Principles of Biochemistry

Foundations of Biochemistry

Learning goals:
• Distinguishing features of living organisms
• Structure and function of the parts of the cell
• Roles of small and large biomolecules
• Energy transformation in living organisms
• Regulation of metabolism and catalysis
• Coding of genetic information in DNA
 Biochemistry is
the Chemistry of Living Matter
Living Matter is characterized by:

• High degree of complexity and organization

• Extraction, transformation, and systematic use of
energy to create and maintain structures and to do
work
• Interactions of individual components are dynamic
and coordinated
• Ability to sense and respond to changes in
surrounding
• A capacity for fairly precise self-replication while
allowing enough change for evolution
Living organisms must intake nutrients
Living organisms must
accurately reproduce
 Cell: The Universal Building Block

• Living organisms are made of cells

• Simplest living organisms are single-celled

• Larger organisms consist of many cells with different

functions

• Not all of the cells are the same

All cells share some common features
Three Distinct Domains of Life Defined by:
Cellular and Molecular Differences
 Six Kingdoms of Life Defined by:
Organism, Cellular, and Molecular Differences

Six kingdoms Cellular organization

• Archaea Unicellular prokaryote
• Bacteria Unicellular prokaryote
• Protista Unicellular eukaryote
• Fungi Uni- or Multicellular eukaryote
• Plantae Multicellular eukaryote
• Animalia Multicellular eukaryote
Bacterial Cell Structure
 Components of Bacterial Cell

Structure Composition Function

Cell wall Peptidoglycan Mechanical support
Cell membrane Lipid + protein Permeability barrier
Nucleoid DNA + protein Genetic information
Ribosomes RNA + protein Protein synthesis
Pili Protein Adhesion, conjugation
Flagella Protein Motility
Cytoplasm Aqueous solution Site of metabolism
 Eukaryote Cells: More Complexity
• Have nucleus by definition
– protection for DNA; site of DNA metabolism
– selective import and export via nuclear membrane pores
– some cells become anuclear (red blood cells)
• Have membrane-enclosed organelles
– Mitochondria for energy in animals, plants, and fungi
– Chloroplasts for energy in plant
– Lysosome for digestion of un-needed molecules
• Spatial separation of energy-yielding and energy-
consuming reactions helps cells to maintain
homeostasis and stay away from equilibrium
Bacterial, animal, and plant cells
are different
Bacterial, animal, and plant cells
are different
 Cytoplasm and Cytoskeleton
• Cytoplasm is highly viscous solution where many
reactions take place

• Cytoskeleton consists of microtubules, actin

filaments, and intermediate filaments
– Cell shape
– Intracellular organization
– Intracellular transport paths
– Cellular movement
 Biochemistry is
the Chemistry of Living Matter
• The basis of all life is the chemical reactions that take place
within the cell.

Chemistry allows for:

• A high degree of complexity and organization
• Extraction, transformation, and systematic use of energy to
create and maintain structures and to do work
• The interactions of individual components to be dynamic and
coordinated.
• The ability to sense and respond to changes in surrounding
• A capacity for fairly precise self-replication while allowing
enough change for evolution
 The Molecular Logic of Life

We look at the chemistry that is behind:

• Accelerating reactions
• Organization of metabolism and signaling
• Storage and transfer of information
 30 Elements Essential for Life
• Other than carbon, elements H, O, N, P, S are also common
• Metal ions (e.g., K+, Na+, Ca++, Mg++, Zn++, Fe++) play important roles
in metabolism
The Molecular Hierarchy of Structure
 Interactions between
biomolecules are specific

• Macromolecules have unique binding pockets

• Only certain molecules fit in well and can bind

• Binding of chiral biomolecules is stereospecific

 Interactions between
biomolecules are specific
ATP: Chemical Currency of Energy
 How to speed reactions up
Higher temperatures
Stability of macromolecules is limiting

Higher concentration of reactants

Costly as more valuable starting material is needed

Change the reaction by coupling to a fast one

Universally used by living organisms

Lower activation barrier by catalysis

Universally used by living organisms
 Catalysis

• A catalyst is a compound that increases the rate of a

chemical reaction
• Catalysts lower the activation free energy G
‡

• Catalysts does not alter G°

• Enzymatic catalysis offers:
– acceleration under mild conditions
– high specificity
– possibility for regulation
Enzymes lower the activation energy to
increase the reaction rate
Series of related enzymatically catalyzed
reactions forms a pathway

Metabolic Pathway
• produces energy or valuable materials

Signal Transduction Pathway

• transmits information
Pathways are controlled in order to
regulate levels of metabolites

Example of a negative regulation:

Product of enzyme 5 inhibits enzyme 1
Genetic and Evolutionary Foundations

• Life on Earth arose 3.5–3.8 billion years ago

• Formation of self-replicating molecules a key step
• Could it been DNA?
• Could it been proteins?
The Central “Dogma” of Biochemistry:
DNA → RNA → Protein
 Natural selection
favors some mutations

• Mutations occur more or less randomly

• Mutations that give organisms an advantage in a
given environment are more likely to be propagated
 Summary

• To understand what defines living organisms

• To relate structure and function of the cell
• To realize that the structure of biomolecules
often gives them specific functions
• To grasp principles of bioenergetics
 Amino Acids, Peptides, Proteins

Learning goals:
• Structure and naming of amino acids

• Structure and properties of peptides

• Ionization behavior of amino acids and peptides

• Methods to characterize peptides and proteins

 Proteins:
Main Agents of Biological Function
• Catalysis
– enolase (in the glycolytic pathway)
– DNA polymerase (in DNA replication)

• Transport
– hemoglobin (transports O2 in the blood)
– lactose permease (transports lactose across the cell membrane)

• Structure
– collagen (connective tissue)
– keratin (hair, nails, feathers, horns)

• Motion
– myosin (muscle tissue)
– actin (muscle tissue, cell motility)
 Amino Acids:
Building Blocks of Protein
• Proteins are linear heteropolymers of -amino acids

• Amino acids have properties that are well-suited to carry

out a variety of biological functions
– Capacity to polymerize
– Useful acid-base properties
– Varied physical properties
– Varied chemical functionality
Amino acids share many features,
differing only at the R substituent
 Most -amino acids are chiral
• The -carbon always has four substituents and is
tetrahedral
• All (except proline) have:
– an acidic carboxyl group
– a basic amino group
– an -hydrogen connected to the -carbon
• The fourth substituent (R) is unique
– In glycine, the fourth substituent is also hydrogen
 Amino Acids: Atom Naming
• Organic nomenclature: start from one end
• Biochemical designation:
– start from -carbon and go down the R-group
Amino Acids
a simple organic compound containing both a carboxyl
(—COOH) and an amino (—NH2) group
All amino acids are chiral (except glycine)
Proteins only contain L amino acids
 Amino Acids: Classification

Common amino acids can be placed in five

basic groups depending on their R substituents:
• Nonpolar, aliphatic (7)
• Aromatic (3)
• Polar, uncharged (5)
• Positively charged (3)
• Negatively charged (2)
These amino acid side chains absorb UV light at 270–280 nm
These amino acids side chains can form hydrogen bonds.
Cysteine can form disulfide bonds.
 Uncommon Amino Acids in Proteins

• Not incorporated by ribosomes

 except for Selenocysteine
• Arise by post-translational modifications of
proteins
• Reversible modifications, especially
phosphorylation, are important in regulation and
signaling
 Ionization of Amino Acids

• At acidic pH, the carboxyl group is protonated

and the amino acid is in the cationic form.
• At neutral pH, the carboxyl group is
deprotonated but the amino group is protonated.
The net charge is zero; such ions are called
Zwitterions.
• At alkaline pH, the amino group is neutral –NH2
and the amino acid is in the anionic form.
Cation  Zwitterion  Anion

E
D

B
A
Amino acids carry a net charge of zero
at a specific pH (the pI)

• Zwitterions predominate at pH values between the pKa

values of the amino and carboxyl groups
• For amino acids without ionizable side chains, the Isoelectric
Point (equivalence point, pI) is
pK1  pK 2
pI 
2

• At this point, the net charge is zero

– AA is least soluble in water
– AA does not migrate in electric field
 Formation of Peptides
• Peptides are small condensation products of amino acids
• They are “small” compared to proteins (Mw < 10 kDa)
 Peptide ends are not the same
Numbering (and naming) starts from the amino terminus
AA1 AA2 AA3 AA4 AA5
Peptides: A Variety of Functions
• Hormones and pheromones
– insulin (think sugar)
– oxytocin (think childbirth)
– sex-peptide (think fruit fly mating)
• Neuropeptides
– substance P (pain mediator)
• Antibiotics
– polymyxin B (for Gram – bacteria)
– bacitracin (for Gram + bacteria)
• Protection, e.g., toxins
– amanitin (mushrooms)
– conotoxin (cone snails)
– chlorotoxin (scorpions)
 Proteins are:
• Polypeptides (covalently linked -amino acids) + possibly:
• cofactors
 functional non-amino acid component
 metal ions or organic molecules
• coenzymes
 organic cofactors
 NAD+ in lactate dehydrogenase
• prosthetic groups
 covalently attached cofactors
 heme in myoglobin
• other modifications
 Hydrogen Bonds
• Strong dipole-dipole or charge-dipole interaction that arises
between an acid (proton donor) and a base (proton acceptor)
• Typically 4–6 kJ/mol for bonds with neutral atoms,
and 6–10 kJ/mol for bonds with one charged atom
• Typically involves two electronegative atoms (frequently
nitrogen and oxygen)
• Hydrogen bonds are strongest when the bonded molecules are
oriented to maximize electrostatic interaction
• Ideally the three atoms involved are in a line
What to Study about Peptides and
Proteins

What is its sequence and composition?

What is its three-dimensional structure?
How does it find its native fold?
How does it achieve its biochemical role?
How is its function regulated?
How does it interacts with other macromolecules?
How is it related to other proteins?
Where is it localized within the cell?
What are its physico-chemical properties?
A mixture of proteins can be separated
• Separation relies on differences in physical and
chemical properties
– Charge
– Size
– Affinity for a ligand
– Solubility
– Hydrophobicity
– Thermal stability
• Chromatography is commonly used for
preparative separation
Column Chromatography
Separation by Charge
Separation by Size
Separation by Affinity
 Electrophoresis for Protein Analysis

Separation in analytical scale is commonly done by

electrophoresis
– Electric field pulls proteins according to their charge
– Gel matrix hinders mobility of proteins according to their
size and shape
 SDS PAGE: Molecular Weight
• SDS – sodium dodecyl sulfate – a detergent

• SDS micelles bind to and unfold all the

proteins
– SDS gives all proteins an uniformly negative
charge
– The native shape of proteins does not matter
– Rate of movement will only depend on size: small
proteins will move faster
SDS-PAGE can be used to calculate the
molecular weight of a protein
Isoelectric focusing can be used to
determine the pI of a protein
Isoelectric focusing and SDS-PAGE are
combined in 2D electrophoresis
 Spectroscopic Detection of
Aromatic Amino Acids
• The aromatic amino acids absorb light in the UV region
• Proteins typically have UV absorbance maxima around
275–280 nm
• Tryptophan and tyrosine are the strongest
chromophores
• Concentration can be determined by UV-visible
spectrophotometry using Beers law: A = ·c·l
Specific activity (activity/total protein)
can be used to assess protein purity
 Protein Sequencing
• It is essential to further biochemical analysis that we know
the sequence of the protein we are studying
• Actual sequence generally determined from DNA sequence
• Edman Degradation (Classical method)
– Successive rounds of N-terminal modification, cleavage, and
identification
– Can be used to identify protein with known sequence
• Mass Spectrometry (Modern method)
– MALDI MS and ESI MS can precisely identify the mass of a
peptide, and thus the amino acid sequence
– Can be used to determine post-translational modifications
Edman’s Degradation
MS Procedures for Sequence IDs
 Protein Sequences as Clues to
Evolutionary Relationships
• Sequences of homologous proteins from a wide range
of species can be aligned and analyzed for differences

• Differences indicate evolutionary divergences

• Analysis of multiple protein families can indicate

evolutionary relationships between organisms,
ultimately the history of life on Earth
 Chapter : Summary

In this chapter, we learned about:

• The many biological functions of peptides and proteins

• The structures and names of amino acids found in
proteins
• The ionization properties of amino acids and peptides
• The methods for separation and analysis of proteins
 Proteins: Structure, Function, Folding

Learning goals:
– Structure and properties of the peptide bond
– Structural hierarchy in proteins
– Structure and function of fibrous proteins
– Structure analysis of globular proteins
– Protein folding and denaturation
 Structure of Proteins
• Unlike most organic polymers, protein molecules
adopt a specific three-dimensional conformation.
• This structure is able to fulfill a specific biological
function
• This structure is called the native fold
• The native fold has a large number of favorable
interactions within the protein
• There is a cost in conformational entropy of folding
the protein into one specific native fold
 Favorable Interactions in Proteins
• Hydrophobic effect
– Release of water molecules from the structured solvation layer around
the molecule as protein folds increases the net entropy
• Hydrogen bonds
– Interaction of N-H and C=O of the peptide bond leads to local regular
structures such as -helices and -sheets
• London dispersion
– Medium-range weak attraction between all atoms contributes
significantly to the stability in the interior of the protein
• Electrostatic interactions
– Long-range strong interactions between permanently charged groups
– Salt-bridges, esp. buried in the hydrophobic environment strongly
stabilize the protein
4 Levels of Protein Structure
 Structure of the Peptide Bond
• Structure of the protein is partially dictated by
the properties of the peptide bond
• The peptide bond is a resonance hybrid of two
canonical structures
• The resonance causes the peptide bonds
– to be less reactive compared to esters, for
example
– to be quite rigid and nearly planar
– to exhibit a large dipole moment in the
favored trans configuration
Resonance in the Peptide Bond
 The Rigid Peptide Plane and
the Partially Free Rotations
• Rotation around the peptide bond is not permitted
• Rotation around bonds connected to the alpha carbon
is permitted
• f (phi): angle around the -carbon—amide nitrogen
bond
• y (psi): angle around the -carbon—carbonyl carbon
bond
• In a fully extended polypeptide, both y and f are
180°
The polypeptide is made up of a series of
planes linked at α carbons
Distribution of f and y Dihedral Angles
• Some f and y combinations are very unfavorable because of
steric crowding of backbone atoms with other atoms in the
backbone or side chains

• Some f and y combinations are more favorable because of

chance to form favorable H-bonding interactions along the
backbone

• A Ramachandran plot shows the distribution of f and y

dihedral angles that are found in a protein

• shows the common secondary structure elements

• reveals regions with unusual backbone structure

 Secondary Structures
• Secondary structure refers to a local spatial
arrangement of the polypeptide backbone
• Two regular arrangements are common:
• The  helix
– stabilized by hydrogen bonds between nearby residues
• The  sheet
– stabilized by hydrogen bonds between adjacent
segments that may not be nearby
• Irregular arrangement of the polypeptide chain is
called the random coil
 The  Helix
• Helical backbone is held together by hydrogen
bonds between the backbone amides of an n and
n+4 amino acids
• Right-handed helix with 3.6 residues (5.4 Å) per turn
• Peptide bonds are aligned roughly parallel with the
helical axis
• Side chains point out and are roughly perpendicular
with the helical axis
What is a right-handed helix?
 The  Helix: Top View
• The inner diameter of the helix (no side chains) is
about 4–5 Å
• Too small for anything to fit “inside”
• The outer diameter of the helix (with side chains) is
10–12 Å
• Happens to fit well into the major groove of dsDNA
• Residues 1 and 8 align nicely on top of each other
• What kind of sequence gives an  helix with one
hydrophobic face?
 Sequence affects helix stability
• Not all polypeptide sequences adopt -helical structures
• Small hydrophobic residues such as Ala and Leu are
strong helix formers
• Pro acts as a helix breaker because the rotation around
the N-Ca bond is impossible
• Gly acts as a helix breaker because the tiny R-group
supports other conformations
• Attractive or repulsive interactions between side chains
3–4 amino acids apart will affect formation
 The Helix Dipole
• Recall that the peptide bond has a strong dipole
moment
– Carbonyl O negative
– Amide H positive
• All peptide bonds in the  helix have a similar
orientation
• The  helix has a large macroscopic dipole moment
• Negatively charged residues often occur near the
positive end of the helix dipole
  Sheets
• The planarity of the peptide bond and tetrahedral
geometry of the -carbon create a pleated sheet-like
structure
• Sheet-like arrangement of backbone is held together by
hydrogen bonds between the backbone amides in
different strands
• Side chains protrude from the sheet alternating in up
and down direction
 Parallel and Antiparallel  Sheets
• Parallel or antiparallel orientation of two chains
within a sheet are possible
• In parallel  sheets the H-bonded strands run in
the same direction
– Resulting in bent H-bonds (weaker)
• In antiparallel  sheets the H-bonded strands run
in opposite directions
– Resulting in linear H-bonds (stronger)
  Turns
•  turns occur frequently whenever strands in  sheets
change the direction
• The 180° turn is accomplished over four amino acids
• The turn is stabilized by a hydrogen bond from a
carbonyl oxygen to amide proton three residues down
the sequence
• Proline in position 2 or glycine in position 3 are
common in  turns
 Proline Isomers
• Most peptide bonds not involving proline are in the trans
configuration (>99.95%)
• For peptide bonds involving proline, about 6% are in the
cis configuration. Most of this 6% involve β-turns
• Proline isomerization is catalyzed by proline isomerases
 Circular Dichroism (CD) Analysis
• CD measures the molar absorption difference  of left-
and right-circularly polarized light:  = L – R
• Chromophores in the chiral environment produce
characteristic signals
• CD signals from peptide bonds depend on the chain
conformation
 Protein Tertiary Structure
• Tertiary structure refers to the overall spatial
arrangement of atoms in a protein

• Stabilized by numerous weak interactions between

amino acid side chains.
 Largely hydrophobic and polar interactions
 Can be stabilized by disulfide bonds

• Interacting amino acids are not necessarily next to

each other in the primary sequence.

• Two major classes

– Fibrous and globular (water or lipid soluble)
 Fibrous Proteins:
From Structure to Function
Structure of -Keratin in Hair
Chemistry of Permanent Waving
 Structure of Collagen
• Collagen is an important constituent of connective
tissue: tendons, cartilage, bones, cornea of the eye
• Each collagen chain is a long Gly- and Pro-rich left-
handed helix
• Three collagen chains intertwine into a right-handed
superhelical triple helix
• The triple helix has higher tensile strength than a steel
wire of equal cross section
• Many triple-helices assemble into a collagen fibril
Collagen Fibrils
 4-Hydroxyproline in Collagen
• Forces the proline ring into a favorable pucker
• Offer more hydrogen bonds between the three strands
of collagen
• The post-translational processing is catalyzed by prolyl
hydroxylase and requires α-ketoglutarate, molecular
oxygen, and ascorbate (vitamin C)
Vitamin C in prolyl 4-hydroxylase
restores Fe2+ state
 Silk Fibroin
• Fibroin is the main protein in silk from moths and
spiders
• Antiparallel  sheet structure
• Small side chains (Ala and Gly) allow the close
packing of sheets
• Structure is stabilized by
– hydrogen bonding within sheets
– London dispersion interactions between sheets
 Spider Silk
• Used for webs, egg sacks, and wrapping the
prey
• Extremely strong material
– stronger than steel
– can stretch a lot before breaking
• A composite material
– crystalline parts (fibroin-rich)
– rubber-like stretchy parts
 Motifs (folds)

• Specific arrangement of several secondary

structure elements
– All alpha-helix
– All beta-sheet
– Both
• Motifs can be found as reoccurring structures in
numerous proteins
• Proteins are made of different motifs folded
together
 Domain

• Part of a polypeptide chain

– Independently stable
– Conserved part of a given protein sequence
– Molecular evolution uses domains as building blocks
– Domains vary in length from 25 A. A. to 500 A. A.
– Domains can be swapped by genetic engineering
between protein and another to make chimeric protein
 Quaternary Structure
• Quaternary structure is formed by the assembly of
individual polypeptides into a larger functional cluster
Protein Structure Methods:
X-Ray Crystallography
Steps needed
• Purify the protein
• Crystallize the protein
• Collect diffraction data
• Calculate electron density
• Fit residues into density
Pros
• No size limits
• Well-established
Cons
• Difficult for membrane proteins
• Cannot see hydrogens
 Structure Methods:
Biomolecular NMR
Steps needed
• Purify the protein
• Dissolve the protein
• Collect NMR data
• Assign NMR signals
• Calculate the structure
Pros
• No need to crystallize the protein
• Can see many hydrogens
Cons
• Difficult for insoluble proteins
• Works best with small proteins
 Intrinsically Disordered Proteins
• Contain protein segments that lack definable
structure
• Composed of amino acids whose higher
concentration forces less-defined structure
– Lys, Arg, Glu, and Pro
• Disordered regions can conform to many
different proteins, facilitating interaction with
numerous different partner proteins
Intrinsically Disordered Proteins
 Protein Stability and Folding
• A protein’s function depends on its 3D-structure
• Loss of structural integrity with accompanying loss of
activity is called denaturation
• Proteins can be denatured by:
• heat or cold
• pH extremes
• organic solvents
• chaotropic agents: urea and guanidinium
hydrochloride
 summary
In this chapter, we learned about:
• the two most important secondary structures
–  helices
–  sheets
• how properties and function of fibrous proteins are
related
• how to determine three-dimensional structures of
proteins
• one of the largest unsolved puzzles in modern
biochemistry: how proteins fold
 Enzymes

Key topics about enzyme function:

– Physiological significance of enzymes
– Origin of catalytic power of enzymes
– Chemical mechanisms of catalysis
– Mechanisms of chymotrypsin and lysozyme
– Description of enzyme kinetics and inhibition
 What are enzymes?
•Enzymes are catalysts
• Increase reaction rates without being used up
• Most enzymes are globular proteins
• However, some RNA (ribozymes and ribosomal RNA) also
catalyze reactions
• Study of enzymatic processes is the oldest field of
biochemistry, dating back to late 1700s
• Study of enzymes has dominated biochemistry in the
past and continues to do so
Why biocatalysis over inorganic catalysts?
• Greater reaction specificity: avoids side products
• Milder reaction conditions: conducive to conditions in cells
• Higher reaction rates: in a biologically useful timeframe
• Capacity for regulation: control of biological pathways
- -
COO COO
NH2

-
• Metabolites have many
-
O COO
potential pathways of
OH COO
decomposition
-
O COO -
- Chorismate
COO OH - COO
mutase
OOC • Enzymes make the
O
desired one most
favorable
NH2 OH
 Enzymatic Substrate Selectivity

OH
H
H
- +
OOC NH3 - +
OOC NH3

H
-
OOC
+
NH3
No binding
OH

HO OH
H

H
H Binding but no reaction
NH
CH3

Example: Phenylalanine hydroxylase

Reaction Conditions Compatible with Life

37˚C

pH ≈7
 Six Classes of Enzymes:
Defined by the Reactions Catalyzed
Enzyme Commission Number
EC 1.1.1.1 → alcohol dehydrogenase

http://enzyme.expasy.org/
Group Typical Reaction Enzyme examples
AH + B → BH (reduced)
EC1 Oxidoreductases Dehydrogenase, oxidase
A + O → AO (oxidized)
EC2 Transferases AB + C → A + BC Transaminase, kinase
EC3 Hydrolases AB + H2O → APH + BH Lipase, amylase, peptidase
RCOCOOH → RCOH + CO2
EC4 Lyases Decarbxylase
X-A-B-Y → A=B + X-Y
EC5 Isomerases ABC → BCA Isomerase, mutase
EC6 Ligases X + Y + ATP → XY + ADP + Pi Synthetase
 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 ]
v
K m  [S ]
Enzyme-Substrate Complex
 Enzymatic Catalysis
• Enzymes do not affect equilibrium (ΔG)
• Slow reactions face significant activation
barriers (ΔG‡) that must be surmounted
during the reaction
• Enzymes increase reaction rates (k) by
decreasing ΔG‡


kB T  G 

k exp 
 h   RT 
Reaction Coordinate Diagram
Enzymes Decrease ΔG‡
Illustration of TS Stabilization Idea:
Imaginary Stickase
 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 ]
• Ideal rate: v
Km  S

• Deviations due to:

– limitation of measurements
– substrate inhibition
– substrate prep contains inhibitors
– enzyme prep contains inhibitors
Effect of Substrate Concentration
 Saturation Kinetics:
At high [S] velocity does not depend on [S]
Determination of Kinetic Parameters

Nonlinear Michaelis-Menten plot should be used to

calculate parameters Km and Vmax.

Linearized double-reciprocal plot is good for analysis

of two-substrate data or inhibition.
 Lineweaver-Burk Plot:
Linearized, Double-Reciprocal
Derivation of Enzyme Kinetics Equations
• Start with a model mechanism
• Identify constraints and assumptions
• Carry out algebra ...
– ... or graph theory for complex reactions

• Simplest Model Mechanism: E + S  ES  E + P

– One reactant, one product, no inhibitors
Identify Constraints and Assumptions
• Total enzyme concentration is constant
– Mass balance equation for enzyme: ETot = [E] + [ES]
– It is also implicitly assumed that: STot = [S] + [ES] ≈ [S]

• Steady state assumption

d [ ES ]
 rate of formation of ES rate of breakdown of ES  0
dt

• What is the observed rate? dP

v net   k[ES]
– Rate of product formation dt
 Carry out the algebra
• The final form in case of a single substrate is
kcat [ Etot ][ S ]
v
K m  [S ]
• kcat (turnover number): how many substrate molecules
can one enzyme molecule convert per second
• Km (Michaelis constant): an approximate measure of
substrate’s affinity for enzyme
• Microscopic meaning of Km and kcat depends on the details
of the mechanism
 Sequential Kinetic Mechanism
• We cannot easily distinguish random from ordered
• Random mechanisms in equilibrium will give
intersection point at y-axis
• Lineweaver-Burk: lines intersect
 Ping-Pong Kinetic Mechanism
Lineweaver-Burk: lines are parallel
 Enzyme Inhibition
Inhibitors are compounds that decrease enzyme’s activity

•Irreversible inhibitors (inactivators) react with the enzyme

• One inhibitor molecule can permanently shut off one enzyme molecule
• They are often powerful toxins but also may be used as drugs

•Reversible inhibitors bind to and can dissociate from the enzyme

• They are often structural analogs of substrates or products
• They are often used as drugs to slow down a specific enzyme

•Reversible inhibitor can bind:

• to the free enzyme and prevent the binding of the substrate
• to the enzyme-substrate complex and prevent the reaction
 Competitive Inhibition

• Competes with substrate for binding

– Binds active site
– Does not affect catalysis

• No change in Vmax; apparent increase in KM

• Lineweaver-Burk: lines intersect at the y-axis
Competitive Inhibition
Competitive Inhibition
 Uncompetitive Inhibition

• Only binds to ES complex

• Does not affect substrate binding
• Inhibits catalytic function

• Decrease in Vmax; apparent decrease in KM

• No change in KM/Vmax
• Lineweaver-Burk: lines are parallel
Uncompetitive Inhibition
Uncompetitive Inhibition
 Mixed Inhibition

•Binds enzyme with or without substrate

― Binds to regulatory site
― Inhibits both substrate binding and catalysis
•Decrease in Vmax; apparent change in KM
•Lineweaver-Burk: lines intersect left from the y-axis
•Noncompetitive inhibitors are mixed inhibitors such
that there is no change in KM
Mixed Inhibition
Mixed Inhibition
Enzyme activity can be regulated

• Regulation can be:

– noncovalent modification
– covalent modification
– irreversible
– reversible
 Summary
In this chapter, we learned:

• why nature needs enzyme catalysis

• how enzymes can accelerate chemical reactions
• how chymotrypsin breaks down peptide bonds
• how to perform and analyze kinetic studies
• how to characterize enzyme inhibitors
• how enzyme activity can be regulated
Central Dogma of Biology

DNA→RNA→Protein
 Introduction
• DNA info is in the form of specific sequences
of bases along the DNA strands.
• The DNA leads to specific traits by dictating
the synthesis of proteins.
• Proteins are the links between genotype and
phenotype.
– For example, Mendel’s dwarf pea plants lack a
functioning copy of the gene that specifies the
synthesis of a key protein, gibberellins.
– Gibberellins stimulate the normal elongation of
stems.
 One Gene - One Polypeptide
• George Beadle and Edward Tatum were to
establish the link between genes and
enzymes in their exploration of the
metabolism of a bread mold, Neurospora
crassa.
• Their results provided strong evidence for the
one gene - one enzyme hypothesis.
• Later research refined the one gene - one enzyme
hypothesis.
• First, it became clear that not all proteins are
enzymes and yet their synthesis depends on
specific genes.
– This tweaked the hypothesis to one gene - one
protein.
• Later research demonstrated that many proteins
are composed of several polypeptides, each of
which has its own gene.
• Therefore, Beadle and Tatum’s idea has been
restated as the one gene - one polypeptide
hypothesis.
 DNA vs RNA
• Components of DNA
– Sugar (deoxyribose)
– Base (A,G,C,T)
– Phosphate group
• Components of RNA
– Sugar (ribose)
– Base (A,G,C,Uracil)
• RNA does not contain thymine
– Phosphate group
 DNA vs RNA continued
• Structural Characteristics of DNA
– Double stranded
– Base-pairing rules apply (A:T & G:C)
• Structural Characteristics of RNA
– Primarily single stranded
– Limited base-pairing (G:C & A:U)
 Types of RNA
• Messenger RNA (mRNA)
– Complementary to info in DNA strand
– Variable in length
– Contains specific structural info for the
sequence of amino acids
– Processed before using
 Types of RNA continued
• Transfer RNA (tRNA)
– Multiple varieties, each specific for a specific
amino acid
– Relatively small, with a consistent 3-d shape
– Specificity for each amino acid is
accomplished by a triplet base-pairing
relationship between codon on mRNA and
anti-codon on tRNA
Transcription and translation are the two
main processes linking gene to protein:
an overview
• Genes provide the instructions for making
specific proteins.
• The bridge between DNA and protein synthesis
is RNA.
• RNA is chemically similar to DNA, except that it
contains ribose as its sugar and substitutes the
nitrogenous base uracil for thymine.
– An RNA molecules almost always consists of a single
strand.
 DNA→RNA→Protein
• DNA is TRANSCRIBED to messenger
RNA (mRNA)
• mRNA carries the message to tranfer RNA
(tRNA)
• tRNA is TRANSLATED to an amino acid
chain, which makes up proteins
• In DNA or RNA, the four nucleotide
monomers act like the letters of the
alphabet to communicate information.
• The specific sequence of hundreds or
thousands of nucleotides in each gene
carries the information for the primary
structure of a protein (the linear order of the
20 possible amino acids)
• To get from DNA, written in one chemical
language, to protein, written in another,
requires two major stages, transcription
and translation.
• During transcription, a DNA strand provides a
template for the synthesis of a complementary
RNA strand.
– This process is used to synthesize any type of RNA
from a DNA template.
– Transcription is from the 3’→5’ strand (template
strand)
• Transcription of a gene produces a messenger
RNA (mRNA) molecule.
– mRNA carries the message from the nucleus to the
ribosomes
• During translation, the information contained in
the order of nucleotides in mRNA is used to
determine the amino acid sequence of a
polypeptide.
– Translation occurs at ribosomes.
• The basic mechanics of transcription and
translation are similar in eukaryotes and
prokaryotes.
• Because bacteria lack nuclei, transcription
and translation are coupled.
• Ribosomes attach to the leading end of a
mRNA molecule while transcription is still in
progress.

Fig. 17.2a
• In a eukaryotic cell, almost all transcription occurs
in the nucleus and translation occurs mainly at
ribosomes in the cytoplasm.
• In addition, before the
primary transcript
can leave the nucleus
it is modified in
various ways during
RNA processing
before the finished
mRNA is exported
to the cytoplasm.
– Introns are removed
Fig. 17.2b
 RNA Packaging
• Introns are removed (only exons contain
genetic info)
• Addition of a 5’ cap on mRNA (for
orientation purposes)
• Addition of a 3’ tail on mRNA (allows it to
last longer)
• To summarize, genes program protein
synthesis via genetic messenger RNA.
• The molecular chain of command in a cell is

DNA → RNA → protein.

This is referred to as the Central Dogma of

Biology
Defining Cloning
“Cloning” is a loaded term that can be
used to mean very different things.
 Cutting a piece of DNA from one
organism and inserting it into a vector
where it can be replicated by a host
organism. (Sometimes called subcloning,
because only part of the organism’s DNA
is being cloned.)
 Using nuclear DNA from one organism to
create a second organism with the same
nuclear DNA
DNA CLONING
 DNA cloning is a technique for
reproducing DNA fragments.
 It can be achieved by two different
approaches:
▪ cell based
▪ using polymerase chain reaction (PCR).
 a vector is required to carry the DNA
fragment of interest into the host cell.
DNA CLONING
 DNA cloning allows a copy of any
specific part of a DNA (or RNA)
sequence to be selected among many
others and produced in an unlimited
amount.
 This technique is the first stage of most
of the genetic engineering experiments:
▪ production of DNA libraries
▪ PCR
▪ DNA sequencing
 DNA CLONING
 Massive amplification of DNA
sequences
 Stable propagation of DNA sequences
 A single DNA molecule can be
amplified allowing it to be:
▪ Studied - Sequenced
▪ Manipulated - Mutagenised or
Engineered
▪ Expressed - Generation of Protein
 CLONING PROCESS
 Gene of interest is cut
out with RE
 Host plasmid is cut
with same RE
 Gene is inserted into
plasmid and ligated
with ligase
 New plasmid inserted
into bacterium
(transform)
 PLASMID CLONING STRATEGY
 Involves five steps:
Enzyme restriction digest of DNA sample.
Enzyme restriction digest of DNA plasmid
vector.
Ligation of DNA sample products and plasmid
vector.
Transformation with the ligation products.
Growth on agar plates with selection for
antibiotic resistance.
STEP 1. RE DIGESTION
OF DNA SAMPLE
STEP 2. RE DIGESTION OF
PLASMID DNA
STEP 3. LIGATION OF DNA
SAMPLE AND PLASMID DNA
 STEP 4. TRANSFORMATION
OF LIGATION PRODUCTS
 The process of transferring exogenous DNA
into cells is call “transformation”
 There are basically two general methods for
transforming bacteria. The first is a chemical
method utilizing CaCl2 and heat shock to
promote DNA entry into cells.
 A second method is called electroporation
based on a short pulse of electric charge to
facilitate DNA uptake.
CHEMICAL TRANSFORMATION
WITH CALCIUM CHLORIDE
TRANSFORMATION BY
ELECTROPORATION
STEP 5. GROWTH ON AGAR
PLATES
STEP 5
 Blue colonies represent Ampicillin-resistant
bacteria that contain pVector and express a
functional alpha fragment from an intact
LacZ alpha coding sequence.

White colonies represent Ampicillin-resistant

bacteria that contain pInsert and do not
produce LacZ alpha fragment
TERMS USED IN CLONING
 DNA recombination.
The DNA fragment to be cloned is inserted
into a vector.
 Transformation.
The recombinant DNA enters into the host
cell and proliferates.
 Selective amplification.
A specific antibiotic is added to kill E. coli
without any protection. The transformed E.
coli is protected by the antibiotic-resistance
gene
 Isolation of desired DNA clones
 CLONING VECTORS
 Cloning vectors are DNA molecules that are used to
"transport" cloned sequences between biological hosts
and the test tube.

Cloning vectors share four common properties:

1. Ability to promote autonomous replication.

2. Contain a genetic marker (usually dominant) for
selection.
3. Unique restriction sites to facilitate cloning of insert
DNA. (MCS)
4. Minimum amount of nonessential DNA to optimize
cloning.
 PLASMIDS
 Bacterial cells may
contain extra-
chromosomal DNA
called plasmids.
 Plasmids are usually
represented by small,
circular DNA.
 Some plasmids are
present in multiple
copies in the cell
 PLASMID VECTORS
 Plasmid vectors are ≈1.2–
3kb and contain:
 replication origin (ORI)
sequence
 a gene that permits
selection,
 Here the selective gene is
ampr; it encodes the
enzyme b-lactamase, which
inactivates ampicillin.
 Exogenous DNA can be
inserted into the bracketed
region .
ORIGIN OF REPLICATION
 Origin of replication
is a DNA segment
recognized by the
cellular DNA-replication
enzymes.
 Without replication
origin, DNA cannot be
replicated in the cell.
 MULTIPLE CLONING SITE
 Many cloning vectors contain a
multiple cloning site or
polylinker: a DNA segment with
several unique sites for
restriction endo- nucleases
located next to each other
 Restriction sites of the polylinker
are not present anywhere else in
the plasmid.
 Cutting plasmids with one of the
restriction enzymes that
recognize a site in the polylinker
does not disrupt any of the
essential features of the vector
Disadvantages using
plasmids
 Cannot accept large fragments
 Sizes range from 0- 10 kb
 Standard methods of
transformation are inefficient
Why Plasmids are Good
Cloning Vectors
 small size (easy to manipulate and
isolate)
 circular (more stable)
 replication independent of host cell
 several copies may be present
(facilitates replication)
 frequently have antibody resistance
(detection easy)
BLUE/WHITE SCREENING
 Colony Selection: finding the rare bacterium
with recombinant DNA
 Only E. coli cells with resistant plasmids grow
on antibiotic medium
 Only plasmids with functional lacZ gene can
grow on Xgal
lacZ(+) => blue colonies
lacZ functional => polylinker intact => nothing
inserted, no clone
lacZ(-) => white colonies polylinker disrupted
=> successful insertion & recombination!
 α -complementation
 The portion of the lacZ gene encoding the
first 146 amino acids (the α -fragment) are on
the plasmid
 The remainder of the lacZ gene is found on
the chromosome of the host.
 If the α -fragment of the lacZ gene on the
plasmid is intact (that is, you have a non-
recombinant plasmid), these two fragments of
the lacZ gene (one on the plasmid and the
other on the chromosome) complement each
other and will produce a functional β -
galactosidase enzyme.
 SCREENING RECOMBINANTS

 In the example shown above, the b-

galactosidase gene is inactivated. The substrate
"X-gal" turns blue if the gene is intact, ie. makes
active enzyme. White colonies in X-gal imply the
presence of recombinant DNA in the plasmid.
ENZYMES USED IN
MOLECULAR BIOLOGY
Removes phosphate groups from 5' ends of
Alkaline phosphatase DNA (prevents unwanted re-ligation of cut
DNA)
Joins compatible ends of DNA fragments
DNA ligase (blunt/blunt or complementary cohesive ends).
Uses ATP
Synthesises DNA complementary to a DNA
DNA polymerase I template in the 5'-to-3'direction. Starts from an
oligonucleotide primer with a 3' OH end
Digests nucleotides progressiviely from a DNA
Exonuclease III
strand in the 3' -to-5' direction
Adds a phosphate group to the 5' end of
Polynucleotide kinase double- or single-stranded DNA or RNA. Uses
ATP
RNase A Nuclease which digests RNA, not DNA

Heat-stable DNA polymerase isolated from a

Taq DNA polymerase
thermostable microbe (Thermus aquaticus)
 T-A Cloning
 When DNA fragments are
generated Taq
polymerase adds 1 or 2
extra adenines onto the
end of 3’ end of blunt ds
DNA
 Several commercially
available kits take
advantage of this ability
 Use a plasmid vector with
thymidine residues linked
onto the 3’ ends of
linearised plasmid DNA
 Nucleotides and Nucleic Acids

Key topics:
– Biological function of nucleotides and nucleic acids
– Structures of common nucleotides
– Structure of double-stranded DNA
– Structures of ribonucleic acids
– Denaturation and annealing of DNA
– Chemistry of nucleic acids; mutagenesis
 Functions of
Nucleotides and Nucleic Acids
• Nucleotide Functions:
– Energy for metabolism (ATP)
– Enzyme cofactors (NAD+)
– Signal transduction (cAMP)

• Nucleic Acid Functions:

– Storage of genetic info (DNA)
– Transmission of genetic info (mRNA)
– Processing of genetic information (ribozymes)
– Protein synthesis (tRNA and rRNA)
 Nucleotides and Nucleosides
• Nucleotide =
– Nitrogeneous base
– Pentose
– Phosphate

• Nucleoside =
– Nitrogeneous base
– Pentose

• Nucleobase =
– Nitrogeneous base
 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
 Pentose in Nucleotides
• -D-ribofuranose in RNA
• -2’-deoxy-D-ribofuranose in DNA

• Different puckered conformations of the sugar ring

are possible
 Nucleobases

• Derivatives of pyrimidine or purine

• Nitrogen-containing heteroaromatic molecules
• Planar or almost planar structures
• Absorb UV light around 250–270 nm
 Pyrimidine Bases

• Cytosine is found in both DNA and RNA

• Thymine is found only in DNA
• Uracil is found only in RNA
• All are good H-bond donors and acceptors
•Neutral molecules at pH 7
 Purine Bases

• Adenine and guanine are found in both RNA

and DNA
• Also good H-bond donors and acceptors
• Neutral molecules at pH 7
 -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 N1 in pyrimidines
– to position N9 in purines
• 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
 UV Absorption of Nucleobases
• Absorption of UV light at 250–270 nm is due to  
* electronic transitions
• Excited states of common nucleobases decay rapidly
via radiationless transitions
– Effective photoprotection of genetic material
– No fluorescence from nucleic acids
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)
 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’
 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
AT and GC Base Pairs
 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
Thymine C5H7O isolates “nuclein” from
O cell nuclei
HO P O

Structure of DNA: O

1929 Adenine C5H7O • Hydrolysis of nuclein

(Levene & London) O
– phosphate
OH
– pentose
HO P O – and a nucleobase
H O

H H
H H

Thymine CH2O
OH

P O
• Chemical analysis
O
H O – phosphodiester linkages
Structure of DNA:
1935 H
H H H – pentose is ribofuranoside
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

that the phosphate-
(major & minor groove)
sugar backbone
– Hydrogen bonding:
is outside
A pairs with T
– 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
Other Forms of DNA
 Complementarity of DNA Strands

• Two chains differ in sequence

(sequence is read from 5’ to 3’)
• Two chains are complementary
• Two chains run antiparallel
 Replication of Genetic Code
• Strand separation occurs first
• Each strand serves as a template
for the synthesis of a new strand
• Synthesis is catalyzed by enzymes
known as DNA polymerases
• Newly made DNA molecule has one
daughter strand and one parent strand.

“It has not escaped our notice that the specific pairing
we have postulated immediately suggests a possible
copying mechanism for the genetic material.”
―Watson and Crick, Nature, 1953
Replication of Genetic Code
 Messenger RNA:
Code Carrier for the Sequence of Proteins
• Is synthesized using DNA template
• Contains ribose instead of deoxyribose
• Contains uracil instead of thymine
•Together with transfer RNA (tRNA) transfers genetic
information from DNA to proteins
 Palindromic sequences can form
hairpins and cruciforms

Palindromes: words or phases that are the same when

read backward or forward:

Saippuakuppinippukauppias
(Finnish word for “soap cup batch trader”)
RNA molecules have quite complex structures
 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 (PCR)
- site-directed mutagenesis
• Evolutionary relationships
• Antisense therapy
 Molecular Mechanisms of
Radiation-Induced Mutagenesis
• UV light induces dimerization of pyrimidines; this may be the
main mechanism for skin cancers.

• Ionizing radiation (X-rays and -rays) causes ring opening and

strand breaking .
• These are difficult to fix.

• Cells can repair some of these modifications, but others cause

mutations. Accumulation of mutations is linked to aging and
carcinogenesis.
Other Functions of Nucleotides:
Energy Source
Other Functions of Nucleotides:
Coenzymes
Other Functions of Nucleotides:
Regulatory Molecules
 Summary
In this chapter, we learned about:

• Function of nucleotides and nucleic acids

• Names and structures of common nucleotides
• Structural basis of DNA function
• Reversible denaturation of nucleic acids
• Chemical basis of mutagenesis
Plant cell wall
Understanding biomass biology is critical for
biofuels production because…

Cell wall architectures impact plant stature and form (biomass

quantity)

The architecture of cell walls limits their deconstruction to substrates

for biofuel production (biomass quality)

Plant cell walls may be optimized for their end-use in biofuel

conversion processes (tailored biomass)
Plant Anatomy & Physiology
 What is plant anatomy?
• ANATOMY: study of the structure of
organisms… looking at cells, tissues
• (Morphology: Study of form)

What is plant physiology?

• PHYSIOLOGY: study of the function of
cells, tissues, organs of living things;
and the physics/chemistry of these
functions…
 Major Plant Parts

• Roots
• Stems
• Leaves
• Flowers
 storage high
transport &
energy carbs. storage: pigments,
modification contains DNA
acids, ergastic
substances
structural support

respiration

cell recognition,
transport

photosynthesis

protein synthesis ribosomes: site of

& transport of protein
materials synthesis
Leaf Layers
Vascular bundles
 Cell Theory
All of life is composed of 1 or more cells.
Cells arise only from pre-existing cells, via
cell division or cell fusion.
Cells are units of metabolic processes.
Each cell contains set of hereditary
information (DNA), transferred from cell
to cell, coding for structural & functional
features.
 Always keep in mind that in plant
anatomy, morphology & physiology…

“Structure correlates to
function”
• How can water
move from
the ground
all the way
to the top
of a 100 m
tall redwood
tree?
 Plant Anatomy: Cells
• Plant cells are basic building blocks
• Can specialize in form and function
• By working together, forming tissues, they
can support each other and survive
• Levels of organization
atoms > molecules > cells > tissues > organs > whole plant
 Plant Tissues Types
All plant organs (roots, stems, leaves) are
composed of the same tissue types.
There are three types of tissue:

• 1. Dermal – outermost layer

• 2. Vascular – conducting tissue, transport

• 3. Ground – bulk of inner layers

Organization of primary tissues in young stems
Organization of primary tissues in roots
 1. Dermal tissue
• Epidermis is the outermost layer of
cells
• Like the “skin” of animals
• In stems and leaves,
epidermis has cuticle,
a waxy layer that prevents
water loss.
• Some have trichomes, hairs.
• Root epidermis has root hairs, for
water and nutrient absorption
 2. Vascular tissue
• Transports water and organic materials
(sugars) throughout the plant
• Xylem – transports water and
dissolved ions from the root
to the stem and leaves.
• Phloem – carries dissolved
sugars
from leaves to rest of the plant
 Xylem
• Transports water and dissolved minerals
• Tracheids: long, thin tube like structures without
perforations at the ends
• Vessel elements: short, wide tubes perforated at
the ends (together form a pipe, called vessel).
• Both cells have pits (thin sections) on the walls

Tracheids Vessel elements

 Xylem cells
• Xylem cells are dead!
• They are hollow cells
and consist only of
cell wall
 Phloem
• Cells that transport organic materials (sugars)
• Phloem cells are ALIVE! (unlike xylem)
 Phloem: transports sugars
• Phloem composed of cells called sieve tube
members (STM)
• Companion cells join sieve tube members, are
related, and help to load materials into STM
• End walls of STM have large pores called
sieve plates

Companion cells

Sieve tube member Sieve plates

 3. Ground tissue
• Makes up the bulk of plant organs.
• Functions: Metabolism, storage and support.

Root Stem Leaf

 Plant Organs
Organs: tissues that act together to
serve a specific function

Dermal
• Roots Vascular
Ground

Dermal
• Stems Vascular
Ground

Dermal
• Leaves Vascular
Ground
 Functions of plant organs:
• ROOTS: Anchorage, water/nutrient
absorption from soil, storage, water/nutrient
transport

• STEMS: Support, water/nutrient transport

• LEAVES: Photosynthesis (food production)

 ROOTS

• ROOTS “the hidden half”

• Functions of roots:
• Ancorage
• Absorption of water & dissolved
minerals
• Storage (surplus sugars, starch)
• Conduction water/nutrients
Anatomy of a root

epidermis
cortex
vascular
 Root Epidermis
• Outermost, single layer of cells that:
– Protects (from diseases)
– Absorbs water and nutrients

• ROOT HAIRS: tubular extensions

of epidermal cells.
• Increase surface area of root,
for better water/nutrient
absorption
Root Hairs: water and mineral
absorption

Root hairs
increase surface
area for better
absorption
 Root Cortex
• Stores starch, sugars and other substances
 Root Ground tissue
• In roots, ground tissue provides support,
and often stores sugars and starch
(for example: yams, sweet potato, etc.)

You’re not a
yam, you’re a Hey!
sweetpotato! I yam
what I
cortex yam,
man!
 Root Cortex: Endodermis
• Endodermis: the innermost layer of the
cortex
 Root cortex: Casparian strip
• The Casparian strip is a water-impermeable
strip of waxy material found in the endodermis
(innermost layer of the cortex).
• The Casparian strip helps to control the uptake
of minerals into the xylem: they have to go
through the cytoplasm of the cell!
 STEMS
• Above-ground organs (usually)

• Support leaves and fruits

• Conduct water and sugars

throughout plant (xylem and phloem)
 Stem anatomy
• Dermal, ground and vascular tissues…

epidermis
cortex

Vascular
pith
bundles
Organization of primary tissues in young stems
 Types of stems
• Herbaceous vs. Woody
stems
 Tissues of stems
• Epidermis (Dermal tissue type)
• Provides protection
• Has cuticle (wax) prevents water loss
• Trichomes (hairs) for protection, to release
scents, oils, etc.
 Stem Vascular tissue
• Vascular bundles – composed of both
xylem and phloem
• Xylem
– Conducts water
– Support
• Phloem
– Conducts food Vascular
– Support cambium
 Vascular cambium
• Occurs in woody stems
• Vascular cambium located in the
middle of the vascular bundle, between
xylem and phloem
 Vascular tissue: Trees
• Vascular tissue is located on the outer
layers of the tree.

bark

phloem

Vascular
cambium wood

xylem
 Vascular tissue forms rings in trees
• Annual rings: xylem formed by the
vascular cambium during one growing
season
• One ring = one year
 History of the tree: annual rings
Dendrochronology : tree time-keeping

1917 & 1945: Tree

Survives two World
1776: Declaration Wars 1969: Man
of US independence lands on Moon
1492: Columbus lands in
the Americas

1620: Pilgrims land 1861: Start of

in Plymouth, Mass. Civil War
1489: Tree is planted 1971: Birth Year
by Native American
of the IDIOT
who cut down
this tree!!!
 Ground tissue: Cortex & pith
• Stores food (e.g. potato)
• Site of Photosynthesis (when green)
• Support cells

cortex

pith
 LEAVES:
• ‘Photosynthetic factories’ of the plant…
• Function: Photosynthesis – food
production for the whole plant
• Blade: Flat expanded area
• Petiole: stalk that connects
BLADE
leaf blade to stem, and
transports materials
 Leaf Anatomy
• Leaf anatomy is correlated to
photosynthesis:
Carbon dioxide + Water  sugars + oxygen
dermal
ground

vascular

dermal
 Leaf epidermis
• Is transparent – so that sun light can go through.
• Waxy cuticle protects against drying out
• Lower epidermis: stomata with guard cells – for
gas exchange (CO2 in; O2, H2O out)
 Leaf epidermis

• Trichomes (give fuzzy texture)

(“Panda plant”)
 Leaf vascular tissue
• VEINS  vascular tissue of leaves.
• Veins are composed of xylem (water transport)
phloem (food transport)
and bundle sheaths,
cells surrounding the
xylem/phloem for
strength & support
 Leaf Mesophyll
• Middle of the leaf (meso-phyll)
• Composed of photosynthetic ground cells:
• Palisade parenchyma
(long columns below epidermis;
have lots chloroplasts for
photosynthesis)
Spongy parenchyma
(spherical cells)
with air spaces around,
(for gas exchange)
 Plant water transport
• How can water move from
the ground
all the way
to the top
of a 100 m
tall redwood
tree?
 Water transport in plants:
• The same way we drink soda
from a straw!

• Water’s great
cohesive forces (molecules
sticking to each other)
and adhesive forces
(attaching to walls of xylem cells)
Transpiration-cohesion Theory
for water transport in the xylem
• Evaporation of water in the leaves
(through stomates) generates the
‘sucking force’ that pulls adjacent water
molecules up the leaf surface
 Water transport (cont.)
• Like a long chain, water molecules pull
each other up the column.
• The column goes from roots  leaves.

• What’s amazing is that the

water moves up by using the sun’s
evaporative energy…
• Plants control transpiration by
opening/closing stomata
 Sugar translocation
• 1. Sugars made in leaf mesophyll cells (source)
diffuse to phloem cells in the vascular bundles.
• 2. Companion cells load dissolved sugars into
the phloem STM using energy (ATP).
• 3. Water moves into cells with high sugar
concentration.
• 4. Osmotic water flow generates a high hydraulic
pressure that moves dissolved sugars through
the phloem to the rest of the plant (sink).
 Pressure flow in phloem
• Sugars made in the
leaves are loaded into
companion cells and
into phloem STM.

• Water (from xylem)

moves in by osmosis,
creating pressure flow
down the phloem.
 Plant Hormones
• Chemical compounds produced by
plants
• Effective at very low concentrations
 What is a hormone?
• Biochemical which regulates growth based
on biological and environmental influences
• Synonyms: Plant hormones, plant growth
regulators (PGRs), phytohormones
• Regulate growth and development
• Mobile throughout plant
• Environment and stress responsive
 Major plant hormones

• Auxin
• Cytokinin
• Abscisic acid
• Gibberellic acid
• Ethylene
• Jasmonic acid
• Brassinosteroids
Hormones & environmental signals
involve signal transduction
pathways
Internal and external signals
 Hormones influence gene
expression

• Gene expression
regulated by
– microRNAs
– transcription factors
Plant hormones & growth

(abscisic acid)
 General hormone biochemistry
• Present in all cells at various levels
• Classes of hormones work in signal cascades
– Hormone-receptor interactions
– Respond to a host of factors and biological needs
• Abiotic
– Water stress
– Light
– Nutrient deficiency
• Biotic
– Growth
– Development
– Herbivore stress
 Hormone biosynthesis
Made from four biosynthetic pathways:
– Terpenoids
• AMP + IPP (cytokinins)
• Carotenoid breakdown (abscisic acid)
• Diterpene (gibberellic acid)
• Triterpene (brassinosteroids)
– Fatty acids (jasmonic acid)
– Tryptophan (auxins)
– Methionine (ethylene)
 Auxins
• Greek: auxein; to grow or increase
• Apical dominance growth
• Cell elongation
• Hormone level very important
 AUXINS
• Promote cell growth
• Involved in
gravitropism
and phototropism

• Control fruit development

 Cytokinin
• Cytokinesis (cell division)
• Accidently added degraded DNA to
medium
• Organization and development of xylem
tissue
• Response to light
• Lateral growth of shoots
• Open stomata
 Auxin and cytokinin ratio
importance
• Auxin alone = Large cells (no division)
• Cytokinin alone = Cells have no change
• Auxin + Cytokinin = Normal cell growth
and division
• Auxin + >Cytokinin = Shoot growth
• >Auxin = Cytokinin = Root growth
 Auxin

Cytokinin
 Abscisic acid (ABA)

• Originally implicated in leaf and fruit

abscission
• Involved in leaf senescence
• Maintains seed dormancy (opposed to GA)
• Involved in stomata regulation (closes)
• Single hormone unit
 Gibberellic acid
• Originally found in Gibberella (rice pathogen)
– Responsible for ‘foolish seedling’
phenomenon
– Uninhibited growth until breaking
• Involved in cell elongation
• Flowering and seed germination
 Ethylene: the cell phone of
PGRs
• A hydrocarbon gas
• Involved in fruit ripening, stress response
• Inhibition of growth in dark conditions
• Excess ethylene inhibits callus growth
 Conversational Plants?

• Ethylene production increases during

stress
– Drought
– Heat

• Perceived by neighboring plants

• Unstressed plants induce stress

pathways
 Jasmonic acid
• First identified in jasmine oil
• Response to biotic stress
– Wounding induces JA biosynthesis
– Microbial and fungal invasion

• Plant growth effects similar to auxin

– Specialty growth structures
 Brassinosteroids
• Stress responses
– Switchgrass suspension cells have minor
amounts of lignin (small amounts of H
monolignols)
– Addition of brassinolide induces normal lignin
formation and composition
• Stem elongation
• Seed germination
• Pollen tube growth
• Cell differentiation control
Hormones (PGR) pathways
 summary
• Hormones are biochemicals that
regulate plant growth based on
biological and environmental cues
• Auxin and cytokinin are key for plant
growth
• Abiotic and biotic stress response is
regulated by hormones
• Cell signaling is regulated by specific
receptors on cell membranes
 Plant physiology
• PHYSIOLOGY: study of the function of cells,
tissues, organs of living things;
and the physics/chemistry of these functions…
 photosynthesis
• Photo means ‘light’ and synthesis
means ‘to make’
• Process in which plants convert carbon
dioxide and water into sugars using
solar energy
• Occurs in chloroplast
Photosynthesis:

6 CO2 + 6 H2O C6 H12 O6 + 6 O2

carbon dioxide + water = sugar + oxygen

photosynthetic
products often
stored as
starch
•Starch =
glucose polymer
• Plant Cell
– Plant Cell structure and function

• Different Bio-Physico-Chemical Phenomena

– Colloids
– Diffusion
– Osmosis
– Imbibitions

• The Enzymes
– Structure
– Mode of action
– Inhibitors
– Factors affecting enzymatic activity
• Metabolism

• Respiration
– Glycolysis, TAC, Fermentation
– Factors affecting rate of respiration

• Amino acid and protein metabolism

• Biotechnology

• -What is biotechnology?

• Biotechnology benefit
1- Plant Cell (Structure and Function)

• Cells are the basic unit of life.

• Cells carryout all the necessary
functions for life such as: reproduction,
taking in nutrients and excreting
wastes.
• Cells are made up of chemicals and
molecules. Human cells contain mostly
water.
 The Cell
• Cells are classified as prokaryotic and
eukaryotic.
• Prokaryotic cells lack a well-defined
nucleus and many organelles. Bacteria
are prokaryotic cells.
• Eukaryotic cells include the protists,
fungi, plant and animal cells. We will
study these cells during this lab.
 The Cell
• Animal Cells • Plant Cells
• Have centrioles • Lack centrioles
• Store glucose as • Store glucose as
glycogen cellulose or starch
• Do not have a cell wall • Have a cell wall
• Contain mitochondria • Have large vacuoles
• Contain chloroplasts
as well as
mitochondria
Animal Cell

100
Plant Cell

101
 Cell is a unit or module of plant
organization

- Supports cells & plant

- Controls cell growth &
shape
 Cell Wall

• Only found in plant cells

• Surrounds cell membrane
• Composed of lignin and cellulose
• Function to strengthen and protect plant
cells.

103
 Cell Wall
• Structural functions
– Provides rigidity to plant
– Determines size and shape of cell
– Determines size and shape of plant
• Other functions
– Absorption
– Transport
– Secretion
– Cell-cell communication
 Structure of cell wall
• Cellulose is principal component
– Repeated monomers of glucose attached end to end
– Bundled into microfibrils
– Crystalline properties
• Cellulose matrix is embedded in matrix of noncellulosic
molecules
– Provide cross-links between cellulose microfibrils
– Pectins = hydophilic polysaccharides
– Glycoproteins- extensins

105
Structure of cell wall continued
• Primary wall
– Formed first while cells growing and dividing
– Enriched in pectins and water (to allow expansion)
• Middle lamella
– Joins adjacent cells
– Mostly pectins

106
 Cell wall structure continued
• Secondary wall
– Not all cells form
– Deposited after cell has stopped growing
– Inside primary wall
– More cellulose
– Pectins lacking
– Rigid
– Formed by cells that provide strength or transport
Cell walls (yellow) separated by middle
lamella (black)

108
109
Middle lamella (black) separates walls from
2 different cells

Cell
membrane
contacts
inner part of
wall

110
Cellulose is polymer of glucose that aggregates
into threads (microfibrils)

111
 Plant physiology
• PHYSIOLOGY: study of the function of cells,
tissues, organs of living things;
and the physics/chemistry of these functions…
 photosynthesis
• Photo means ‘light’ and synthesis
means ‘to make’
• Process in which plants convert carbon
dioxide and water into sugars using
solar energy
• Occurs in chloroplast
Photosynthesis:

6 CO2 + 6 H2O C6 H12 O6 + 6 O2

carbon dioxide + water = sugar + oxygen

photosynthetic
products often
stored as
starch
•Starch =
glucose polymer
 Plant cell walls provide
important durable materials

Wood is
primarily
composed of
plant cell
walls.
 Cell Wall

• Only found in plant cells

• Surrounds cell membrane
• Composed of lignin, cellulose and
hemicellulose
• Function to strengthen and protect plant
cells.
 Cell Wall
• Structural functions
– Provides rigidity to plant
– Determines size and shape of cell
– Determines size and shape of plant
• Other functions
– Absorption
– Transport
– Secretion
– Cell-cell communication
 Structure of cell wall
• Cellulose is principal component
– Repeated monomers of glucose attached
end to end
– Bundled into microfibrils
– Crystalline properties
• Cellulose matrix is embedded in matrix of
noncellulosic molecules
– Provide cross-links between cellulose
microfibrils
– Pectins = hydophilic polysaccharides
– Glycoproteins- extensins
13
 Structure of cell wall
• Primary wall
– Formed first while cells growing and
dividing
– Enriched in pectins and water (to allow
expansion)

• Middle lamella
– Joins adjacent cells
– Mostly pectins
 Cell wall structure
continued
• Secondary wall
– Not all cells form
– Deposited after cell has stopped growing
– Inside primary wall
– More cellulose
– Pectins lacking
– Rigid
– Formed by cells that provide strength or
transport
Cell walls (yellow) separated by middle
lamella (black)

16
Cell Wall Structure

secondary cell wall

primary cell wall

middle lamella
 Components of plant cell walls

Cellulose
Fermentable sugars
obtained from
cellulose in 1819

Lignin

Hemicellulose Extractives
(need special yeast Ash
to convert to ethanol)
Plant cell walls
Primary cell walls
Primary plant cell walls are
composed mainly of carbohydrates
and proteins.

Some cells produce a

rigid secondary wall that
incorporates lignin, an
insoluble cross-linking
compound.
Secondary cell walls
Cellulose is polymer of glucose that aggregates
into threads (microfibrils)

24
 Cellulose is the principal scaffolding
component of all plant cell walls.
• Cellulose in primary and secondary cell walls
• Made of (1→4)β-D-glucan chains hydrogen
bonded to one another along their length
• Groups of 30 to 40 of these chains laterally
hydrogen bond to form crystalline or para-
crystalline microfibrils
Current updates and evolving
concepts on Cellulose
Biosynthesis in plants
 Plant cell wall
biosynthesis
• Plant cell wall is
made of cellulose
microfibrils, which
is consisted of
about 36 chains of
cellulose, a
polymer of
b(14)glucose.
 Outline
• What genes involved in cellulose
biosynthesis?
• Which compartment is involved in cellulose
biosynthesis?
• What are the protein components of the
cellulose syntheses machinery and how they
are coupled in the cytoskeleton?
• What is the first committed step in cellulose
polymerization?
• How trafficking of cellulose synthase occurs?
• What are the approaches in studying
cellulose synthase?
Aaron H. Liepman, Raymond Wightman, Naomi Geshi, Simon R. Turner and
Henrik Vibe Scheller (2010) Arabidopsis – a powerful model system for plant cell
wall research The Plant Journal 61, 1107–1121.
 • The CESA proteins that make up the CSC responsible
for primary cell wall formation consist of CESA1 and
CESA3, together with some combination of CESA2,
CESA5, CESA6, or CESA9. ACesA2 and CesA5, which
have been reported to bepartially redundant with CesA6,
may compete for the same position in the enzyme
complex
• Cellulose biosynthesis at the secondary wall requires
CESA4, CESA7, and CESA8.

Illustration of the structure of CESA3, a typical CESA protein.

Cellulose biosynthesis
• Cellulose is
synthesized by
terminal complexes
(rosettes), consisting
of cellulose synthase
and associated
enzymes.
Terminal complex (rosette)

p.777
 Cellulose synthase
• Cellulose synthase has not been isolated in
its active form, but from the hydropathy
plots deduced from its amino acid sequence
it was predicted to have eight
transmembrane segments, connected by
short loops on the outside, and several
longer loops exposed to the cytosol.
Initiation of new
cellulose chain
synthesis
• Glucose is
transferred from
UDP-glucose to a
membrane lipid
(probably sitosterol)
on the inner face of
the plasma
membrane.
 New cellulose
chain synthesis (1)
• Intracellular cellulose synthase
adds several more glucose
residues to the first one, in
(b14) linkage, forming a
short oligosacchairde chain
attached to the sitosterol
(sitosterol dextrin).
New cellulose chain
synthesis (2)
• Next, the whole sitosterol
dextrin flips across to the
outer face of the plasma
membrane, where most of
the polysaccharide chain is
removed by endo-1,4-b-
glucanase.
New cellulose chain
synthesis (3)
• The dextrin
primer (removed
from sitosterol by
endo-1,4-b-
glucanase) is now
(covalently)
attached to
another form of
cellulose synthase.
New cellulose chain synthesis (4)

• The UDP-glucose used

for cellulose synthesis
is generated from
sucrose produced from
photosynthesis, by the
reaction catalyzed by
sucrose synthase (this
enzyme is wrongly
named).
New cellulose chain synthesis (5)

• The glucose associated

with UDP is a-linked.
• Its configuration will
be converted by
glycosyltransferases so
the product (cellulose)
is b-linked.
 What are Hemicelluloses?
• Hemicelluloses are also sugar polymers but
different from cellulose because they are:
– Made up glucose and other sugars.
– Contain some molecules other than sugars.
– Branched little polymers
– Much smaller than cellulose as they are made up of
between 50-300 sugars
– There are lots of varieties of hemicelluloses.
– Not very resistant to chemical attack – many easily
break down to simple sugars
• Hemicellulose belong to a group of heterogeneous
polysaccharides which are formed through biosynthetic
routes different from that of cellulose.

• Like cellulose most hemicellulose function as supporting

material in the cell wall.

• Most hemicellulose have a degree of polymerization of

only 200.

• The amount of hemicellulose of the dry weight of

wood is usually between 20 and 30%.
• The composition and structure of the hemicellulose
in the softwood differ in a characteristic way from
those in the heartwood.

• Considerable differences also exist in the

hemicellulose content and composition between the
stem, branches, roots, and bark.

•Mannose is the most important hemicellulosic monomer

followed by xylose, glucose, galactose and arabinose.
 Hemicelluloses
Structurally, hemicelluloses are co-polymers of
two or more sugars and sugar acids
– glucose, mannose, galactose, xylose, arabinose and
4-O-methylglucuronic acid
They are of made up of 120 – 200 sugars with
short branching chains, making them amorphous
heteropolysaccharides
 Hemicelluloses
These are Heteropolysaccharides
Supporting material in cell walls that
vary from plant to plant and from one
plant part to another.
In woody plants, there are two basic
types
• D glucomannans
• D glucuronoxylans
The composition and amount of each is
species dependent
• Softwood vs Hardwood
 Hardwood VS Softwood
Classifying wood as either a hardwood or softwood comes
down to its physical structure and makeup, and so it is overly
simple to think of hardwoods as being hard and durable
compared to soft and workable softwoods.

Hardwood Softwood

Cellulose

Hemicellulose

Lignin

Extractives
 Softwood Hemicelluloses (major)
The principle hemicellulose of softwoods is the
galactoglucomannans (~ 20% of woody material)
Note: Galactose C4 OH is axial Alternating Glucose & Mannose
Mannose C2 OH is axial along the main chain; Galactose
branches off; Random acetates at
a-D-galactopyranose
C5 & C3 of main chain
HO OH
O
a(1-6) OH b-D-mannopyranose
O
O OH OH b(1-4) OH
O OCCH3
O HO O O HO O O
OH OH OH
HO O O O O O HO O
OH OH CH3C O
b-D-glucopyranose O OH a(1-6)
HO
acetyl group
O
OH OH
a-D-galactopyranose
Note β-1-4 links along the main chain
Softwood Hemicelluloses (major)
The principle hemicellulose of softwoods is the
galactoglucomannans (~ 20% of woody material)
They are subdivided as :
High Galactose content:
Galactose 1/Glucose 1/Mannose/4

Low Galactose content

Galactose 0.1/Glucose 1/Mannose/3
Softwood Hemicelluloses (minor)
The minor hemicellulose of softwoods is the
Arabinoglucuronoxylans (~5-10% of wood)
4-O-methyl-a-D-glucuronopyranose

Note: Xylan has no C6 primary OH CO2H

O OCH3
OH
b-D-xylopyranose a(1-2) OH
OH b(1-4) O
O HO O HO O
O O O
O O O HO O
HO O OH O
OH OH
a(1-3) OH
O Note: Arabinose is a Furan; 5 member
HO ring sugar structure with a C4 primary
CH2OH OH
Note β-1-4 links
a-L-arabinofuranose
along the main chain
Xylanose linked along the
main chain; arabinose and
glucuronic acid branches off
Harwood Hemicelluloses (major)
The principle hemicellulose of hardwoods is
the glucuronoxylans (15-30% of woody material)
COOH 4-O-methyl-a-D-glucuronopyranose
O OCH3
OH
a(1-2) OH
O OH
O O O O HO O
HO O
HO O O O O O HO O
OH OH
b(1-4) O CCH3 O
O
CCH3 b-D-xylopyranose
7
acetyl group

Note β-1-4 links

along the main chain
Hardwood Hemicelluloses (minor)
The minor hemicellulose of hardwoods is the
glucomannans (2-5% of wood)

OH b(1-4) OH OH
O OH
O HO O O HO O O
OH OH OH
HO O O HO O O HO O
OH OH OH
b-D-mannopyranose b-D-glucopyranose

Note β-1-4 links

along the main chain
Lignin
 What is lignin ?
• An aromatic heteropolymer

• Monomers Synthesized from Phenylalanine in the cytoplasm

• Deposition of lignin occurs both within the secondary cell wall

and in the middle lamella

• Mechanical support

• Water impermeable surface

• Unusually stable
contribute to the long life of tree species
terrestrial carbon cycle

• Protection from pathogen or fungi degradation

Bonawitz, N.D. and C. Chapple, The Genetics of Lignin Biosynthesis: Connecting Genotype to Phenotype. Annu Rev Genet, 2010.
 Monomers of lignin

( H lignin )

( G lignin ) ( S lignin )
Three major monomers
Synthesis pathway of lignin monomers
PAL: phenylalanine ammonia lyase
C4H: cinnamate 4-hydroxylase
4CL: 4-coumarate CoA ligase
CHS: chalcone synthase
HCT: hydroxycinnamoyl CoA:shikimate/
quinate hydroxycinnamoyl transferase
C3’H: p-coumarate 3-hydroxylase
CCR: cinnamoyl CoA reductase
CCoAOMT:caffeoyl CoA 3-O-
methyltransferase
CAD: cinnamyl alcohol dehydrogenase
F5H: cinnamoyl CoA reductase
The linkages of lignin monomers

peroxidases

laccase

Coniferyl alcohol radicals

The linkages of lignin monomers
The linkages of lignin monomers
Example of the polymer structure of Lignin
 Characterization
•Difficult to analyze due to heterogeneous linkages

•Highly hydrophobic

•Not exacted from plant tissues by any organic solvent

 RNAi-mediated suppression of p-coumaroyl-CoA 3'-hydroxylase

Autofluorescence UV fluorescence
light microscopy microscopy

WT

RNAi-
C3H
 Research trends

• Improvement of lignin degradation by copolymerizing

alternative units that derived from the incomplete
monomer biosynthesis pathway

• More fundamental research on the biosynthesis

pathway.

• Lignin engineering on end-use applications (pulping,

saccharification…)
Effect of lignin content on sugar and biomass yields
 KAZAKH NATIONAL AGRARIAN UNIVERSITY
FACULTY OF AGROBIOLOGY AND PHYTOSANITARY

DEPARTMENT OF SOIL SCIENCE AND AGROCHEMISTRY

Work teaching program with discipline of

Soil biology – Soil biology plays a vital role in determining many soil
characteristics. The decomposition of organic matter by soil organisms has an
immense influence on soil fertility, plant growth, soil structure, and carbon storage.

Amount of credits: 2

ALMATY, 2015
 Author: Professor Won Chan Kim

Educational methodical complex of discipline was discussed at a meeting of

the Department "Soil Science and Agrochemistry" "29" 09.2015, Protocol №2.

Head of Department A. Balgabayev

"Soil Science and Agrochemistry"

Educational methodical complex of discipline of Professor Won Chan

Kimwas discussed and recommended by the teaching-methodological commission
of Agrobiology and Phytosanitary Faculty

Chairman of Faculty UMK A. Salykova

 CONTENTS
1. Report
2. Form of teaching
3. Materials
4. Main topics

1. REPORT
As a result of this course a student should obtain an in-depth knowledge of the
mechanisms of water, amino acids and proteins, enzymes, nucleotides, nucleic acid, DNA-based
information technologies, plant cell walls, genetic information of plant cell wall biosynthesis,
regulation of plant cell wall biosynthesis by transcription factors. Accordingly, the student will
gain a deeper understanding of fundamental soil biology.

2. FORM OF TEACHING
The course consisted of lectures and practices. Demonstrations of lectures were given with
the help of power point and black board presentations and interactive oral discussions. Students
were encouraged to participate actively in the lectures. Practices were held in a lecture room.
During the practices student had to discuss about what they had learned in lectures. They were
given homework to practice what they had learned and they had to write a brief report. The final
grade is made up of two components: the results of oral test and the reports.

3. MATERIALS
The following materials are available for all students:
1. Lecture notes in the form of PowerPoint slides
2. Book references such as:
- David L. Nelson & Michael M. Cox (2013). Lehninger Principles of Biochemistry (6th
Edition).
- Lincoln Taiz, Eduardo Zeiger and Lan M. Møller (2015). Plant Physiology and
Development

4. MAIN TOPICS
The following main topics were discussed and studied:
1. The foundation of biochemistry.
2. Amino acids, peptides and proteins.
3. Protein function and enzymes.
4. Nucleotides and nucleic acids.
5. DNA-based information technologies.
6. General information of biomass
7. Plant secondary cell wall definition.
8. Plant secondary cell wall biosynthesis.
9. On/off switches for secondary cell wall biosynthesis
10. Regulation of secondary cell wall biosynthesis by transcription factors.
 Types of lessons for students in the topic of “Soil biology”

Amount of credits- 2
Lectures: 10
Practical lessons: 20

Types of lessons Amount

(hours*)
Lectures 10

Practical lessons 20
Final exam (oral test and report) 1

*one academic hour= 50 minutes

Completed by: Professor Won Chan Kim

The teaching program (Syllabus) with discipline of “Soil biology” for Master students in
2015/2016.
1. Basic information

Faculty Faculty of Agrobıology and

Phytosanıtary
Amount of credits 2
Auditorium Building 4, Auditorium 403
Lecturer Professor Won Chan Kim
Teacher of Practical Lessons Professor Won Chan Kim

2. Pro- properties and Post- properties

Pro- properties Biology
Post- properties Molecular biology

3. Aims and requirements of course

Aim of this course was to introduce to the students in-depth knowledge of the mechanisms of
water, amino acids and proteins, enzymes, nucleotides, nucleic acid, DNA-based information
technologies, plant cell walls, genetic information of plant cell wall biosynthesis, regulation of
plant cell wall biosynthesis by transcription factors.

Requirements: oral exam and report

4. Workload for students

Total(3 credits)
Lectures Practical Lessons
Hours length 50 minutes 50 minutes
Number of hours 10 20

5. Content of lessons

1. The foundation of biochemistry.

2. Amino acids, peptides and proteins.

3. Protein function and enzymes.

4. Nucleotides and nucleic acids.

5. DNA-based information technologies.

6. General information of biomass

7. Plant secondary cell wall definition.

8. Plant secondary cell wall biosynthesis.

9. On/off switches for secondary cell wall biosynthesis

10. Regulation of secondary cell wall biosynthesis by transcription factors.

6. Literature (required and recommended)
- David L. Nelson & Michael M. Cox (2013). Lehninger Principles of Biochemistry (6th Edition).
- Lincoln Taiz, Eduardo Zeiger and Lan M. Møller (2015). Plant Physiology and Development

Completed by: Professor Won Chan Kim

List of auditoriums, materials and equipment

Room 403 (lecture and practice), laptop, LCD projector

Head of program: Professor Won Chan Kim

 The teaching – methodological presence map of course (TMPRC)

Code of discipline Number Textbook and Lecturer and teacher of

of teaching aids Practical Lessons
students
49 Computer, LCD Professor Won Chan
projector Kim, PhD

Head of program: Professor Won Chan Kim

 The thematic plan of the course Soil biology

Auditorium lessons: 30 hours

Includes lectures: 10 hours and
Practical lessons: 20 hours
Exam: 1 hour

Lectures- 10 hours
Presentation
No Lecture topics Hours Literature for students
Material
Power Point Lehninger Principles of
1 The foundation of biochemistry 1
Presentation Biochemistry
Amino acids, peptides and Lehninger Principles of
Power Point
2 proteins 1 Biochemistry
Presentation
Lehninger Principles of
Protein function and enzymes Power Point
3 1 Biochemistry
Presentation
Lehninger Principles of
Nucleotides and nucleic acids Power Point
4 1 Biochemistry
Presentation
DNA-based information Lehninger Principles of
Power Point
5 technologies 1 Biochemistry
Presentation
Plant Physiology and
General information of biomass Power Point
6 1 Development
Presentation
Plant secondary cell wall Plant Physiology and
Power Point
7 definition 1 Development
Presentation
Plant secondary cell wall Plant Physiology and
Power Point
8 biosynthesis 1 Development
Presentation
On/off switches for secondary Plant Physiology and
Power Point
9 cell wall biosynthesis 1 Development
Presentation
Regulation of secondary cell
Power Point Plant Physiology and
10 wall biosynthesis by 1
Presentation Development
transcription factors.
 Practical Lessons – 20 hours
Topics of Practical Equipment and Literature for
No Hours
Seminars material students
The foundation of
Power Point
1 biochemistry 2 -
Presentation
Amino acids, peptides and
Power Point
2 proteins 2 -
Presentation
Protein function and
Power Point
3 enzymes 2 -
Presentation
Nucleotides and nucleic
Power Point
4 acids 2 -
Presentation
DNA-based information
Power Point
5 technologies 2 -
Presentation
General information of
Power Point
6 biomass 2 -
Presentation
Plant secondary cell wall
Power Point
7 definition 2 -
Presentation
Plant secondary cell wall
Power Point
8 biosynthesis 2 -
Presentation
On/off switches for
secondary cell wall Power Point
9 2 -
biosynthesis Presentation

Regulation of secondary
Power Point
10 cell wall biosynthesis by 2 -
Presentation
transcription factors.

The thematic plan of Soil biology viewpoint was discussed and approved on a meeting of
department of soil sience, agro chemistry and ecology.

Completed by: Professor Won Chan Kim