http://smtom.lecture.ub.ac.

id/
Password: s1-biochem

Plant Biochemistry
Lecture 1: General Introduction

Science without religion is lame,

religion without science is blind.
--Albert Einstein
S.M. Sitompul
 LEARNING OUTCOMES
Students on completion of this course would be
able
1. to explain plant biochemistry
2. to identify the basic molecules which make up
plant
3. to describe the way in which chemical
components are synthesized and utilized by
plants in the life process
4. to describe the process of pant life on a
chemical level
5. to initiate ways from the standpoint of
biochemistry to improve the growth of plants or
to solve problems in plant growth
 LECTURE FLOW
LEARNING OUTCOMES
1.Definition
2.Course Plan
 Subjects
 References
3.Introduction
Basic Principle
Biomolecules
What Is Biochemistry Used For?
The Core of Plant Biochemistry
Breakthroughs In Biochemistry
4.Examples of Biochemistry
 1. COURSE PLAN
NO. TOPICS WEEK
1. INTRODUCTION 1
2. ENZYME 2-4
3. CARBOHYDRATE 5-6
4. BIOSYNTHESIZE OF 7
NUCLEOTIDE
MID SMESTER EXAM 8
5. LIPID 9-10

6. NITROGEN METABOLISM 11

7. BIOLOGICAL N FIXATION 12

8. AMINO ACIDS 13
9. NUCLEIC ACID 14
10. PROTEIN 15

END SMESTER EXAM 16

 REFERENCES
1. Conn, E.E. & Stumpf, P.K., 1976. Outlines of
Biochemistry. John Wiley & Sons, New York.
2. Goodwin, T.W. & Mercer, E.I., Introduction to Plant
Biochemistry. Pergamon Press, Oxford.
3. Stryer, L., 1975. Biochemistry. W.H. Freeman and
Company, San Francisco
4. Wood, W.B., Wilson, J.H., Benbow, R.M., & Hood,
L. E., 1981. Biochemistry A Problems Approach.
5. Wood, J.H, Keehan, C.W., Bull, W.E. and Bowman,
N.S., 1963. Fundamentals of College Chemistry. A
Harper International edition, Harper & Row, NY,
Evanston & London and John Weatherhill, Inc.,
Tokyo
 2. Definition
What is Plant Biochemistry ?
Plant Biochemistry, or the chemistry of living
plants, is
1. the study of the chemistry of living things (types,
structures & reactions)
2. the study of the process of plant life on a
chemical level
3. the study of molecular basis of plant life or the
study of the way in which chemical components
are synthesized and utilized by plants in the life
process (growth & development).
 3. INTRODUCTION
1. Basic Principle
• Living organisms, whether they are plants,
animals or microbes, are made up basically
of the same chemical components
• Biochemical Reactions
Urease catalyzes the hydrolysis of urea
H2N
Urease
C O + 3 H2O 2NH4++OH-+HCO3-
H2N
Urease from jack beans (Canavalia
ensiformis) was the first enzyme ever
purified and crystallised, an
achievement of James B. Sumner in
1926 who earned a Nobel Prize in
Chemistry in 1946
2. Biomolecules
What are Types of Molecules studied in Biochemistry?
 The principal types of biological molecules, or
biomolecules are:
 carbohydrates
 lipids
 proteins
 nucleic acids

 Many of these molecules are complex

molecules called polymers which are made up
of monomer subunits
 Biochemical molecules are principally based
on carbon.
 carbo lipids proteins nucleic acids
monomer glucose fatty acid amino acid nucleotide

polymer cellulose phospholipid protein subunit DNA

supramolecular
structure cell wall membrane protein complex chromosome
 monomer glucose

polymer cellulose

supramolecular
structure cell wall

• Cellulose is the major

structural material of
plants. Wood is largely
cellulose, and cotton is
almost pure cellulose.
 One Fatty Acid

 The “head” of the molecule is a carboxyl group

which is hydrophilic.
 The “tail” of a fatty acid is a long hydrocarbon
chain, making it hydrophobic.
 Fatty acids are the main component of soap, where
their tails are soluble in oily dirt and their heads are
soluble in water to emulsify and wash away the oily
dirt. However, when the head end is attached to
glycerol to form a fat, that whole molecule is
hydrophobic.
 monomer fatty acid

polymer phospholipid

supramolecular
membrane
structure
 Description Saturated Fats Unsaturated Fats
Saturated fats are fats with a Unsaturated fats are fats with
Definition: single bond between the one or more double bonds
carbon atoms of the fatty acids between the fatty acids
Excessive consumption is not
good because of their Unsaturated fats are
Health: association with considered good to eat if you
atherosclerosis and heart are watching your cholesterol
diseases.
Saturated fats increase LDL Unsaturated fats increase HDL
Cholesterol: (bad cholesterol) and decrease (good cholesterol) and
the HDL decrease LDL
Form: Solid at room temperature Liquid at room temperature
Derived from: Mostly from animal products Plants
contains one or more double
contains only single bonds
Hydrocarbon bonds between carbon atoms
between carbon atoms, no
chain: -monounsaturated -
double bonds (ex: stearic acis)
polyunsaturated
Commonly Butter, coconut oil, breast milk, Avocado, soybean oil, canola
found in: meat oil, olive oil
These are long lasting and do
Life: not get spoiled quickly
These get spoiled quickly

Recommended Not more than 10% of total Not more than 30% of total
consumption: calories per day. calories per day
lipids
Saturated
Formula Common Name Melting Point
CH3(CH2)10CO2 H lauric acid 45 ºC
CH3(CH2)12CO2 H myristic acid 55 ºC
CH3(CH2)14CO2 H palmitic acid 63 ºC
CH3(CH2)16CO2 H stearic acid 69 ºC
CH3(CH2)18CO2 H arachidic acid 76 ºC
Unsaturated
Formula Common Name Melting Point
CH3(CH2)5CH=CH(CH2)7 CO2 H palmitoleic acid 0 ºC
CH3(CH2)7CH=CH(CH2)7 CO2 H oleic acid 13 ºC
CH3(CH2)4CH=CHCH2CH=CH
linoleic acid -5 ºC
(CH2)7CO2H
CH3CH2CH=CHCH2CH=CHC
linolenic acid -11 ºC
H2CH=CH(CH2)7CO2 H
CH3(CH2)4(CH=CHCH2)4 (CH2)
arachidonic acid -49 ºC
2CO2H
Omega-3 and Omega-6 Fatty Acids
Cis and Trans Bonds
 monomer amino acid

polymer protein subunit

supramolecular
structure Enzyme complex
 monomer nucleotide

polymer DNA

supramolecular
chromatin
structure
3. Importance of Plant Biochemistry
What Is Biochemistry Used For?
1. Biochemistry is used to learn about the biological
processes which take place in cells and organisms.
2. Biochemistry may be used to study the properties
of biological molecules, for a variety of purposes.
For example, a biochemist may study the
characteristics of the keratin in hair so that a
shampoo may be developed that enhances
curliness or softness.
3. Biochemists find uses for biomolecules. For
example, a biochemist may use a certain lipid as a
food additive.
 4. Alternatively, a biochemist might find a substitute
for a usual biomolecule. For example, biochemists
help to develop artificial sweeteners.
5. Biochemists can help cells to produce new
products. Gene therapy is within the realm of
biochemistry. The development of biological
machinery falls within the realm of biochemistry.

4. The core of plant biochemistry

The core of biochemistry is the conversion of
substrates to products through biochemical
reactions which catalyzed by enzymes in
most cases.
 Isolation and Identification
1. Biochemistry is firstly concerned with the isolation
and identification of all different substances which
make up plant and animal organisms
A living organism is composed of more than just fasts,
carbohydrates and protein. Hundreds of other
substances are necessary to the proper functioning of
the organisms

 Chemical Changes
2.Secondly, biochemistry is concerned with all chemical
changes which take place in the cells to provide for
energy, growth, reproduction, and aging.
Protoplasm is an aqueous solution of certain substances
with other colloidally dispersed substances
 PLANT
BIOCHEMISTRY
 5. Breakthroughs in Biochemistry
Two notable breakthroughs in the history of
biochemistry
1. Discovery of the role of enzymes as
catalysts
2. Identification of nucleic acids as
information molecules

Flow of information: from nucleic acids to

proteins

At lunch Francis [Crick] winged into the Eagle to tell everyone within
hearing distance that we had found the secret of life. — James Watson
• Two polynucleotides
associate to form a
double helix
• Genetic information is
carried by the sequence
of base pairs
 What is Cloning?
 Cloning is to make a genetically identical
organism through non-sexual means.
 Cloning of African violets:

 Take a leaf from a plant

 Immerse the stalk in water

• Roots start to form after a

week
• Pot the plant
• A new plant is produced
 How Dolly was cloned?
Egg cell

Parent cell

Sel telur dengan inti dari induk yang berkembang

menjadi anak domba yang sama dengan induknya
How Dolly was cloned?
4. EXAMPLES OF
PLANT
BIOCHEMISTRY
a. a-Amylase

b. Cellulose
c. PIGMENT ADENIUM OBESUM ' CHERRY'
Grafted Desert Rose
Family : Apocynaceae
Origin : East Africa
Size : 5'
Light Requirements : Full Sun/Light
Shade
Water Requirements : Keep Dry
Min. Temp. : 35°
Flower : Year Round

Pigment Class Compound Type Colors

Porphyrin chlorophyll green
Carotenoid carotene and yellow, orange, red
lycopene yellow
xanthophyll
Flavonoid flavone yellow
flavonol yellow
anthocyanin red, blue, purple,
magenta
 d. Gugur Daun
 Perusakan dinding sel Auxin
pada lapisan absisi
oleh aktivitas enzim
Ethylene
Cellulase dan
Polygalacturonase
 Sintesis kedua enzim
tersebut terhambat Cellulase/Polygalacturonase
jika kadar hormon
tumbuh auxin cukup
tinggi

• Transpor auxin dari tempat pembentukan pada

bagian ujung daun ke lapisan absisi dihambat
oleh hormon ethylene
 e. The Narcotic Analgesics
 Narcotics block the transmission of the
nerve signal across nerve gaps, [the minor
analgesics blocked prostaglandin
synthesis]
 The more important ones:
 Morphine, codeine,
 oxycodone (PERCODAN), hydromorphone (DILAUDID),
methadone, + heroin [ = not legal]
 meperidine (DEMEROL), pentazocine (TALWIN),
 fentanyl (SUBLIMAZE), buprenorphine (BUPRENEX)
 Morphine:
 Opium [est. ~ 10,000 tons] extracted
from the poppy Papaver somniferum,
Afghanistan spring 06 6100 tons
alone.
 Morphine goes to receptors (opiate receptors) which
control passage of Ca2+ and K + through channels, which in
turn control acetylcholine (nerve transmitter) flow across
synapses.

DEPRESSES RESPIRATORY SYSTEM - usual overdose

effect; some euphoria - plus is addictive

Komunikasi saraf (neuron & nerve cells) antara satu dengan yang
lain, atau dengan yang lain (kelenjar, otot & organ tubuh lain) terjadi
melalui pelepasan zat, “neurotransmitters”, pada reseptor dari neuron
atau organ bersangkutan. Suatu zat yang secara mengyakinkan
berfungsi sebagai neurotransmitter adalah Acetylcholine.
 f. Cyanide Poisoning
 Disrupts metabolism by inhibiting metal
containing enzymes, most notably,
cytochrome oxidase.
 Cytochrome A3 catalyzes O2  H2O
 Blocks ability of mitochondria to use O 2
 O2 saturation may be normal
 Poisoning can occur through
percutaneous absorption and inhalation.
 Degree of symptoms depends on
severity of exposure.
Antidote
Specific antidotes available
Sodium Nitrate injected
Cytochrome
Oxyhemoglobin Metemoglobin oxidase

Sodium Thiosulfate injected CN

Cyano-methemoglobin
Rodonase (low toxicity)

Thiocyanate Kidneys

1. Sodium nitrite reacts with hemoglobin to form

methemoglobin that removes cyanide ions from various
tissues to form cyanmethemoglobin (relatively low toxicity).
2. The function of Sodium thiosulfate is to convert cyanide to
thiocyanate, by an hepatic enzyme known as rhodanese
 g. Methanol Poisoning
 Methanol itself has a relatively low degree of toxicity,
but it is metabolized to formic acid which is responsible
for the acidosis and blindness that characterizes
methanol poisoning.
 The initial step in the metabolism of methanol occurs by
the action of alcohol dehydrogenase (ADH).
 h. What is Biodiesel?
 Alternative fuel for diesel engines
 Made from vegetable oil or animal fat
 Meets health effect testing (CAA)
 Lower emissions, High flash point (>300F), Safer
 Biodegradable, Essentially non-toxic.
 Chemically, biodiesel molecules are mono-alkyl esters
produced usually from triglyceride esters
Biodiesel Samples
 Chemistry of Triglycerides
 Biodiesel is made from the combination of a triglyceride
with a monohydroxy alcohol (i.e. methanol, ethanol…).
 What is a triglyceride? Made from a combination of
glycerol and three fatty acids:
 Transesterification
While actually a multi-step process, the overall reaction looks like this:

CH2OOR1 catalyst CH2OH

|  |
CHOOR2 + 3CH3OH  3CH3OORx + CHOH
| |
CH2OOR3 CH2OH
Triglyceride 3 Methanols Biodiesel Glycerin

R1, R2, and R3 are fatty acid alkyl groups (could be different, or the
same), and depend on the type of oil. The fatty acids involved
determine the final properties of the biodiesel (cetane number, cold
flow properties, etc.)
 Individual step of Transesterification
First step, triglyceride turned into diglyceride, methoxide (minus Na) joins
freed FA to make biodiesel, Na joins OH from water (from methoxide
formation) to make NaOH. Other H joins the diglyceride.

H O H
| | |
HCOR1 H HCO H O
| | | | |
HCOOR2 + HCONa +H2O  CHOOR2 + HCOR1 + NaOH
| | | |
HCOR3 H HCOR3 H
| | | |
H O H O

Triglyceride + Methoxide + H2O  Diglyceride + Biodiesel + NaOH

 Penyakit encok (gout) yang mengakibatkan
radang pada persendian adalah akibat akumulasi
asam urat
 Radang sendi dipicu
oleh presipitasi kristal
urat natrium (sodium
urate crystals)
 Penyakit Ginjal dapat
juga terjadi karena
deposisi kristal urat
dalam organ tersebu
THANK YOU
Спасибо
 LECTURE 2:
INTRODUCTION TO ENZYME

A model for the structure of rubisco in chloroplasts from higher plants

 LECTURE FLOW
Competency
1.DEFINITION
2.INTRODUCTION
1. History of Enzyme
2. Papain
3.ENZYME PROPERTIES
A. Enzyme Structure
B. Enzyme Characteristics
 1. DEFINITION
● Enzymes are metabolic catalysts which
increase the rate of chemical reactions by
lowering the activation energy of that
reactions
● An enzymes (in yeast) is made up of protein
that acts as a catalyst to cause certain
desired reaction
● Enzymes are the largest and most highly
specialized catalysts in the body for the
reactions involved in metabolism
 2. INTRODUCTION
 Discovery of enzymes
 Eduard Buchner
 Buchner discovered in 1897 that yeast extracts
could ferment sugar to alcohol:
C6H12O6 → 2C2H5OH + 2CO2 + 2 ATP
 Buchner’s finding showed that fermentation was
promoted by molecules that continued to function
when removed from cells.
 James Sumner
 In 1926, Sumner isolated and crystallized jack
bean urease, which catalyzes the reaction:
CH4N2O + H2O → 2NH3 + CO2
  The
urease crystals contained only protein, leading
Sumner to propose that all enzymes are proteins.

 PAPAIN
What is papain?
 Papain is a protein-cleaving
enzyme derived from papaya
and certain other plants.
 Enzymes are complex
molecules produced in living
organisms to catalyze (speed
up) chemical reactions within
the cell
PAPAIN

 Why is meat tough?

 Muscles have to endure a lot of mechanical stress, and
are made of strong fibers that are hard to cut, and tough
connective tissue holds them together
 Individual muscle cells contain microscopic fibrils that
give them their structural integrity and allow them to
contract.
 The fibrils have a complex internal structure bound
together by long protein chains. The connective tissue
that holds the muscle together is also mostly protein.
 How does papain tenderize meat?
 Papain cuts the protein chains in the fibrils and also in
the connective tissue, disrupting the structural integrity
of the muscle fiber, and tenderizing the meat.
b. Enzyme Nomenclature
 Most other enzymes are named for their
substrates and for the reactions that they catalyze,
with the suffix "ase" added. (ATPase )
 Six major classes of enzymes (Superfamilies:
EC 1.1.3.4)

1. Transferases 4. Lyases
 Transfer functional  Elimination reactions
groups between to form double bonds
molecules 5. Isomerases
2. Oxidoreductases  Intramolecular
 Transfer electrons rearangements
(RedOx reactions) 6. Ligases
3. Hydrolases  Join molecules with
 Break bonds by new bonds
adding H2O
 Enzyme Commission Number
(EC Number)
 Every enzyme code consists of the letters "EC" followed by four
numbers separated by periods. Those numbers represent a
progressively finer classification of the enzyme. Preliminary EC
numbers exist and have an 'n' as part of the fourth (serial) digit (e.g.
EC 3.5.1.n3).
 For example, the tripeptide aminopeptidases have the code "EC
3.4.11.4", whose components indicate the following groups of
enzymes:
EC 3 enzymes are hydrolases (enzymes that use water to break up
some other molecule)
EC 3.4 are hydrolases that act on peptide bonds
EC 3.4.11 are those hydrolases that cleave off the amino-
terminal amino acid from a polypeptide
EC 3.4.11.4 are those that cleave off the amino-terminal end from
a tripeptide
 Transferases
Aminotransferases: transferases catalyzing the amino acid degradation by
removing amino groups.
 Oxidoreductases
Alcohol dehydrogenase: an oxidoreductase converting alcohols to
aldehydes/ ketones.
 Hydrolases
Glucose-6-phosphatase: a hydrolase that removes the phosphate group
from glucose-6-phosphate, leaving glucose and H3PO4.
 Lyases
Pyruvate decarboxylase: a lyase that removes CO2 from pyruvate.
 Isomerases
Ribulose phosphate epimerase: an isomerase that catalyzes the
interconversion of ribulose-5-phosphate and xylulose-5-phosphate.
 Ligases
Hexokinase: a ligase that catalyzes the interconversion of glucose and
ATP with glucose-6-phosphate and ADP.
 2. ENZYME PROPERTIES
A. Enzyme Structure
1. Proteins
2. Cofactors
3. Active Site
B. Enzyme Characteristics
a. Critical components of cells
Almost all reactions in living organism/plants are catalyzed
by enzymes)
b. Very efficient
c. Reduce G for reaction (by binding the transition
state)
d. Enzymes are very specific
e. Subject to regulatory control of various sorts
 A. Enzyme Structure NADH

Activator
(Metal ion)
Protein
M+
molecule
Organic

Active
site Substrate

Apoenzyme Coenzyme

Apoenzyme + Coenzyme = Holoenzyme

1. Protein
 All known enzymes are high molecular weight
compounds that are made up principally of
proteins (except for catalytic RNA)
 Apoenzyme: The polypeptide or
protein part of the enzyme which
may be inactive in its original
synthesized structure
 The inactive form of the
apoenzyme is known as a
proenzyme or zymogen.
 The proenzyme may contain several extra amino
acids in the protein which are removed, and allows
the final specific tertiary structure to be formed before
it is activated as an apoenzyme.
2. Cofactors
 Many enzymes require the presence of other
compounds - cofactors - before their catalytic activity
can be exerted
 This entire active complex is referred to as the
holoenzyme; apoenzyme (protein portion) + the
cofactor (coenzyme, prosthetic group or metal-ion-
activator)
Apoenzyme + Cofactor = Holoenzyme
The cofactor may be:
1. A coenzyme - a non-protein organic substance which is
loosely bound to the protein part, dialyzable, and
thermostable
2. A prosthetic group - an organic substance which is
firmly bound to the protein or apoenzyme portion,
dialyzable and thermostable.
3. A metal-ion-activator - these include K+, Fe++, Fe+++,
Cu++, Co++, Zn++, Mn++, Mg++, Ca++, and Mo+++
3. Enzyme Active Sites
● The active site is
 the specific area of the enzyme to which the
substrate attaches during the reaction
 part of the conformation of the enzyme molecule
arranged to create a special pocket or cleft
whose three-dimensional structure is
complementary to the structure of the substrate
● The enzyme and the substrate molecules
"recognize" each other through this structural
complementarity
● The substrate binds to the enzyme through
relatively weak forces -H bonds, ionic bonds (salt
bridges), and van der Waals interactions between
sterically complementary clusters of atoms.
 Sucrase
active site

 Lysozyme active site: Green shows

substrate contacts and orange are
catalytic residues
B. Characteristics
a. Critical Components
Enzymes are critical for every aspect of cellular life (cell
metabolism & biological processes)
b. Very efficient catalysts
A very small quantity of an enzyme can catalyze
the transformation of vastly lager quantity of the
substrate
 Sucrase
 Catalase

 Sucrase (invertase)
can effect the
hydrolysis of at least
1,000,000 times its
own weight of sucrose
without exhibiting any
appreciable diminution
in its activity
  Catalase is one of the more efficient enzymes,
one molecule of this enzyme being able to
catalyze the conversion 5,000,000 molecules of
H202 per minute (the reduction of hydrogen
peroxide to water and molecular oxygen) when
conditions are favorable

Reaction Function of Rate

Enzyme
catalyzed reaction *
removes toxic H2O2
Catalase 2H2O2  2H2O + O2 1015
from cell
Carbonic hydrates CO2 gas
CO2 + H2O H2CO3 107
anhydrase for transport
* rate of enzyme catalyzed compared to rate uncatalyzed
c. Activation Energy
 Enzymes reduce the activation energy of a
reaction so that the kinetic energy of most
molecules exceeds the activation energy
required and so they can react.
 For example, for the catalase reaction
2H2O2 → 2H2O + O2
The activation energy is
86 kJ mol-1 with no catalyst
62 kJ mol-1 with an inorganic catalyst
1 kJ mol-1 with the enzyme catalase
 RATE of reaction is affected by enzyme
 RATE depends on ΔG‡, the Arrhenius activation
energy (i.e., the free energy of activation for the
reaction).
G‡ = activation energy

- enzyme
G‡ –enzyme
ENERGY

G‡
+enzyme
+ enzyme

REACTANT Overall energy

e.g. CO2 + H2O released

PRODUCT
H2CO3

REACTION PROGESS
Gibb’s Free Energy Change (G)
 Enzymes decrease activation energy
(ΔG‡) for reactions catalyzed. G'0
K’eq (KJ/mol)
 K’°eq = [P]/[S] 10-6 34.2
 From thermodynamics, the 10-5 28.5
equilibrium constant and the free 10-4 22.8
energy are related by the expression 10-3 17.1
ΔG’° = -RTln K’°eq 10-2 11.4
= -2.3RTlog K’°eq 10-1 5.7
1 0
 ΔG = overall difference in free energy
10 -5.7
between final (P) and starting (S), not
102 -11.4
affected by enzyme.
103 -17.1
Enzymatic catalysts have much higher rates
than non-enzymatic catalysts do, and even at
relatively low temperatures
 UREA FERTILIZER
 The optimum conditions for enzyme catalysis are almost
invariably moderate temperatures, and pHs which are not
extreme
 The contrast between a reaction catalysed by an enzyme
and by a non-enzymatic catalyst is well illustrated by the
process of nitrogen fixation (i.e. reduction of N2 to
ammonia). Nitrogenase catalyses this reaction at
temperatures around 300 K and at neutral pH. The enzyme
is a complex system comprising two dissociating protein
components one of which contains iron and the other iron
and molybdenum. Several molecules of ATP are
hydrolyzed during the reduction
 By contrast, in the industrial synthesis of ammonia from
nitrogen and hydrogen, the conditions used are as follows:
temperature 700 - 900 K, pressure 100 - 900 atmospheres,
and the presence of an iron catalyst, often promoted by
traces of oxides of other metals
d. Enzyme Specificity
Enzymes very specific
– for substrate acted upon
– for reaction catalyzed
Example: Proteases are a whole class of enzymes that all
catalyze hydrolysis of peptide bonds:
Specificity class
● Absolute specificity - the enzyme will catalyze only
one reaction.
● Group specificity - the enzyme will act only on
molecules that have specific functional groups, such
as amino, phosphate and methyl groups.
● Linkage specificity - the enzyme will act on a
particular type of chemical bond regardless of the
rest of the molecular structure.
● Stereochemical
specificity - the enzyme
will act on a particular
steric or optical isomer.

Enzyme Specificity
e. Enzyme Regulation
 Enzymes are tightly regulated light switches
 Unregulated enzymes become constitutively
active or inactive (light is always on or off )
 Unregulated enzyme activity disrupts cell
homeostasis and often lead to disease states.
 Unregulated enzyme
activity disrupts cell
homeostasis and
often lead to disease
states.
THANK YOU
Спасибо
 LECTURE 3:
ENZYME KINETICS
V0
V0 = Vmax

V
½Vmax

k1 k3
E+S ES E+P
k2

KM [S]
 LECTURE FLOW
 Enzyme-Substrate Interaction
1. Lock and Key" Hypothesis
2. The "Induced Fit" Hypothesi
 Enzyme Kinetics
 Michalis-Menten Equation
 Double-reciprocal or Lineweaver-Burk
 Eadie-Hofstee
 Hanes-Woolf
 INTRODUCTION

Vmax

½Vmax [O ].s-1
http://www.demochem.de/kinetics.htm 2

KM [H2O2]
 Enzyme-Substrate Interaction
1. Lock and Key" Hypothesis
2. The "Induced Fit" Hypothesis

1. "Lock and Key" Hypothesis

 Emil Fischer in 1890 proposed "Lock and Key"
Hypothesis
active site substrates

 The shape, or configuration, of the active site is

especially designed for the specific substrate
involved.
 Because the configuration is determined by the
amino acid sequence of the enzyme, the native
configuration of the entire enzyme molecule must be
intact for the active site to have the correct
configuration.
 In such a case, the substrate then fits into the active
site of the enzyme in much the same way as a key
fits into a lock.

P1
E S E S E

P2
2. The "Induced Fit" Hypothesis
 Enzymes are highly flexible, conformationally dynamic
molecules, and many of their remarkable properties,
including substrate binding and catalysis, are due to their
structural pliancy.

P1
S
E S E E
P2
Transition conformation

 Realization of the conformational flexibility of proteins led

Daniel Koshland to hypothesize that the binding of a
substrate (S) by an enzyme is an interactive process. The
shape of the enzyme's active site is actually modified upon
binding S, in a process of dynamic recognition between
enzyme and substrate aptly called induced fit.
 ATP

ATP
lyase or
ATPase

Mg(2+)
 In essence, substrate binding alters the conformation
of the protein, so that the protein and the substrate "fit"
each other more precisely. The process is truly
interactive in that the conformation of the substrate
also changes as it adapts to the conformation of the
enzyme.

Induced Fit Model

 Enzyme Kinetics
 Basic Prinsiple
 Enzyme kinetics is the study of the chemical reactions that are
catalyzed by enzymes or the study of the rates of enzyme-
catalyzed reactions
 This study provides information on enzyme specificities and
mechanisms
 The formation of ES complex is the base of enzymic reactions

E+S ES E+P
What is the rate of reaction (V) if

Case 1 Case 2
[S] is constant [S] increases
[E] increases [E] is constant
V = dP/dt
Zero order
SP
dS dP
V  k
dt dt
S  t
dS  kdt  dS  k  dt
S 0
S  kt  C 0

Why are enzyme kinetics important?

1. It provides valuable information for enzyme mechanism
2. It gives an insight into the role of an enzyme under
physiological conditions
3. It can help show how the enzyme activity is controlled and
regulated
The influence of [E]
V
These reactions are said to be
"zero order" because the rates
are independent of substrate
concentration, and are equal to
some constant k [S] is constant

[E]

Order Rate Comments

k rate is independent of substrate
zero concentration
k[S] rate is proportional to the first power of
first substrate concentration
2 rate is proportional to the square of the
second k[S][S]=k[S] substrate concentration
rate is proportional to the first power of
k[S1][S2] each of two reactants
Enzyme Kinetics

 Enzymes follow zero order kinetics when

substrate concentrations are high.
 Zero order means there is no increase in the rate of
the reaction when more substrate is added.
 Given the following breakdown of sucrose to
glucose and fructose

Sucrose + H20 → Glucose + Fructose

H
H H
O
OH
HO H OH
H
OH H
H OH H
HO
H O
HO
HO H
H OH

H OH
 The reaction is reversible which means some
substance can be synthesized from the substrate in the
reaction
 If environmental factors are constant, the rate of
product formation (reaction velocity, V) is dependent
upon the concentration of enzyme and substrate
 [E] = enzyme concentration
 [S] = substrate concentration
 The influence of [S]
 The concentration of substrate [S] greatly
influences the rate of product formation (the
velocity of a reaction, V)
 Studying the effects of [S] on the velocity of a reaction
is complicated by the reversibility of enzyme
reactions, e.g. conversion of product back to
substrate
 To overcome this problem, initial velocity (vo)
measurements are used. At the start of a reaction,
[S] is in large excess of [P], thus the initial velocity of
the reaction will be dependent on substrate
concentration
 When initial velocity is plotted against [S], a
hyperbolic curve results, where
 Vmax represents the V0
V0 = Vmax
maximum reaction velocity
V
 This occurs at [S] >> E, and
all available enzyme is

½Vmax
"saturated" with bound
substrate, meaning only the
ES complex is present.
KM [S]

High [S]
Saturating [E]
Low [S] 50% [S]
Km
 MICHAELIS-MENTEN MODEL
 Lenore Michaelis and Maud L. Menten proposed
a general theory of enzyme action in 1913
1. The formation of ES Complex
 Their theory was based on the assumption that the
enzyme (E) and its substrate (S) associate reversibly
to form an enzyme-substrate complex, ES

k1 k3
E+S ES E+P
k2 k4
E = Enzyme, S = Substrate, P = Product
ES = Enzyme-Substrate complex , and k1, k 2, k3 & k4 = rate
constants. k4 is very small and ignored
 This association/dissociation is assumed to be a
rapid equilibrium, and Ks is the enzyme :
substrate dissociation constant. At equilibrium,
k2[ES] = k1[E][S]
and [E][S] k 2
KS  
[ES] k 1

2. Steady-State Assumption
 The interpretations of Michaelis and Menten were
refined and extended in 1925 by Briggs and Haldane,
by assuming [ES] quickly reaches a constant value in
such a dynamic system.
  That is, ES is formed as rapidly from E + S, and
disappears by its two possible fates
 dissociation to regenerate E + S, and
 reaction to form E + P
 This assumption is termed the
steady-state assumption and is
expressed as d[ES]
0
dt
3. Initial Velocity Assumption
 Because enzymes accelerate
the rate of the reverse reaction
as well as the forward reaction,
the conversion of E+P to ES,
and k4(E][P] = 0
 k1 k3
E+S ES E+P
k2
 However, if we observe only the initial velocity for the
reaction immediately after E and S are mixed in the
absence of P, the rate of any back reaction is
negligible because its rate will be proportional to [P],
and [P] is essentially 0
 Given such simplification, we now analyze the system
described by equation above in order to describe the
initial velocity v as a function of [S] and amount of
enzyme.
4. Total Enzyme
 The total amount of enzyme is fixed and is given by
the formula
[E] = free enzyme and [ES] = the amount of
[E]0 = [E] + [ES] enzyme in the enzyme-substrate complex.
The rate of product formation is dependent upon
[ES] and k3
dP [ES] is the difference between
v  k 3 ES the rates of ES formation minus
dt the rates of its disappearance.

dES
 k1 ES  k 2 ES  k 3 ES
dt
The assumption of steady state gives
The rate of [ES] formation = The rate of [ES] formation
ES = k1[E][S] = ES = (k2 + k3) (ES)
Michaelis-Menten Cosntant
KM = (k2 + k3 )/k1
Michaelis-Menten Equation Vmax [S]
V
Vmax
KM  [S]
V
½Vmax

KM [S]
 The best substrate for enzyme is that which has the
highest Vmax/KM
Enzyme Vmax KM Vmax/KM
Chymotrypsin 100 5000 1/50
Carbonic Anhydrase 600,000 8000 600/8

1. KM
Michaelis-Menten Equation:
V = Vmax[S]/(KM+[S]) This means that
 If V = ½Vmax KM is equal to the
 ½Vmax = Vmax[S]/(KM+[S]) substrate
concentration at
 ½(KM+[S]) = [S]KM+[S] = 2[S]
V = ½ Vmax
 KM = [S]
2. KM = (k2 + k3)/k1
k1 k3
E+S ES E+P
k2
 The rate-determining step of the reaction is k 3, for
the formation of product
so if k2 >> k3  KM = k2/k1
 k2/k1 is known as a dissociation constant for the ES
complex
 k2/k1 reflects a tendency of ES complex to dissociate to
be E and S,
 KM = k2/k1 can be used as a relative measure of the
affinity of a substrate for an enzyme
 Low KM  high apparent affinity of a substrate
for an enzyme
 High KM  low apparent affinity of a substrate
for an enzyme
Enzyme Substrate Km (μM)
pyruvate 400
Pyruvate carboxylase HCO3- 1000
ATP 60
3. Turnover number
k3 = Vmax/[E]0
Moles of substrate transformed per second per mole of
active site, or the number of substrate molecules
converted to P by an E molecule in a unit time when E is
fully saturated.
 Penetuan KM dan Vmax
 Harga KM bervariasi sangat besar, tapi dari
kebanyakan enzim berkisar diantara 10-1 - 10-6
M (Tabel 2.1) tergantung substrat dan
lingkungan seperti suhu dan kuantitas ion
 Untuk mendapatkan harga KM dan Vmax,
analisis langsung persamaan diatas dapat
dilakukan, tapi cara ini membutuhkan waktu
yang lama, dan bantuan komputer sangat
penting untuk mengoptimasi harga parameter
persamaan dengan cepat.
Tabel 2.1 Parameter beberapa enzim
 PENDEKATAN LAIN
 Linierisasi persamaan
Modifikasi persamaan ke bentuk linier
sehingga dapat dianalisis dengan
mudah
1. Persamaan “double-reciprocal”
atau “Lineweaver-Burk”
2. Persamaan “Eadie-Hofstee”
3. Persamaan “Hanes-Woolf”
 Persamaan “double-reciprocal”
atau “Lineweaver-Burk”
Vmax [S]
V
KM  [S]
 Jika ruas kiri dibalik dan demikian juga
ruas kanan, maka
1 KM 1 1
 . 
V Vmax [S] Vmax

 y=a+bx
 Sekarang
y = 1/V ; x = 1/[S]
a = 1/Vmax ; b = KM/Vmax
dapat dianalisis dengan y = a + bx
Jika 1/V dihubungkan
dengan 1/[S], suatu
garis lurus akan 1/V
dihasilkan yang KM/Vmax
memotong sumbu y
pada 1/Vmax dan
sumbu x pada -1/KM
serta membentuk 1/Vmax
sudut terhadap sumbu -1/KM
x sebesar KM/Vmax.

1/[S]
Persamaan “Eadie-Hofstee”
Vmax [S]
V
KM  [S]
V ( K M  [ S ])  V max [ S ]
V[S]   VK M  Vmax [S]
 VK M  Vmax [S]
V
[S]

V
V  K M  Vmax
[S]
 Sekarang
y = V ; x = V/[S]
a = Vmax ; b = -KM
dapat dianalisis dengan y = a + bx
Vmax
Jika V dihubungkan
dengan V/[S], suatu garis
lurus akan dihasilkan yang V
memotong sumbu y pada
Vmax dan sumbu x pada KM
Vmax/KM serta
membentuk sudut
terhadap sumbu x sebesar Vmax/KM
KM

V/[S]
Persamaan “Hanes-Woolf”
Vmax [S]
V
KM  [S]
V(K M  [S])  Vmax[S]
[S] K M  [S]

V Vmax
[S] KM 1
  .[S]
V Vmax Vmax
[S] K M 1
  .[S]
V Vmax Vmax
 Sekarang
y = [S]/V ; x = [S]
a = KM/Vmax ; b = 1/Vmax
dapat dianalisis dengan y = a + bx
 Jika [S]/V dihubungkan
dengan [S], suatu garis
lurus akan dihasilkan [S]/V
yang memotong sumbu 1/Vmax
y pada KM/Vmax dan
sumbu x pada -KM serta
membentuk sudut
KM/Vmax
terhadap sumbu x
-KM
sebesar 1/Vmax.
[S]
STEPS OF MODEL
DERIVATION
 STEPS OF MODEL DERIVATION
1. Pembentukan ES adalah inti dari hipotesis
tersebut
k1 k3
E+S ES E+P (1)
k2 k4
1. Reaksi E dengan S terjadi dengan kecepatan
k1 dan menghasilkan kompleks ES (enzim-
substrat)
2. Kompleks ES dapat berubah menjadi E dan S
bebas kembali dengan kecepatan k2, atau
menjadi E dan P dengan kecepatan k3.
4. Jika k3  k4 , maka reaksi bersifat
“irreversible”, sehingga produk P tidak ada
yang diubah kembali menjadi substrat asal dan
k4 dapat diabaikan.
5. Suatu hal penting yang perlu diingat adalah
bahwa konstanta k1, k2, k3 dan k4 proporsional
dengan G aktivasi substrat dari reaksi yang
bersangkutan
6. Pada [S] yang rendah, kebanyakan enzim
berada dalam bentuk bebas, sehingga
penambahan S akan langsung terikat dengan
E dan diubah menjadi P dengan demikian
kecepatan awal proporsional dengan
peningkatan [S]
7. Pada [S] yang lebih tinggi, kecepatan reaksi
bervariasi dengan peningkatan [S] karena
enzim mulai mengalami kejenuhan
8. Pada [S] yang tinggi, semua enzim dijenuhi
oleh substrat dan karenanya berada dalam
bentuk kompleks ES
9. Jadi enzim dalam suatu reaksi dapat berada
dalam keadaan bebas dan terikat dengan
substrat, sehingga total enzim secara
matematis adalah

[E]0 = [E]+[ES] (2)

10. Penurunan persamaan Michaelis-Menten
tergantung pada asumsi yang disebut
”Briggs-Haldane Steady-State”
11. Keadaan "steady state" adalah suatu
keadaan dimana konsentrasi intermediat
(perantara) ES tetap konstan, sementara
konsentrasi substrat dan produk berubah
12. Keadaan demikian terjadi apabila
kecepatan pembentukan ES sama dengan
kecepatan peruraian ES
13. Keadaan “steady” dapat dinyatakan
secara matematis seperti dengan
persamaan berikut
d[ES]/dt = 0 (3)
dimana t = waktu (menit)
14. Pernyataan [ES]/t dapat ditulis dari
sudut konstanta dan konsentrasi pers (1)
yaitu
Kecepatan pembentukan ES
ES = k1[E][S] (4a)
 Kecepatan peruraian ES

ES = (k2 + k3) (ES) (4b)

15. Dalam keadaan "steady state" kedua
persaman (4a) dan (4b) adalah sama,
sehingga

d[ES]/dt = k1[E][S]-(k2+k3)(ES) = 0
(5)
16. Subsitusi E dari pers (2) kedalam pers (5)
menghasilkan
(2) [E]0 = [E]+[ES] [E] = [E]0-[ES]
(5) d[ES]/dt = k1[S][E]-(k2+k3)(ES)
k1[S][E] = k1[S]([E]0-[ES])
= k1[S][E]0-k1[S][ES]

Hence
k1[S][E]0-k1[S][ES] - (k2+k3)(ES) = 0
k1[S][E]0–(k1[S]+k2+k3)[ES]=0 (6)
17. Pengaturan persamaan lebih lanjut
(k1[S]+k2+k3)[ES] = k1[S][E]0
(7)

18. Persamaan ini dapat dimodifikasi dengan cara

ruas kanan dibagi dengan k1[S],
[E]0
[ES]  (8)
1  (k 2  k 3 ) / k1[S]
19. Karena k1, k2, dan k3 adalah konstanta, maka
ketiga konstanta ini dapat dijadikan satu
konstanta yaitu (k2 + k3 )/k1 = KM yang dikenal
sebagai konstanta Michaelis-Menten
20. Untuk kebanyakan enzim k3  k2, sehingga KM akan
mendekati (k2 + k1), sedang (k2 + k3 )/ k1 adalah Ks
(konstanta dissosiasi kompleks enzim-substrat).
21. Jika KM, yang merupakan ukuran affinitas enzim akan
substrat, disubsitusikan kedalam pers (8), maka

(9)]  [E]0
[ES
1  (K M /[S])
22. Kecepatan reaksi katalisis dapat dinyatakan dengan
jumlah produk yang tebentuk per satuan waktu yaitu
produk dari konsentrasi kompleks ES dengan kapasitas
katalisis enzim k3 (turnover number).

[P]
V
(10)  k 3[ES]
t
23. Subsitusi [ES] dari pers. (10) ke dalam pers (9)
memberikan
k 3 [ E ]0
V (12)
1  (K M /[S])
24. Pada keadaan E dijenuhi S yang berarti semua
enzim terikat dengan substrat dalam kompleks
ES, maka V = Vmax = k3[E]0. Kemudian
persamaan diatas dapat ditulis dalam bentuk
berikut.

Vmax Vmax [S]

V V 
1  ( KM / [S]) atau
KM  [S]
(13)
25. Persamaan diatas dikenal sebagai persamaan
Michaelis-Menten yang digunakan secara luas
26. Stoikiometri pers (13) didasarkan atas satu
substrat dan satu produk (uni-uni), sementara
banyak reaksi enzimatis yang melibatkan
stoikiometri yang lebih kompleks seperti berikut;

S  P1 + P2 (Ui-Bi)
S1 + S2  P (Bi-Uni)
S1 + S2  P1 + P2 (Bi-Bi)
27. Tetapi, persamaan Michaelis-Menten berlaku
untuk reaksi yang lebih kompleks sekalipun
dengan mekanisme yang berbeda.
FIG. 2. Overall structure of Rubisco from C. reinhardtii. The view is down the local
4-fold axis. The L subunits are depicted in two shades of gray, and the S subunits are
in yellow. The S subunit A-B loops are shown in red. The inset shows a close-up view
of the same region in the Spinacia enzyme with the same color coding. Pictures were
produced with MOLSCRIPT (41) modified by Esnouf (42) and rendered with
RASTER3D (43). Taylor et al., 2001, J. Biol. Chem., 276: 48159–48164
Web Figure 8.3.A A model for the structure of rubisco in chloroplasts
from higher plants. Rubisco consists of 8 large (L) and 8 small (S) subunits
arranged as 4 dimers. Small subunits are shown in red (only four of the
small subunits are seen), large subunits are shown in blue and green, in
order to show the boundaries of the dimers. (From Malkin and Niyogi 2000.)
(Click image to enlarge.)
 LECTURE 4:
REACTION MECHANISM &
INHIBITORS
 LECTURE LAYOUT
REACTION MECHANISMS
A. Sequential Reactions
B. Random Bisubstrate Reactions
C. Ping-Pong Reactions
INHIBITORS
1. Irreversible
2. Reversible
 Competitive inhibition,
 Uncompetitive inhibition.
 Noncompetitive inhibition
 Reaction Mechanisms
A: Sequential Reactions
 All substrates must combine with enzyme
before reaction can occur
A B P Q

E EA EABEPQ EQ E
Bisubstrate reactions
B. Random Bisubstrate Reactions
 C. Ping-Pong Reactions
 Group transfer reactions
 One or more products released before
all substrates added

A P B Q

E EAFP F FBEQ E
 2. INHIBITORS
 An important number of compounds have the
ability to combine with certain enzymes in either
a reversible or irreversible manner, and thereby
block catalysis by that enzyme
 Such compounds are called INHIBITORS and
include drugs, antibiotics, poisons, anti
metabolites, as well as products of enzymic
reactions
 Two general classes of inhibitors are recognized;
 Irreversible
 Reversible
 1. IREVERSIBLE INHIBITION
 An irreversible inhibitor forms a covalent bond
with a specific function, usually an amino acid
residue, which may, in some manner, be
associated with the catalytic activity of the
enzyme
 There are many examples of enzyme inhibitors
which covalently bind not at the active site, but
physically block the active site
 The inhibitor cannot be released by dilution or
dialysis; kinetically, the concentration and hence the
velocity of active enzyme is lowered in proportion to
the concentration of the inhibitor and thus the effect
is that of noncompetitive inhibition:
Review of Initiation of Protein Synthesis
1 3
30S 2 GTP

1 2 3 GTP
Initiation Factors
f-met-tRNA
mRNA
Spectinomycin

GDP + Pi
50S
2
P A
1 1
2 GTP

70S Aminoglycosides
30S
Initiation Initiation
Complex Complex
IRREVERSIBLE INHIBITORS
k1 k3
E+S ES E+P
+ k2 k4
I  Examples of irreversible inhibitors include;
 diisopropyl fluorophosphate which reacts
kI irreversibly with serine proteases, chymotrypsin

EI
 iodoacetate which
reacts with essential
sulfhydryl group of an
enzyme such as triose
phosphate
dehydrogenase:
E-SH+ICH2COOH 
E-SCH2COOH+HI
 A unique type of irreversible inhibition has been
recently described as kcat inhibition in that a latent
inhibitor is activated to an active inhibitor by binding to
the active site of the enzyme.
 The newly generated inhibitor now reacts chemically
with the enzyme leading to its irreversible inhibition
 These inhibitors have great potential as drugs in highly
specific probes for active sites since they are not
converted from the latent to the active form except by
their specific target enzymes
 An excellent example is the inhibition of D-3-hydroxyl
decanoyl ACP clehydrase (of E. coli) by the latent
inhibitor 3-decynoyl-N-acetyl cystamine according to
the following sequences of events:
3-decynoyl-N-
acetyl cystamine
 2. REVERSIBLE INHIBITION
 As the term implies, this type of inhibition involves
equilibrium between the enzyme and the inhibitor,
the equilibrium constant (Ki) being a measure of
the affinity of the inhibitor for the enzyme.
 Three distinct types of reversible inhibition are
known;
 Competitive inhibition,
 Uncompetitive inhibition
 Noncompetitive inhibition
 A. Competitive Inhibition
 Compounds that may or may not be
structurally related to the natural substrate
combine reversibly with the enzyme at or near
the active site
Competitive inhibitor
 The inhibitor and the
+I
substrate therefore compete
for the same site according -I

to the reaction:
Vmax [S]
V
 [I ] 
K M  1    [S] -1/KM -1/[KM(1+1/KI)]
 KI 
 k1 k3
E+S ES E+P
Competitive + k2 k4
Inhibitor I
S
kI S
EI
I
ES and EI complexes are
formed, but EIS complexes are I
never produced.
One can conclude that high concentrations of
substrate will overcome the inhibition by causing the
reaction sequence to swing to the right. The velocity
of reaction can be calculated by the following
equation
 Among other enzymes that may undergo competitive
inhibition is succinic dehydrogenase, which readily
oxidizes succinic acid to fumaric acid.
 If increasing concentrations of malonic acid, which
closely resembles succinic acid in structure, are
added, however, succinic dehydrogenase activity falls
markedly Competitive Inhibitor
Substrate
 This inhibition can Product Succinate Glutarate Malonate Oxalate
now be reversed by C-OO- C-OO- C-OO- C-OO- C-OO-
increasing in turn
C-H H-C-H H-C-H H-C-H C-OO-
the concentration of
C-H H-C-H H-C-H C-OO-
the substrate
C-OO- C-OO- H-C-H
succinic acid. -
C-OO
Succinate Dehydrogenase
Competitive Inhibition
 B. Uncompetitive Inhibition
 Compounds that combine only with the ES complex,
not with the free enzyme, are called uncompetitive
inhibitors. The inhibition is not overcome by high
substrate concentrations.
 HIV protease in a complex
with the protease inhibitor
ritonavir
 The structure of the
protease is shown by the
red, blue and yellow
ribbons. The inhibitor is
shown as the smaller ball-
and-stick structure near the
centre. Created from PDB

Peptide-based protease
inhibitor ritonavir

Human immunodeficiency virus

Scanning electron micrograph of HIV-1 (in green) budding from cultured
lymphocyte. Multiple round bumps on cell surface represent sites of assembly
and budding of virions
 KM value is consistently smaller than the KM value
of the uninhibited reaction which implies that S is
more effectively bound to the enzyme in the
presence of the inhibitor.
 The equation used to calculate the velocity of the
noncompetitive inhibition is as follows
Vmax [S]
V Uncompetitive inhibitor
 [I ] 
K M  [S] 1   +I

 KI 
-I

(1+[I]/KI)/Vmax

-1/Vmax

-1/KM
-(1+[I]/KI)/KM
 C. Noncompetitive Inhibition
 Compounds that reversibly bind with either the enzyme
or the enzyme substrate complex are designated as
noncompetitive inhibitors
 Noncompetitive inhibition therefore differs from
competitive inhibition in that the inhibitor can combine
with ES, and S can combine with EI to form in both
instances EIS.
 This type of inhibition is not completely reversed by
high substrate concentration since the closed
sequence will occur regardless of the substrate
concentration
 Since the inhibitor binding site is not identical to nor
does it modify the active site directly, the KM is not
altered.
 Vmax [S]
V
 [I ] 
K M  [S] 1  
 KI 

Noncompetitive
+I

-I

(1+[I]/KI)/Vmax

-1/Vmax
FEEDBACK
INHIBITION
 The switch: Allosteric inhibition
Allosteric means “other site”
Active site

E
Allosteric
site

© 2008 Paul Billiet ODWS

 Switching off
 These enzymes
have two receptor
sites
 One site fits the
substrate like other Inhibitor
enzymes Substrate molecule

 The other site fits cannot fit into

the active site
an inhibitor Inhibitor fits into
molecule allosteric site

© 2008 Paul Billiet ODWS

HOW TO SOLVE THE EQUATIONS
 1. Competitive inhibitor
Vmax [S]
V
 [I ] 
K M  1    [S]
 KI 
 [I ] 
K M  1  
1
  KI  1

1
V Vmax [S] V max
y =1/V; x = 1/[s]
 a = 1/Vmax
 b = KM(1+[I]/KI)/Vmax
 2. Uncompetitive
Vmax [S]
V
 [I ] 
K M  [S] 1  
 KI 

 [I ] 
 1  
1 KM 1  KI 
 
V V max [S] V max

y =1/V; x = 1/[s]
 a = (1+[I]/KI)/Vmax
 b = KM/Vmax
 3. Noncompetitive Inhibition
Vmax [S]
V
 [I ] 
K M  [S] 1  
 KI 

 [I ]   [I ] 
K M  1    1  
1
  KI  1 

KI 
V V max [S] V max

y =1/V; x = 1/[s]
 a = (1+[I]/KI)/Vmax
 b = KM(1+[I]/KI)/Vmax
 I Competitive I Non-competitive I Uncompetitive
Vmax Vmax Vmax
Direct Plots

vo vo
Vmax’ Vmax’
I I I

Km Km’ [S], mM Km = Km’ [S], mM Km’ Km [S], mM

Vmax unchanged Vmax decreased
Double Reciprocal

Km increased Km unchanged Both Vmax & Km decreased

1/vo 1/vo 1/vo

I I I
Two parallel
Intersect lines
at Y axis 1/ Vmax Intersect 1/ Vmax 1/ Vmax
at X axis

1/Km 1/[S] 1/Km 1/[S] 1/Km 1/[S]

Juang RH (2004) BCbasics

 SOAL
[S] V(-I) V(+I)
 Diketahui suatu
reaksi enzimatis 1*10-4 28 17

tanpa dan dengan 1.5*10-4 36 23

inhibitor dengan [I] =
2.0*10-4
2,2.104M. 43 29

 Hitunglah KM dan 5*10-4 65 50

Vmax tanpa dan
7.5*10-4 74 61
dengan I serta KI
 Feedback Inhibition (Allosteric
Effectors)
 The activity of some enzymes is controlled by
certain molecules binding to a specific regulatory
(or allosteric) site on the enzyme, distinct from
the active site.
 Different molecules can either inhibit or activate the
enzyme, allowing sophisticated control of the rate.
Only a few enzymes can do this, and they are often at
the start of a long biochemical pathway.
 They are generally activated by the substrate of the
pathway and inhibited by the product of the pathway,
thus only turning the pathway on when it is needed.
This process is known as feedback inhibition.
http://www.biologymad.com/resources/Ch%204%20-%20Enzymes.pdf
THANK YOU
Спасибо
 QUIZ (10 min)
1. How is enzyme specificity achieved ?
2. Calculate Vmax & KM from the following data, and does the
reaction obey Michaelis-Menten kinetics ?

[DNA] Free nucleotides in solution,

mol total V (pmol/L)
nucleotides/L 0 min 10 min
1.0 x 10-5 0.05 5.1
1.0 x 10-6 0.04 4.5
1.0 x 10-7 0.06 3.2
1.0 x 10-8 0.04 1.4
1.0 x 10-9 0.04 0.23
 ANSWERS
1. The enzyme specificity is achieved
through the characteristic of active site
2. Vmax = 4.36695
KM = 2.2E-08
R2 = 0.999864, so the reaction obeys
Michaelis-Menten kinetics
LECTURE 5: CARBOHYRATE
 WHAT WOULD YOU LIKE TO KNOW
I. Where does it come from? photosynthesis
II. What is Its function many
III. What is carbohydrate? definition
IV. What are characteristics of carbohydrate 
1. Classification?
2. Aldose & Ketose Sugars?
3. Number of carbon atoms?
4. Location of carbonyl group (C=O)?
5. The chirality of carbohydrate & asymmetric carbon?
6. Diastereoisomers & Epimers?
7. Enantiomers & Epimers?
8. D or L designation refers to the asymmetric carbon?
9. Fischer Projections: D-isomer & L-isomer?
10. Number of Stereoisomers ?
 VOCABULARY
1. Derivatives
2. Precursor
3. Intermediates
4. Entities
5. Recognition
6. Attach
7. Encounter
8. Properties
9. referred to
10. Configuration
11. Superimposable
 LECTURE OUTLINE
1. INTRODUCTION
2. DEFINITION
3. CARBOHYDRATE CLASSIFICATION
 Monosaccharides
 Oligosaccharides
 Polysaccharides.
4. MONOSACCHARIDES
 number of carbon atoms in the molecule
 location of the carbonyl group
 the chirality of the carbohydrate
5. EXERCISE
6. POLARIMETRY
 1. INTRODUCTION
Where does it come from?
I. Photosynthesis: D-Fructose 6-phosphate
1. The carbohydrates of plants are derived originally
from atmospheric CO2 which is converted, through
the process of photosynthesis, into PGA (3-
Phosphoglyceric acid) and then to F-6-P (D-Fructose
6-phosphate)
2. A whole range of monosaccharides and
monosaccharide derivatives are synthesized from F-
6-P.
3. Some of these monosaccharide derivatives are the
precursor of oligosaccharides and polysaccharides.
4. It is important to note that sucrose is the type of
carbohydrates (oligosaccharides) transported around
the plant.
INTRODUCTION

5. Polysaccharides can be divided into the structural

polysaccharides (e.g. cellulose, pectins) and storage
polysaccharides (e.g. starch, fructosans).

II. Carbohydrate Functions What is its function?

 Sources of energy
 Intermediates in the biosynthesis of other basic
biochemical entities (i.e. fats and proteins)
 Associated with other entities such as glycosides,
vitamins and antibiotics
 Form structural tissues in plants and in
microorganisms (cellulose, lignin, murein)
 Participate in biological transport, cell-cell recognition,
activation of growth factors, modulation of the immune
system
 2. DEFINITION
What Is a Carbohydrate?
1. Carbohydrates may be defined as polyhydroxy
aldehydes or ketones, or substances that yield one of
these compounds on hydrolysis.
ketone

aldehyde
DEFINITION

aldehyde

ketone

Aldoses (e.g., glucose) have Ketoses (e.g., fructose) have a

an aldehyde at one end. keto group, usually at C #2
In aldehydes, the carbonyl group has a hydrogen
atom (H) attached to it together with either a second
hydrogen atom or, more commonly, a hydrocarbon
group which might be an alkyl group or one containing a
benzene ring.

In ketones, the carbonyl group has two hydrocarbon

(H & C) groups attached. Again, these can be either alkyl
groups (CnH2n+1) or ones containing benzene rings (C6H6).
Notice that ketones never have a hydrogen atom attached
to the carbonyl group.
DEFINITION

2. The formula (CH2O)n where n 3 can be used to

represent many carbohydrates which are
regarded originally as the hydrates of carbon.
 C3H6O3: Dihydroxyacetone, Dimethyl carbonate &
Glyceraldehyde
 C4H8O4: Tetrose (Erythrose, Erythrulose & Threose)

3. The formula is not suitable when other

compounds were encountered that had the
general properties of carbohydrates but
contained N (nitrogen) or S (sulfur) in addition to
carbon, hydrogen and oxygen.
4. Moreover, the important simple sugar
deoxyribose, found in every cell as a component
of deoxyribonucleic acid, has the molecular
formula C5H10O4 rather than C5H10O 5
 3. CARBOHYDRATE CLASSIFICATION
 The most useful classification scheme divides the
carbohydrates into groups according to the number of
individual simple sugar units
 Monosaccharides
 Trioses, tetroses, pentoses, hexoses
 Oligosaccharides
 Di, tri, tetra, penta, up to 9 or 10
 Most important are the disaccharides
 Polysaccharides.
 Homopolysaccharides
 Heteropolysaccharides
 Complex carbohydrates
 In general, the monosaccharides and disaccharides are
commonly referred to as sugars.
 Monosaccharides
 Monosaccharides also known as simple sugars are
classified based on three characteristics:
A. number of carbon atoms in the molecule
B. location of the carbonyl group
C. the chirality of the carbohydrate

A. Number of carbon atoms

 three carbons: triose
 four carbons: tetrose
 five carbons: pentose
 six carbons: hexose
 seven carbons: heptose
 etc.
1. Triose (3 C)

D-Glyceraldehyde L-Glyceraldehyde

2. Tetrose (4 C)

D -Erythrose D-Threose D-Erythrulose

3. Pentose (5 C)

D-Ribose D-Arabinose D-Xylose D-Lyxose

• The ring form of ribose is a component ribonucleic acid

(RNA).
• Deoxyribose, which is missing an oxygen at position 2, is
a component of deoxyribonucleic acid (DNA).
• In nucleic acids, the hydroxyl group attached to carbon
number 1 is replaced with nucleotide bases

Ribose Deoxyribose
4. Hexose (6 C)

D-Allose D-Altrose D-Glucose D-Mannose

D-Gulose D-Idose D-Galactose D-Talose

D-Glucose (an aldose) α-D-Glucose β-D-Glucose

Cyclation of Glucose
5. Heptoses (7C)
Sedoheptulose has the same structure as fructose,
but it has one extra carbon. Sedoheptulose is found
in carrots. Mannoheptulose is a monosaccharide
found in avocados.

D-Sedoheptulose D-Mannoheptulose
 CARBOHYDRATE CLASSIFICATION

B. Location of carbonyl group (C=O)

 an aldose (aldehyde sugar)
 a ketose (ketone sugar)

Structure of a simple aldose and a simple ketose

aldose

ketose
 For example
− glucose is an aldose;
− fructose, a structural isomer of glucose, is a ketose

carbonyl group
Monosaccharides

Aldose sugars
H H H H H

C O C O C O C O C O

(H C OH)n H C OH H C OH H C OH H C OH

CH2OH CH2OH H C OH H C OH H C OH

Aldose Aldotriose CH2OH H C OH H C OH

n=1
Aldotetrose CH2OH
n=2 H C OH
Aldopentose
CH2OH
n=3
Aldohexose
n=4
Monosaccharides

Ketose sugars
CH2OH CH2OH
CH2OH
CH2OH
C O C O CH2OH
C O
C O
(H C OH)n H C OH C O
H C OH
CH2OH
CH2OH H C OH H C OH
CH2OH
CH2OH H OH
Ketose Ketotriose Ketotetrose
n=1 Ketopentose H C OH
n=0
n=2
CH2OH
Ketohexose
n=3
C. The chirality of carbohydrate
 Chirality is a term derived from the Greek word for
hand (χειρ =cheir) or asymmetric carbon atoms
 This causes optical isomerism which is the type of
isomerism commonly found in carbohydrate.
 Isomerism may be divided into structural isomerism
and stereoisomerism.
 Structural isomers have the same molecular
formula but differ from each other by having
different structures, and
 Stereoisomers have the same molecular formula
and the same structure, but they differ in
configuration, that is, in the arrangement of atoms
in space. Stereochemisty is the study of the
arrangement of atoms in three-dimensional space
The chirality of the carbohydrate

 Saccharides with identical functional groups but with different

spatial configurations have different chemical and biological
properties.
 Compounds that are mirror images of each other but are not
identical, comparable to left and right shoes, are called
enantiomers
 Any carbon atom that has four different groups bonded to it
is called asymmetric carbon or a chiral carbon
 Any carbon atom which is connected to four different groups
is called asymmetric carbon or a chiral carbon and will have
two nonsuperimposable mirror images or a center of chirality
 Monosaccharides contain one or more asymmetric C-atoms:
D- and L-forms
 D-form: OH group is attached to the right of the asymmetric
carbon
 L-form: OH group is attached to the left of the asymmetric carbon
The chirality of the carbohydrate

 If there is more than one chiral C-atom:

absolute configuration of chiral C furthest
away from carbonyl group determines whether
D- or L-
 D and L designations are based on the
configuration about the single asymmetric
carbon in glyceraldehyde.
 Glyceraldehyde is a
chiral molecule — it
cannot be superimposed
on its mirror image.
 The two mirror-image
forms of glyceraldehyde
are enantiomers of each
other
Two carbohydrates are said to be enantiomers if the
two carbohydrates are complete mirror images of
one another.

An example of an
enantiomer is the D and L
isomers of glucose

The blue indicates the D-isomer and the red

indicates the L-isomer
 CH2O CH2O
C=O C=O
HO-C-H Enantiomers H-C-OH
H-C-OH Mirror image HO-C-H
configurations
H-C-OH HO-C-H
CH2O CH2O

CH2O
CH2O
C=O
C=O
H-C-OH
H-C-OH
HO-C-H
HO-C-H
HO-C-H
HO-C-H
CH2O
CH2O
 The differing
Diastereoisomers. Two carbohydrates stereogenic centers.
are said to be diastereoisomers if
they have the opposite configuration
at one or more of the chiral centers
present in the carbohydrate but the
two carbohydrates are not mirror
images of one another.

Epimers are a special type of An example of two

carbohydrates that are
diastereoisomers in that diastereoisomers are D-
they only differ at one of the Glucose and D-Altrose
stereogenic centers.

An example of epimers
that differ at one
stereogenic center is D-
Glucose and D-Mannose,
Enantiomers and epimers
The chirality of the carbohydrate

 For sugars with more than one chiral center, the

D or L designation refers to the asymmetric
carbon farthest from the aldehyde or keto group.
 Most naturally
occurring sugars
are D isomers.
 D & L sugars are
mirror images of
one another. They
have the same
name. For example,
D-glucose and L-
glucose are shown
at right.
Fischer Projections
 Fischer projections are a convenient way to
represent mirror images in two dimensions.
 Place the carbonyl group at or near the top and
the last chiral CH2OH at the bottom.
 When there is more than one chiral center in a
carbohydrate, look at the chiral carbon farthest
from the carbonyl group: if the hydroxy group
points to right when the carbonyl is “up” it is the
D-isomer, and when the hydroxy group points to
the left, it is the L-isomer.
 Number of Stereoisomers: When a molecule has
more than one chiral carbon, each carbon can
possibly be arranged in either the right-hand or
left-hand form, thus
if there are n chiral carbons, there are 2n possible
stereoisomers.
Maximum number of possible stereoisomers = 2n
• D-Glucose have four asymmetric carbon atoms (No. 2,
3, 4 & 5)2n = 24 = 16

D-Glucose
(an aldose)
3 examples of chiral Carbon atoms:

from http://ntri.tamuk.edu/cell/carbohydrates.html)
 Exercise
 How many isomers are there of

 How many chiral carbon atoms

are there in
 Ring formation / Ring structure

An aldose: Glucose

from http://ntri.tamuk.edu/cell/carbohydrates.html
 A ketose: Fructose

from
http://ntri.tamuk.edu/cell/carbohydrates.html
 POLARIMETRY
 Measurement of optical activity in chiral or asymmetric
molecules using plane polarized light
 Molecules may be chiral because of certain atoms or
because of chiral axes or chiral planes
• Measurement uses an
instrument called a
polarimeter (Lippich
type)
• Rotation is either
(+) dextrorotatory or
(-) levorotatory
 Polarimetry

– Magnitude of rotation depends upon:

1. the nature of the compound
2. the length of the tube (cell or sample container)
usually expressed in decimeters (dm)
3. the wavelength of the light source employed;
usually either sodium D line at 589.3 nm or
mercury vapor lamp at 546.1 nm
4. temperature of sample
5. concentration of analyte in grams per 100 ml
  observed x 100
[] D
T
=
lxc
D = Na D line
T = temperature oC
obs : observed rotation in degree (specify solvent)
l = length of tube (dm, decimeter)
c = concentration in grams/100ml
[] = specific rotation
 Specific rotation of various carbohydrates at 20 oC

 D-glucose +52.7
 D-fructose -92.4
 D-galactose +80.2
 L-arabinose +104.5
 D-mannose +14.2
 D-arabinose -105.0
 D-xylose +18.8
 Lactose +55.4
 Sucrose +66.5
 Maltose+ +130.4
 Invert sugar -19.8
 Dextrin +195
THANK YOU
Спасибо
 A. REFRESHING
1. D & L Designation
 The arrangement of the OH's and H's on these
atoms is very important.
 Structural formulas for sugar molecules are often
written in the vertical arrangement with the
aldehyde or the ketone group at or near the top.
 The sugar is of D (dextro) form when O H
1C
the position of the OH (hydroxyl group)
on the last asymmetric carbon atom H 2C OH
(No. 5) is on the right, the reverse is of HO 3C H
L (levo) form H 4C OH
 D-glucose is an aldohexose with H 5C OH
aldehyde group (red), and the the 6CH 2OH
asymmetric center (blue) furthest from D-glucose
the aldehyde
 1CH2OH H O
2. Asymmetric Carbon O
1C
2C
 Monosaccharides contain H 2C OH
asymmetric carbon atoms H 3C OH
HO 3C H
(e.g. 4 in glucose & 2 in H 4C OH
H 4C OH
ribulose) 5CH2OH
H 5C OH
D-ribulose
6CH2OH
D-glucose

 The number of stereoisomers is 2n, where

n is the number of asymmetric centers.
 The number of stereoisomers of
 D-ribulose = 22 = 4
 D-glucose = 24 = 16
3. Pyranose and Furanose
● If the carbon chain is long enough, the alcohol at
one end of a monosaccharide can attack the
carbonyl group at the other end to form a cyclic
compound.
 When a six-
membered ring is
formed, the product
of this reaction is
called a pyranose
 When a five- 2
membered ring is
formed, it is called 3
1
a furanose

4
5
 ● Glucose can exist in both a
straight-chain and ring form.

O H
1C
HO

H 2C OH
H
H

HO 3C H
O H
6CH 2OH

5C

4C

2C

1C
3C

H 4C OH
OH
OH
H
OH

H 5C OH
6CH 2OH

D-glucose can cyclize in two ways forming either furanose or

pyranose structures
 The reactions that lead to the formation of a pyranose
or a furanose are reversible
 For example -D-glucopyranose or b-D-
glucopyranose, these anomers are interconverted to
give an equilibrium mixture that is 63.6% of the b-
anomer and 36.4% of the -anomer.
 The 2:1 preference for the b-anomer can be
understood by comparing the structures of these
molecules.
 In the b-anomer, all of the bulky -OH or -CH2OH substituents
lie more or less within the plane of the six-membered ring.
 In the a-anomer, one of the -OH groups is perpendicular to the
plane of the six-membered ring, in a region where it feels
strong repulsive forces from the hydrogen atoms that lie in
similar positions around the ring.
 As a result, the b-anomer is slightly more stable than the a-
anomer
4. The  and b anomers
 There are two possible structures for the pyranose and
furanose forms of a monosaccharide, which are called
the - and b-anomers
 Cyclization of glucose produces a new asymmetric
center at C1. The 2 stereoisomers are called anomers,
 & b.
6 CH2OH 6 CH2OH
5 O 5 O
H H H OH
H H
4 H 1 4 H 1
OH OH
OH OH OH H
3 2 3 2
H OH H OH
-D-glucose b-D-glucose

 (OH below the ring) & b (OH above the ring)

Fructose forms either
 a 6-member pyranose ring, by reaction of the C2 keto
group with the OH on C6, or
 a 5-member furanose ring, by reaction of the C2 keto
group with the OH on C5.

1
CH2OH

2C O

HO C H
1 CH2OH
3 HOH2C 6 O
H C OH
4 5 H HO 2

H C OH H 4 3 OH
5
OH H
6
CH2OH

D-fructose (linear) -D-fructofuranose

 +

D-ribose -D-ribofuransoe b-D-ribofuranose

D-fructose -D-fructofuranose b-D-fructofuranose

 The  and b anomers of glucose.
 The position of the anomeric carbon (green or red)
relative to the CH2OH group bound to carbon 5:
they are either on the opposite sides (α), or the
same side (β).
5. Glycosidic Linkages
 The anomeric hydroxyl and a hydroxyl of
another sugar or some other compound can
join together, splitting out water to form a
glycosidic bond:
R-OH + HO-R'  R-O-R' + H2O
E.g., methanol reacts with the anomeric OH
on glucose to form methyl glucoside (methyl-
glucopyranose).
Alternative Glycosidic Linkages: Maltose and
Lactose
● Maltose contains two molecules of D-glucose, and
Lactose is comprised of one molecule of D-glucose
and one of D-galactose.
● In both cases the monosaccharide units are
connected by 1,4-glycosidic linkages, i.e. they both
contain a C1-O-C4' link.
● In maltose the C1-O bond is in the  position, while
in lactose it is oriented b to the D-galactose ring.
 B. Carbohydrate synthesis
 CHO’s are made in the chloroplast and
cytosol
 Starch = chloroplast
 Sucrose = cytosol

 Chloroplast organelles are

found in plants and algae
 Enclosed by a double
membrane
 Have their own small
genome
 The inner membrane is
impermeable to ions
such as H+, and to polar
and charged molecules
 Glyceraldehyde-3 Phosphate (G 3-P)
 Glyceraldehyde-3 Phosphate (3-phosphoglycerate) is
the first product of photosynthesis through three
rounds of the Calvin cycle to fix three CO2 molecules to
produce one molecule of G 3-P
● G 3-P is converted to
starch in the chloroplast,
and to sucrose in the
cytosol for export
● G 3-P synthesis is
balanced by phosphate
levels & triose phosphate
levels in the compartments
 If cytosol Pi is high, Pi exchanges for trioses;
sucrose is made
 If cytosolic Pi
is low, trioses
stay in
plastid; starch
is made
High cytosol Pi, Triosescytosol sucrose
Low cytosol Pi, Trioses (Chloroplast) Starch
 LECTURE 6
Oligo- and Polysaccharides
lactose 15
maltose 32
galactose 32
regular corn syrup 40
trehalose 45
mannitol 50
sorbitol 55
high-DE corn syrup 70
glucose 74
tagatose 92
xylitol 100
sucrose 100
HFCS (42% fructose) 120
invert sugar* 120
fructose 173

0 50 100 150 200

Relative sweetness

Lactose is a disaccharide found in milk. It is composed of a molecule of D-galactose and a

molecule of D-glucose bonded by a β-1-4 glycosidic linkage.
 LECTURE OUTLINE
 INTRODUCTION  POLYSACCHARIDES
 QUESTIONS  Starch
 OLIGOSACCHARIDES  Cellulose
 Number of C Atoms  Glycogen
 disaccharides,  Inulin
 trisaccharides,  Chitin
 tetrasaccharides  Dextrans
 Examples  Dextrins
 Sucrose
 Lactose
 Pectins
 Maltose  RESIN
 Lactulose  Bacterial cell wall
 INTRODUCTION
How much energy you need everyday

W =3,943,595 cal
=3,943 Cal = kcal

http://firstyear.chem.usyd.edu.au/calculators/food_energy.shtml
356-380 g/day/capita
 BMI = weight (kg)/(height,m) 2

SMR 1.0, then there is a higher number of

deaths than is expected
Monosaccharide has just one ring, a disaccharide has
two, and a polysaccharide has many
 In reality, an aqueous sugar solution
contains only 0.02% of the glucose in
the chain form, the majority of the
structure is in the cyclic chair form
Since carbohydrates contain both
alcohol and aldehyde or ketone
functional groups, the straight-chain
form is easily converted into the chair
form - hemiacetal ring structure.

Due to the tetrahedral geometry of carbons that ultimately

make a 6 membered stable ring , the -OH on carbon #5
is converted into the ether linkage to close the ring with
carbon #1. This makes a 6 member ring - five carbons
and one oxygen
Steps in the ring closure (hemiacetal synthesis):
1. The electrons on the alcohol oxygen are used to bond the
carbon #1 to make an ether (red oxygen atom).
2. The hydrogen (green) is transferred to the carbonyl oxygen
(green) to make a new alcohol group (green).
The chair structures are always written with the orientation depicted
on the left to avoid confusion.
Hemiacetal
Functional Group:
● Carbon # 1 is now
called the anomeric
carbon and is the
center of a
hemiacetal functional
group.
● A carbon that has
both an ether oxygen
and an alcohol group
is a hemiacetal.
 QUESTIONS
1. What are the most common disaccharides
2. What are the products resulting from the hydrolysis of
sucrose
3. What is the carbohydrate used for the production of
penicillin?
4. What is the carbohydrate produced by soybean causing
flatulence?
5. How many groups are carbohydrates classified as
polysaccharides?
6. What are the components of starch?
7. What is the color of amylose reacting with iodine?
8. What is the difference between starch and cellulose in
term of each glucose constituent?
9. What is the product resulting from the partial and
complete hydrolysis of cellulose?
10. How much is cellulose found in wood in general
QUESTIONS

11. What is the cellulose product used for plastic

12. What is the animal starch
13. What is the carbohydrate polymer found in the
exoskeletons of insects and spiders
14. What are the products of the reaction of glucose and
the enzyme transglucosidase
15. What carbohydrate is the components of dental
plaques
16. What is the carbohydrate used for glues
17. What is the heteropolysaccharides found in the pulp of
fruits (citrus, apples)
18. What is the polysaccharide found in the bacterial cell
wall providing the strength and rigidity for the organism
19. What is the polysaccharide produced by garlic
20. What is the oligosaccharide that causes flatulence
 Oligosaccharides
 Oligosaccharides are compounds in which
monosaccharide units are joined by glycosidic
linkages.
 According to the number of units, they are called
 disaccharides, trisaccharides, tetrasaccharides,
pentasaccharides etc.

 The borderline with polysaccharides cannot be

drawn strictly.
 However the term 'oligosaccharide' is commonly used to
refer to a defined structure as opposed to a polymer of
unspecified length or a homologous mixture. When the
linkages are of other types, the compounds are regarded
as oligosaccharide analogues.
Oligosaccharides

 An extremely important biochemical reaction is the

condensation of two (or more) monosaccharides by the
elimination of water from an OH group present on each
of the two sugars.
 Most commonly the reaction occurs between the OH
present on C1 of one monosaccharide and that present
on C4 of the second to form 1  4 GLYCOSIDIC
linkage.
 Because the reaction involves C1, which can exist in
either a- or b- forms, we can obtain either an a (1 4) or
a b (1 4) glycoside
Glycoside links to other carbon atoms are fairly
common, notably 1 2 and 1 6

Glycosidic bonds:
 A glycosidic bond is a type of covalent bond that
joins a carbohydrate (sugar) molecule to another
group (carbohydrate or not)
 The anomeric hydroxyl group and a hydroxyl group of
another sugar or some other compound can join
together, splitting out water to form a glycosidic bond

R-OH + HO-R' --> R-O-R' + H2O

For example, methanol reacts with the anomeric
hydroxyl on glucose to form methyl glucoside
(methyl-glucopyranose).

Maltose, a cleavage product of starch (e.g., amylose,

see below), is a disaccharide with an a(1-4) glycosidic
linkage between the C1
hydroxyl of one glucose and
the C4 hydroxyl of a second
glucose. Maltose is the a
anomer, because the O at
C1 points down from the ring.
 Most common Oligosaccharides (oligo- "several")
 disaccharides: Sucrose, lactose, and maltose
 Trisaccharide: raffinose (glucose, galactose and
fructose)
 Tetrasaccharide: stachyose (2 galactoses, glucose
and fructose)
 Pentasaccharide: verbascose (3 galactoses,
glucose and fructose)
 Hexasaccharide: ajugose (4 galactoses, glucose
and fructose)
 Maltose hydrolyzes to 2 molecules of D-glucose
 Lactose hydrolyzes to a molecule of glucose and a
molecule of galactose
 Sucrose hydrolyzes to a moledule of glucose and a
molecule of fructose
Sucrose (table sugar, cane sugar, 6
1

saccharose, or beet sugar) 4

5
1 5
is glucose-a (1 → 2) -b-fructose.
2
3 2 3 4
6

Raffinose, also called melitose, is a trisaccharide that is

widely found in legumes and cruciferous vegetables,
including beans, peas, cabbage, brussels sprouts, and
broccoli.
 Disaccha-
Disaccharide Unit 1 Unit 2 Bond
ridase
Sucrose glucose fructose α(1→2) sucrase
Lactose galactose glucose β(1→4) lactase
Maltose glucose glucose α(1→4) maltase
Trehalose glucose glucose α(1→1) trehalase
Cellobiose glucose glucose β(1→4) cellobiase
Sucrose (table sugar, cane sugar, saccharose, or beet sugar)
Lactose (milk sugar)
Disaccharides are formed by condensing a pair of monosaccharides. The structures of
three important disaccharides with the formula C 12H22O11 are shown in the figure
below
Sucrose
 Sucrose, commonly called table sugar, is a
disaccharide of glucose (left) and fructose (right)
 Hydrolysis yield glucose and fructose (invert
sugar) (sucrose: +66.5o ; glucose +52.5o;
fructose –92o) Sugar cane

IUPAC name β-D-fructofuranosyl-(2→1)-α-D-glucopyranoside

Other names sugar, saccharose, sugar beet

Molecular formula C12H22O11

Molar mass 342.30 g/mol
IUPAC = International Union of Pure and Applied Chemistry
Lactose (milk sugar)
 b-D-galactose joined to a-D-
glucose via b (1,4) linkage
 milk contains the a and b-
anomers in a 2:3 ratio
 b-lactose is sweeter and more soluble than ordinary a-
lactose
 used in infant
formulations,
medium for
penicillin
production and as
a diluent in
pharmaceuticals
Maltose
 2-glucose molecules joined via a(1,4) linkage
 known as malt sugar
 produced by the partial hydrolysis of starch (either
salivary amylase or pancreatic amylase)
 used as a nutrient (malt extract; Hordeum vulgare); as a
sweetener and as a fermentative reagent
Lactulose
 galactose-b-(1,4)-fructose
 a semi-synthetic disaccharide (not naturally occurring)
 not absorbed in the GI tract
 used either as a laxative (Chronulac) or in the
management of portal systemic encephalopathy
(Cephulac)
 metabolized in distal ileum and colon by bacteria to lactic
acid, formic acid and acetic acid (remove ammonia)
Structures of some oligosaccharides
Structures of some oligosaccharides

An enzymatic product (Beano) can be used to prevent the flatulence

 Sweet potato contains raffinose, one of the sugars
responsible for flatulence.
Raffinose consists of
glactose, glucose and
fructose (from the top).
The enzyme invertase
can split the bond
between glucose and
fructose to create (2)
melibiose and (3)
fructose
 Three of the sugars which occur in plant tissues,
raffinose, stachyose and verbascose are not digested
in the upper digestive tract, and so are fermented by
colon bacteria to yield the flatus gases, hydrogen and
carbon dioxide.
Stachyose
Oligosaccharides occur widely as components of
antibiotics derived from various sources

Honey also contains glucose and

fructose along with
some volatile oils
 POLYSACCHARIDES
 Polysaccharide (glycan) is a macromolecule
consisting of a large number of monosaccharide
(glycose) residues joined to each other by
glycosidic linkages.
 The most important compounds in this class are
starch, cellulose and glycogen
 Polysaccharides can be divided into two groups:
homopolysaccharides (homoglycans) and
heteropolysaccharide (heteroglycan)
 Homoglycans are composed of only one kind of
monosaccharide (starch, cellulose, glycogen, inulin)
  Heteroglycans are composed of two or more different
kinds of monosaccharide (gums,
mucopolysaccharides).

 Characteristics:
 Polymers (MW from 200,000)
 White and amorphous products (glassy)
 Not sweet
 Not reducing; do not give the typical aldose
or ketose reactions)
 Form colloidal solutions or suspensions
 Starch
 Starch is the most common storage polysaccharide in
plants, and composed of 10 – 30% a-amylose and 70-
90% amylopectin depending on the source
 The chains are of varying length, having molecular
weights from several thousands to half a million
 Amylopectin

The side branching chains are clustered together within the

amylopectin molecule
Amylopectin is a
highly branched
structure, with
branches occurring
every 12 to 30
residues

suspensions of amylose in water

adopt a helical conformation

Iodine (I2) can insert in

the middle of the amylose helix to
give a blue color that is characteristic
and diagnostic for starch
 Cellulose
 Polymer of b-D-glucose attached by b(1,4) linkages
 Yields glucose upon complete hydrolysis
 Partial hydrolysis yields cellobiose
 Most abundant of all carbohydrates
 Cotton flax: 97-99%
cellulose
 Wood: ~ 50%
cellulose
 Gives no color
with iodine
 Held together
with lignin in
woody plant
tissues

a (-OH group below the ring) & b (-OH group above the ring)
Structure of cellulose
Linear structures of cellulose and chitin
(2 most abundant polysaccharides)
 Products obtained from cellulose
 Microcrystalline cellulose : used as binder-
disintegrant in tablets
 Methylcellulose: suspending agent and bulk
laxative
 Oxidized cellulose: hemostat
 Sodium carboxymethyl cellulose: laxative
 Cellulose acetate: rayon; photographic film;
plastics
 Cellulose acetate phthalate: enteric coating
 Nitrocellulose: explosives; collodion
(pyroxylin)
 Glycogen
 Glycogen is the storage form of glucose in animals and humans
which is analogous to the starch in plants.
 Glycogen is synthesized and stored mainly in the liver and the
muscles.
 Structurally, glycogen is very similar to amylopectin with alpha
acetal linkages, however, it has even more branching and more
glucose units are present than in amylopectin.
 Various samples of glycogen have been measured at 1,700-
600,000 units of glucose.
 also known as animal starch
 stored in muscle and liver
 present in cells as granules (high MW)
 contains both a(1,4) links and a(1,6) branches at every 8 to 12
glucose unit
 complete hydrolysis yields glucose
 glycogen and iodine gives a red-violet color
 hydrolyzed by both a and b-amylases and by glycogen
phosphorylase
 Glycogen

 Glycogen contains both a(1,4) links and

a(1,6) branches at every 8 to 12 glucose unit
 A complete hydrolysis of glycogen yields
glucose
 Glycogen and iodine gives a red-violet color,
and hydrolyzed by both a and b-amylases
and by glycogen phosphorylase
 Inulin Jerusalem
artichokes
 Some plants store carbohydrates in the form of
inulin as an alternative, or in addition, to starch.
 Inulins are present in many vegetables and
fruits, including onions, leeks, garlic, bananas,
asparagus, chicory, and Jerusalem artichokes.
 Inulins, also called fructans, are polymers
consisting of fructose units that typically have a
terminal glucose.
 Oligofructose has the same structure as inulin,
but the chains consist of 10 or fewer fructose
units.
 Inulin and oligofructose are nondigestible by
human intestinal enzymes, but they are totally
fermented by colonic microflora. The short-chain
fatty acids and lactate produced by fermentation
contribute 1.5 kcal per gram of inulin or
oligofructose.
 Other characteristics of Inulin are as follows
 b-(1,2) linked fructofuranoses
 linear only; no branching
 lower molecular weight than starch
 colors yellow with iodine
 hydrolysis yields fructose
 used as diagnostic agent for the
evaluation of glomerular filtration
rate (renal function test) Inulin n = approx. 35
 Chitin

 chitin is the second most abundant carbohydrate

polymer
 present in the cell wall of fungi and in the exoskeletons
of crustaceans, insects and spiders
 chitin is used commercially in coatings (extends the
shelf life of fruits and meats)
 Dextrans
 products of the reaction of glucose and the
enzyme transglucosidase from Leuconostoc
mesenteroides
 contains a (1,4), a (1,6) and a (1,3) linkages
 MW: 40,000; 70,000; 75,000
 used as plasma extenders (treatment of shock)
 also used as molecular sieves to separate
proteins and other large molecules (gel
filtration chromatography)
 components of dental plaques
 Dextrins
 produced by the partial hydrolysis of
starch along with maltose and glucose
 dextrins are often referred to as either
amylodextrins, erythrodextrins or
achrodextrins
 used as mucilages (glues)
 also used in infant formulas (prevent the
curdling of milk in baby’s stomach)
 Pectins
 Pectins are heteropolysaccharides that act as
cementing materials in the cell walls of all plant
tissues.
 Pectins are found in the pulp of fruits (citrus, apples),
and the white portion of the rind of lemons and
oranges contains approximately 30% pectin
 On hydrolysis pectins yield galacturonic acid,
galactose, arabinose, methanol and acetic acid
 Pectins are composed of galactans and arabans and
used as gelling agents (to make jellies)
 RESIN
 The resin produced by most plants
is composed mainly of volatile fluid
terpenes
 Terpenes, (C5H8)n, are derived
biosynthetically from units of
isoprene (C5H8)
Resin of a pine

Isopentenyl
Dimethylallyl
pyrophosphate
pyrophosphate
 Bacterial cell wall
 provide strength and rigidity for the
organism
 consists of a polypeptide-
polysaccharide known as petidoglycan
or murein
 determines the Gram staining
characteristic of the bacteria
Structure of peptidoglycan
Cell wall of Gram-positive
bacteria
Gram-negative bacteria
THANK YOU
Спасибо
THE NUCLEIC
ACIDS
Friedrich Miescher in 1869
 isolated what he called nuclein from the nuclei of
cells
 Nuclein was shown to have acidic properties,
hence it became called nucleic acid

© 2007 Paul Billiet ODWS

Two types of nucleic acid are
found
 Deoxyribonucleic acid (DNA)
 Ribonucleic acid (RNA)

© 2007 Paul Billiet ODWS

The distribution of nucleic
acids in the eukaryotic cell
 DNA is found in the nucleus
with small amounts in mitochondria and chloroplasts

 RNA is found throughout the cell

© 2007 Paul Billiet ODWS

DNA as genetic material: The
circumstantial evidence
1. Present in all cells and virtually restricted to the nucleus
2. The amount of DNA in somatic cells (body cells) of any given
species is constant (like the number of chromosomes)
3. The DNA content of gametes (sex cells) is half that of somatic
cells.
In cases of polyploidy (multiple sets of chromosomes) the DNA
content increases by a proportional factor
4. The mutagenic effect of UV light peaks at 253.7nm. The peak for
the absorption of UV light by DNA

© 2007 Paul Billiet ODWS

NUCLEIC ACID STRUCTURE
 Nucleic acids are polynucleotides
 Their building blocks are nucleotides

© 2007 Paul Billiet ODWS

 Asam nukleat merupakan
makromolekul dlm inti sel yg
mengandung semua informasi untuk
aktivitas maupun reproduksi sel.

Berperan:
a) Menyimpan informasi genetik
b) Proses replikasi dan transkripsi

DEPARTEMEN BIOKIMIA
FMIPA IPB
FUNGSI NUKLEOTIDA
 Penting untuk semua sel
 Komponen cofaktor enzim :
CoA; FAD; NAD; NADP
 Pembawa energi kimia (Energy Carriers):
ATP, ADP, GTP
 Precursor sintesis DNA
 Precursor sintesis RNA
-- Except
for some animal virus, DNA is genetic material.
-- RNAs have more diverged functions:
-- structural RNAs: e.g. rRNA
-- enzymatic RNAs: e.g. ribozymes
-- genetic materials: e.g. HIV virus.
-- others: e.g. tRNA, gRNA, etc.
-- (deoxy)ribonucleotides are the building blocks of nucleic acids:
3 characteristic components:
 -- due to the acidity of phosphate＝＞
called nucleic “acid”
 -- also due to phosphate: DNA and RNA
are highly negatively charged.
 -- phosphate and ribose are hydrophilic
NUCLEOTIDE STRUCTURE

PHOSPATE SUGAR BASE

PURINES PYRIMIDINES
Ribose or
Deoxyribose Adenine (A) Cytocine (C)
Guanine(G) Thymine (T)
Uracil (U)

NUCLEOTIDE
© 2007 Paul Billiet ODWS
Ribose is a pentose
C5

C4 C1

C3 C2

© 2007 Paul Billiet ODWS

 Spot the difference
RIBOSE DEOXYRIBOSE

CH2OH CH2OH
O OH O OH

C C C C

H H H H H H H H

C C C C

OH OH OH H
© 2007 Paul Billiet ODWS
 P

THE SUGAR-PHOSPHATE
BACKBONE P

 The nucleotides are all

orientated in the same direction P

 The phosphate group joins the

3rd Carbon of one sugar to the 5th P

Carbon of the next in line.

P

© 2007 Paul Billiet ODWS

 P
G

ADDING IN THE BASES P

C

 The bases are attached

P
to the 1st Carbon C
 Their order is important
It determines the P
A
genetic information of
the molecule P
T

P
© 2007 Paul Billiet ODWS
T
 Hydrogen bonds
P
G C
DNA IS MADE OF P
TWO STRANDS OF P
C
POLYNUCLEOTIDE G
P
P
C G
P
P
A T
P
P
T A
P
P
T A
© 2007 Paul Billiet ODWS
P
DNA IS MADE OF TWO STRANDS OF
POLYNUCLEOTIDE
 The sister strands of the DNA molecule run in opposite
directions (antiparallel)
 They are joined by the bases
 Each base is paired with a specific partner:
A is always paired with T
G is always paired with C
Purine with Pyrimidine
 This the sister strands are complementary but not
identical
 The bases are joined by hydrogen bonds, individually
weak but collectively strong
© 2007 Paul Billiet ODWS
Erwin Chargaff’s Data (1950-51)
Wilkins & Franklin (1952): X-ray
crystallography

© Norman Collection on the History of Molecular Biology in Novato, CA

 Purines & Pyrimidines

Adenine Thymine

Guanine Cytosine
© 2007 Paul Billiet ODWS
Watson & Crick Base pairing

© 2007 Paul Billiet ODWS

The Double Helix (1953)

Public Domain image

© Dr Kalju Kahn USBC Chemistry and Biochemistry
RNA
 mRNA (messenger RNA)
 Membawa informasi genetik dari DNA ke
ribosom untuk sintesis protein

 rRNA (ribosomal RNA)

 Penyusun 65 % ribosom, sisanya protein
 Mengenali mRNA

 tRNA (transfer RNA)

 Menterjemahkan kode genetik dari mRNA
menjadi asam amino tertentu
RNA
Replikasi DNA
Semikonservatif

DEPARTEMEN
BIOKIMIA FMIPA IPB
Metabolisme Asam Nukleat
1. Anabolisme Asam Nukleat
 Biosintesis Purin
 Biosintesis Pirimidin
2. Katabolisme Asam Nukleat
 Degradasi Purin
 Degradasi Pirimidin

DEPARTEMEN
Created by WARAS NURCHOLIS BIOKIMIA FMIPA IPB
5-Phospho- -D-ribosyl-1-
pyrophosphate (PRPP)
 Intermediet untuk baik proses de novo and
salvage pathway
 Berasal dari ribosa 5 phosphat
Biosintesis De
Novo Purines
 IMP synthase

GAR synthetase

AICAR
transformylase

GAR
transformylase

SAICAR lyase

FGAR amidotransferase

SAICAR synthetase
FGAM cyclase AIR karboksilase
Hal-hal penting dalam sintesis de novo purin:

1. Sangat tergantung pada “pool” ribosa

2. Gugus amina  didonor oleh glutamin dgn enzim
amidotransferase
3. Glisin dan fumarat  donor ring dlm nukleotida
4. Daur reaksi  dikontrol secara alosterik dgn AMP, ADP,
GMP dan GDP.  bekerja pada PRPP amidotransferase
 Daur diawali dgn perubahan
PRPP  IMP

 IMP = Inosine monofosfat

mrpkn bentuk nukleotida
purin yang pertama dibentuk
dlm daur ini

 Sebagai basa adalah

hypoxanthin
 Adenilosuksinat
synthetase

IMP dehidrogenase

XMP aminase

Adenilosuksinat lyase

DAUR dr
IMP 
AMP &
GMP
Metabolisme de novo nukleotida
pirimidine

CP synthetase

Aspartat
transcarbomoylase

Dihidrooratate DH
dihydrorotase
 Orotat
fosforibosiltransferase

Orotidilate
CTP synthetase dekarboksilase

UMP kinase
Nukleosida diphosphat kinase
Hal-hal penting dalam sintesis de novo pirimidine:

 cincin pirimidine disintesis terpisah dr gula ribosa nya

 Daur pirimidine de novo tidak bercabang produk akhir dr
daur adalah UMP yang mrpkn bahan dari CMP
 Reaksi pertama  pembtkan karbamoyl aspartate dr asp
dan carbomoyl-P titik regulasi yg penting dlm daur tsb
 Aspartat transcarbomoylase (ATCase)  diaktivasi oleh
ATP dan dihambat oleh CTP sbg produk akhir
 Degradasi purine

 Produk akhir katabolisme purin : asam urat

Vertebrata terestrial 
urea  ureotelic
Burung & reptil 
asam urat  uricotelic
Binatang di air 
ammonia
ammonotelic
Degradasi pirimidin