acid, has been found in 25 human
selenoproteins and selenoenzymes important for fundamental
cellular processes ranging from selenium homeostasis maintenance to
the regulation of the overall metabolic rate
• Hydroxyproline is a major component of the protein collagen,
comprising roughly 13.5% of mammalian collagen. Hydroxyproline
and proline play key roles for collagen stability. They permit the sharp
twisting of the collagen helix
• The 4-hydroxylation of proline residues on collagen enhance the
stability of the triple helix and influence on collagen fibril formation.
Proline hydroxylation may regulate the flexibility of collagen
molecules and make functional sites available for interacting proteins
and receptors
• In the absence of vitamin C, cells cannot hydroxylate the Pro at the Y
positions. This leads to collagen instability and the connective tissue
problems seen in scurvy.
• prolyl 4-hydroxylase
• Glutathione is a substance made from the amino acids glycine,
cysteine, and glutamic acid. It is produced by the liver and involved in
many body processes. Glutathione is involved in tissue building and
repair, making chemicals and proteins needed in the body, and in
immune system function.
• 5 millimolar concentration
• Glutathione exists in cells in 2 states: reduced (GSH) and oxidized
(GSSG)
• Aspartame is an artificial sweetener has been in use in the United
States since the early 1980s. It is used in many foods and beverages
because it is much sweeter than sugar, so much less of it can be used
to give the same level of sweetness
• Aspartame is a methyl ester of a dipeptide consisting of two amino
acids, aspartic acid, and phenylalanine
• The normal function of myoglobin is to bind oxygen reversibly and
facilitate oxygen diffusion from blood capillaries to mitochondria in
the muscle. It also appears that another main function of myoglobin is
to scavenge nitric oxide, preventing inhibition of the mitochondrial
enzyme cytochrome c oxidase.
• Myoglobin is a low-molecular weight protein of 16,000 Da that
contains one heme and binds one molecule of O2 per molecule of
protein. Tissue content of myoglobin depends on the tissue and the
species. Highly oxidative muscle fibers contain a lot of myoglobin.
Because it consists of a single polypeptide chain, myoglobin does not
have subunits that can interact to produce cooperative binding.
• To do this, it uses a special chemical tool to capture slippery oxygen
molecules: a heme group. Heme is a disk-shaped molecule that has a
hole in the center that is perfect for holding an iron ion. The iron then
forms a strong interaction with the oxygen molecule
 Hemoglobin Structure (I)
Hb (Mr 64,500) is a tetrameric protein that is roughly
spherical and has a diameter of about 5.5 nm. Each
subunit contains a bound heme group that has the same
structure as the heme of myoglobin. There are two types
of chains in adult Hb, two  chains (141 residues each),
and two ß chains (146 residues each). Although fewer
than half of the amino acid residues in the two chains are
identical, the structures of the  and ß chains are nearly
similar to that of Furthermore their structures are very
superimposable.
myoglobin (Fig. 5-6), which
has a more dissimilar
amino acid sequence. Note
that the helix naming
conventions are the same
for the Mb and Hb chains,
except that the  subunit
lacks the short D helix.
Like Mb, the heme-binding
pocket is made up of the E
and F helices in the  and
 Hemoglobin Structure (II)
Despite the fact that the amino acid
sequences of the three polypeptides
are identical at only 27 positions
(Fig. 5-7), the structures of the
myoglobin and Hb chains are nearly
identical. This illustrates how similar
amino acids can form similar
secondary and tertiary structures.
 Hemoglobin Structure (III)
Strong interactions involving more than 30 residues occur
within the 1ß1 and 2ß2 protomers of the Hb tetramer (Fig.
5-8). Fewer interactions involving 19 amino acids connect
the 1-ß2 and 2-ß1 contact interfaces. Hydrophobic
interactions predominate at all interfaces, but there are
also hydrogen bonds and a few ion pairs that provide
connections between subunits. When O2 binds to Hb,
contacts within the protomers change little. However,
there are large changes between the 1-ß2 and 2-ß1
interfaces, with several ion pairs being broken.
Insulin is composed of two peptide chains referred to as the A chain and B chain. A and B chains are linked
together by two disulfide bonds, and an additional disulfide is formed within the A chain. In most species, the A
chain consists of 21 amino acids and the B chain of 30 amino acids.

Although the amino acid sequence of insulin varies among species, certain segments of the molecule are highly
conserved, including the positions of the three disulfide bonds, both ends of the A chain and the C-terminal
residues of the B chain. These similarities in the amino acid sequence of insulin lead to a three dimensional
conformation of insulin that is very similar among species, and insulin from one animal is very likely biologically
active in other species. Indeed, pig insulin has been widely used to treat human patients.

Insulin molecules have a tendency to form dimers in solution due to hydrogen-bonding between the C-termini of B
chains. Additionally, in the presence of zinc ions, insulin dimers associate into hexamers.
• This enzyme catalyzes the first unique step in glycolysis, converting
fructose-6-phosphate to fructose-1,6-bisphosphate. This step is
catalyzed by phosphofructokinase 1 (PFK1). The second isoform,
phosphofructokinase 2 (PFK2) catalyzes the conversion of fructose-6-
phosphate to fructose-2,6-bisphosphate.
• PFK-2 or phosphofructokinase-2 is the enzyme that catalyzes the
synthesis of fructose 2,6-bisphosphate from fructose 6-phosphate.
Similar to PFK-1, PFK-2 acts on the same substrate. However, unlike
PFK-1, PFK-2 activity is not affected by ATP concentration.
Phosphoenolpyruvate and citrate can inhibit this enzyme, while
inorganic orthophosphate can stimulate the action of PFK-2.
Structurally, PFK-2 exists with fructose-2,6-bisphosphatase as a
bifunctional enzyme abbreviated as PFK-2/FBPase-2. PFK-2 phosphorylates
fructose 6-phosphate using ATP. On the other hand, FBPase-2
dephosphorylates fructose 2,6-bisphosphate to produce fructose 6-
phosphate and Pi. Hence, PFK-2 has both kinase and phosphatase
activities. When glucose level is high, insulin increases the kinase activity
of PFK-2 enzyme to drive the increased synthesis of fructose 2,6-
bisphosphate. It stimulates glycolysis due to the activation of PFK-1 by
fructose 2,6-bisphosphate. In contrast, when phosphatase activity of PFK-2
is expressed, it breaks fructose 2,6-bisphosphate back into fructose 6-
phosphate, stimulating gluconeogenesis and inhibiting glycolysis
Pyruvate Dehydrogenase Complex
This multienzyme complex pyruvate and coenzyme A (CoA) conversion to acetyl CoA.
There are four stages in this pathway, which are catalyzed by three enzymes:

"E1" - pyruvate dehydrogenase

This enzyme catalyzes the decarboxylation of pyruvate. This involves the prosthetic group thiamine
pyrophosphate or TPP.

"E2" - dihydrolipoyl transacetylase

Two steps of the pathway are catalyzed by this enzyme:

ओxidation of the 2-carbon (acetyl) unit, which is transferred to the lipoamide prosthetic group of the enzyme,
giving an acetyllipoamide group

transfer of the acetyl group from the lipoamide to CoA, giving acetyl CoA

"E3" - dihydrolipoyl dehydrogenase

Finally, this enzyme regenerates the oxidized form of lipoamide. This involves the FAD prosthetic group.

Note that TPP, lipoamide and FAD are catalytic cofactors which remain unaltered by the net reaction, whereas
CoA and NAD+ are stoichiometric cofactors; the overall reaction is:

pyruvate + CoA + NAD+ ----> acetyl CoA + carbon dioxide + NADH

Lactate dehydrogenase isoenzymes (LDH) were used widely in the past for
diagnosis of myocardial infarction, but more recently, due to availability of
troponin immunoassays, lactate dehydrogenase isoenzyme assay has been
mostly discontinued in the clinical setting for diagnosis of myocardial
infarction.
• LDH-1: Present primarily in cardiac myocytes and erythrocytes.

• LDH-2: Present mostly in white blood cells.

• LDH-3: Present in the highest quantity in lung tissue.

• LDH-4: Highest amounts found in pancreas, kidney, and placenta.

• LDH-5: Highest amounts found in liver and skeletal muscle.

• Isozymes are variations of the same enzyme that come from different
genes. Isozymes have different amino acid sequences and therefore
different shapes, sizes, and electrical charges, but the same function.
The hammerhead ribozyme is a small catalytic RNA motif capable of
endonucleolytic (self-) cleavage. It is composed of a catalytic core of
conserved nucleotides flanked by three helices, two of which form
essential tertiary interactions for fast self-scission under physiological
conditions