➢ We can determine which genes are
problematic, and we can attempt to
1.1. BIOTECHBOLOGY & BIOPROCESS
ENGINEERING
✓ We have come a long way with genetic
manipulation. For thousands of years,
genetic engineering has been practiced at
the level of breeding and selection (trial
and error procedure to generate best
offspring)
✓ Now, genetic engineering is done in a
purposeful, predetermined manner with
the advent of techniques to manipulate
DNA at the molecular level.
Biotechnology
✓ In the figure, the plant is modified by
inserting genetic material using a vector.
✓ With this revolution, new visions and
hopes have emerged:
➢ Large-scale culture of bacteria that
can degrade plastics.
➢ Genetically modified plants that are
safe to eat and resistant to
pestilence or even changes in
weather conditions (natural
pesticides)
➢ Develop new means of treating
people with rare genetic diseases
(Called gene therapy)
correct those defective genes.
➢ Identify stem cells for progenitors
of all differentiated tissue; this
allowed us to make artificial organs
in bioreactors starting with these
stem cells.
➢ Use of biological molecules to make
up biological sensors the tests for
diabetes tests for prostate cancer
that's for liver damage.
▪ microchips have these
biological molecules
implanted on them; they
make up of what is called as
biosensor
✓ Biological systems are very complex and
beautifully constructed, but they obey the
rules of chemistry and physics and are
thus susceptible to engineering analysis.
✓ Living cells are, to a degree, predictable
processes to use them can be rationally
designed and constructed on commercial
(large) scales.
✓ This is the job of the bioprocess engineer.
✓ Biotechnology implies the use or
development of methods or direct genetic
manipulation for a socially desirable goal.
➢ The “desirable” goals:
▪ Production of a particular
chemical
▪ Production of better plants
or seeds
▪ Gene therapy
▪ Use of specially designed
organisms to degrade
wastes
✓ Biotechnology is correlated to the use of
sophisticated techniques outside the cell
for genetic manipulation.
✓ the use of living systems and organisms to
develop or make product
 ✓ Biotechnology is also interpreted in a
broader sense and is equated with applied
biology.
➢ This might include engineering as a
subcomponent of biotechnology.
Bioengineering
✓ Bioengineering is a broad title and
includes agricultural, electrical,
mechanical, industrial, environmental,
chemical engineers, etc.
Bioengineers or Biological Engineering
✓ Emphasizes on the applications
animals and plants.
Biochemical Engineering
✓ It is usually meant the extension of
chemical engineering principles to
systems using a biological catalyst to bring
about desired chemical transformations.
✓ It is focused on fermentation engineering,
application of engineering principles to
microscopic biological systems that are
used to create new products by synthesis,
including the production of protein from
suitable raw materials.
✓ It is usually divided into two:
➢ Bioreaction engineering
➢ Bioseparations
Biomedical Engineering
✓ Considered to be completely separate
from biochemical engineering.
✓ Mostly focused on biomedical devices or
implants
✓ The boundary between biomedical and
biochemical engineering is increasingly
vague specifically in:
➢ The areas of cell surface receptors
➢ Animal cell culture
Biomolecular Engineering
✓ Defined by the National Institutes of
Health as “…research at the interface of
biology and chemical engineering and is
focused at the molecular level.”
✓ Application of engineering principles and
deign concepts to medicine and biology
✓ Under its umbrella:
➢ Tissue engineering
➢ Genetic engineering
➢ Neural engineering
➢ Pharmaceutical engineering
➢ Clinical engineering
➢ Bioinformatics
➢ Biomechanics
Biological Systems Engineering
✓ develops technology to monitor the
to conditions of where the process of making
pharmaceuticals takes place
✓ Ex: bioprocess design, biocatalysis,
bioseparation, bioinformatics, bioenergy
Environmental Health Engineering
✓ application of engineering principles to
the control of the environment for the
health, comfort, and safety of human
beings. It includes the field of life-support
systems for the exploration of outer space
and the ocean
Biomimetics
✓ the imitation of models, systems, and
elements of nature for the purpose of
solving complex human problems.
✓ Ex: velcro, designed after George de
Mestral noticed how easily burs stuck to a
dog's hair
Bionics
✓ an integration of Biomedical, focused more
on the robotics and assisted technologies.
✓ Ex: prosthetics
Bioprinting
✓ utilizing biomaterials to print organs and
new tissues
Systems Biology
✓ The study of biological systems.
Other Related Fields:
✓ Bioelectrical Engineering
✓ Biomechanical Engineering
✓ Biorobotics
Human Factors and Ergonomics Engineering
✓ application of engineering, physiology,
and psychology to the optimization of the
human–machine relationship.
✓ Ex: physical ergonomics, cognitive
ergonomics, human–computer interaction
Difference between Bioprocess Engineering
and Biochemical Engineering
✓ In addition to chemical engineering,
bioprocess engineering, include the work
of:
➢ Mechanical;
➢ Electrical;
➢ And industrial engineers
to apply the principles of their disciplines
to processes based on using living cells or
subcomponents of such cells.
✓ Problems of detailed equipment design,
sensor development, control algorithms,
and manufacturing strategies can be
solved through the mentioned disciplines.
✓ Meanwhile, biochemical engineering is
➢ More limited in the sense that it
primarily draws from chemical
engineering principles
➢ Yet more broader in the sense that
it is not restricted to well-defined
artificially constructed processes;
can be applied to natural systems
1.2. BIOLOGISTS & ENGINEERS DIFFER IN
THEIR APPROACH TO RESEARCH
✓ Trainings of biologists and engineers are
distinctly different.
On biology:
✓ In life sciences, mathematical theories and
quantitative methods (except statistics)
play secondary roles
✓ Most progress in biology are due to
experimental tools
➢ Results are qualitative and
descriptive models are formulated
and tested
✓ Biologists often have incomplete
backgrounds in mathematics but are very
strong in terms of laboratory tools and on
the interpretation of laboratory data from
complex systems.
✓ Biologists are better at the formation of
testable hypotheses, experimental design,
and data interpretation from complex
systems.
On Engineering:
✓ Engineers usually have a background in
physical and mathematical sciences.
✓ Often, theory leads to mathematical
formulations.
➢ The validity of the theory is tested
by comparing predicted response
to those in experiments.
➢ Quantitative models and
approaches, even to complex
systems, are strengths.
✓ Engineers are typically unfamiliar with
experimental techniques and strategies
used by life scientists.
Complementary
✓ The skills of engineers and life scientists
are complementary.
✓ To convert the promises of molecular
biology into new processes to make new
products require the integration of these
skills.
✓ Typical route for the development of a
bioprocess:

✓ STEPS 1 TO 12: Handled by life scientist

✓ A broad range of disciplines is involved in
bioprocessing.
✓ STEPS 12 TO 13: Handled by bioprocess
engineer; the one responsible for the
design of a bioreactor; determine if batch
or semi-batch; on step 12, one has to know
the maximum possible anticipated agitate
or speeed
✓ STEPS 14 ONWARD: No life scientist
involved, only bioprocess engineer
✓ Scientists working in this area are
constantly confronted with biological,
chemical, physical, engineering, and
sometimes medical questions.
✓ In bioprocessing, we have involvement of
a broad range of disciplines.
 bacteria grew near the invading
substance.
➢ He recognized that the cell killing
must be due to antibacterial agent.
➢ He recovered the foreign particle
and found that it was a common
mold of the Penicillium genus (later
identified as Penicillium notatum)
➢ Fleming nurtured the mod to grow
though crude extraction methods.
➢ He obtained a tiny quantity of
secreted material.
▪ He discovered that this
material had powerful
antimicrobial properties
and named it penicillin.
➢ He preserved the culture, but its
discover lay dormant for over a
decade.
➢ In World War II, the discovery of
penicillin was resurrected due to
the need of an antibiotic with
minima side effects and broader
compatibility that sulfa drugs.
➢ Howard Florey and Ernst Chain of
Oxford build on Fleming’s
observations.
➢ Norman Heatley produced
sufficient material for Chain and
Florey to test penicillin’s effectivity.
▪ Though Heatley is a
biochemist, he performed as
a bioprocess engineer.
▪ He developed an assay to
1.3. PENCILLIN HISTORY
monitor the penicillin
amount made as so to
✓ In September 1928, Alexander Fleming is at
determine the kinetics of
St. Mary’s Hospital in London
fermentation.
➢ He is trying to isolate the
▪ He developed a culture
bacterium, Staphylococcus aureus,
technique that could be
which causes boils
easily implemented.
➢ The technique in use was to grow
▪ He devised a novel back-
the bacterium on the surface of a
extraction process to
nutrient solution.
recover very delicate
➢ One of the dishes was
product.
contaminated inadvertently with a
foreign particle; he noticed that no
 ▪ After months, they penicillin program like the
produced enough penicillin following:
to treat laboratory animals. ▪ The development of a corn
➢ 18 months after the project, they steep liquor-lactose based
treated a London bobby for a blood medium; this increased the
infection; he recovered. productivity about tenfold.
➢ Penicillin supply was exhausted, ▪ Its worldwide search for
and the man relapsed and died. better producer strains of
➢ Many companies, assisted by Penicillium led to the
universities aided the development isolation of a Penicilium
penicillin including Merck, Pfizer, chrysogenum strain.
Squibb, and the USDA Northern • This strain is isolated
Regional Research Laboratory in from a moldy
Peoria Illinois. cantaloupe at a
➢ Early clinical successes were so Peoria fruit market.
dramatic that in 1943, the War • It is proven to have
Production Board appointed A.L superior to hundreds
Elder to coordinate the activities of of other isolates
producers to increase penicillin tested.
supply. • Its progeny have
▪ The fermentation route was been used in almost
chosen. all commercial
➢ Typical problems in most new fermentations.
fermentation processes:
▪ A valuable product made at ➢ Another problem in penicillin
very low levels. production is on deciding on the
▪ Low rate of production per manufacturing process:
unit volume necessities very ▪ One method involved the
large and inefficient growth of the mold on the
reactors. surface of moist bran.
▪ Also, the low concentration • This is discarded
(titer) made product because of
recovery and purification difficulties in
difficult. temperature control,
➢ In 1939, final concentration of sterilization, and
penicillin fermentation broth was equipment size.
one part per million (ca. 0.001 g/l); ▪ The surface method
gold is more plentiful in sea water. involved growth of the mold
➢ Penicillin is fragile and unstable. on top of a quiescent
▪ This means that there are medium.
significant constrains on the • This used a variety of
approaches used for containers, including
recovery and purification. milk bottles.
➢ Life scientists at the Northern • “bottle plant” is used
Regional Research Laboratory to indicate this
made major contribution to the manufacturing
technique.
 • This method gave ✓ From 1939 to now, the yield of penicillin
relatively high yields gone from 0.001 g/l to over 50 g/l of
but had a long fermentation broth.
growing cycle and ✓ Schematic of penicillin production
was labor intensive. process:
• The first
manufacturing
plants were bottle
plants because the
method worked and
can be implemented
quickly.
• Surface method
would not meet the
full need for
penicillin.
▪ The submerged tank
process was favored by
engineers.
• It presented 1.3 . BIOPROCESSES: REGULATORY
challenges in terms CONSTRAINTS
of both mold
physiology and in ✓ The U.S Food and Drug Administration
tank design and (FDA) and its equivalents in other
operation. countries must ensure the safety and
• Large volume of efficacy of medicines.
absolutely clean oil ✓ There have been tragic examples where a
and dirt-free sterile small process change has allowed a toxic
air were required. trace compound to form or become
• Large agitators are incorporated in the final product, resulting
required to prevent in severe side effects, including death.
organism entry. ✓ Thus, even slight process changes may
require new clinical trials to test the safety
✓ Problems in recovery and purification also of the resulting product. Since clinical
exist. trials are very lengthy and expensive,
➢ A combination of pH shits and rapid process improvements are made under a
liquid-liquid extraction proved limited set of circumstances.
useful. ✓ Bioprocess engineers in the
✓ Soon, tanks of about 10,000 gal were built. pharmaceutical or biotechnology
✓ Pfizer completed in less than six months industry’s primary concern is not the
the first penicillin plant for commercial reduction of manufacturing cost, but the
production. production of consistently high-quality
➢ It has 14 tanks (with 7000-gal amounts to satisfy the medical needs of the
capacity) population.
✓ In World War II, there is enough penicillin ✓ A typical drug undergoes 6.5 years of
for almost 100,000 per year. development from the discovery stage
through preclinical testing in animals.
✓ Human clinical trials are conducted in
three phases:
➢ Phase 1 clinical trials (about 1 year)
are used to test safety; 30 to 80
volunteers involved.
➢ Phase 2 clinical trials (about 2
years) use 100 to 300 patients and
the emphasis is on efficacy (if it
helps the patient) and the
determination of side effects (or if
it exist)
➢ Phase 3 clinical trials (about 3
years) are those with promising
results with about 1000 to 3000
patients.
✓ Clinical trials are presented in the FDA for
review (about 18 months).
✓ The drug is then approved if it is well
design, has statistically significant
improvements, and has acceptable side
effects.
✓ Continued monitoring will be held for
possible adverse effects.
✓ The whole drug discovery-through-
approval process takes an average of 15
years and costs about 400 million
dollars (in 1996).
✓ Only 1 in 10 drugs that enter human
clinical trials receives approval.
✓ Recent FDA reforms decreased the time to
obtain drug approval for life-saving drugs
in treatment of diseases like cancer and
AIDS.
✓ Drugs sold on the marker or used in
clinical trials must come from facilities
that are certified as GMP (Good
Manufacturing Practice)
➢ It concerns the actual
manufacturing facility, design,
layout, equipment and procedures,
training of production personal,
control of process inputs (raw
materials and cultures), and
product handling
➢ The design layout must prevent
contamination of the product and
dictates the flow of material,
personnel, and air.
➢ Equipment and procedures must
be validated.
▪ Procedures also include the
cleaning and sterilization
and not only the operation.
➢ Computer software used to
monitor and control the processes
must be validated.
➢ Offline assays done in the
laboratory must satisfy GLB (Good
Laboratory Practices)
➢ Procedures should be documented
by SOPs (Standard Operating
Procedures)
➢ GMP Guidelines stress the need
for documented procedures to
validate performance.
➢ “Process Validation is
establishing documented evidence
which provides a high degree of
assurance that a specific process
will consistently produce a product
meeting its predetermined
specifications and quality
characteristics” and that “There
shall be written procedures for
production and process-control to
assure that products have identity,
strength, quality, and purity they
purport or are presented to
possess.”
➢ Key concepts in process validation:
▪ Written documentation
▪ Consistency of procedures
▪ Consistency of products
▪ Demonstrable measures of
product quality
(particularly purity and
safety)
✓ The key point is that process changes
cannot be made without considering their
considerable regulatory impact.
 ✓ Some cells grow at -20oC (in brine), while
other can grow at 120oC (under high
pressures).
➢ This can be achieved if there is high
concentration of salts; this allows
freezing point depression
➢ With high pressures, then you can
still have liquid water at this high
temperature
➢ Some cells can grow at very
extreme condition which otherwise
would be very deadly to plants and
animals.
✓ Research is part of the timeline champion
✓ In terms of optimal temperature for
amalgam of discovery only on drug or
growth, cells can be classified as:
quantum product, then there has to be
➢ Psychrophiles: cells that grow
research and this the length of time.
best at low temperatures (below
20oC)
➢ Mesophiles: cells that grow best in
the range of 20oC to 50oC
1.1. CELL DIVERSITY ▪ Plant cells and animal cells
are considered mesophiles
✓ Our focus in microbial diversity or small ➢ Thermophiles: cells that grow
cells. best at temperatures greater than
✓ Living things are not confined to the 50oC
familiar temperate realm of land, water, ▪ for thermophiles, they have
and sunlight inhabited by plants and plant- enzymes that are mostly
eating animals. heat resistant, heat
sensitive; hence, they are
✓ They can be found in the darkest depths of
thoroughly stable
the ocean, in hot volcanic mud, in pools
▪ Some organisms like this are
beneath the frozen surface of the
used in biotechnology: In
Antarctic, and buried kilometers deep in
the Earth’s crust. PCR, we have this special
➢ The aforementioned are some of enzyme polymerase that
the most unique microorganisms. can still function at high
➢ They are inaccessible. temperatures and that
➢ Very microscopic in size. enzyme has been isolated
from a specific thermal
fighter.
✓ Many organisms also have pH optima far
from neutrality, with some growing best
in highly acidic conditions, and some in
highly basic conditions.
➢ Although most organisms grow
only where water activity is high,
some organisms can grow on
 barely moist solid surfaces or in
highly saline solutions.
✓ Cells also differ in oxygen requirements:
➢ Aerobic: require oxygen for
growth and metabolism
▪ like human beings
➢ Anaerobic: growth inhibited by
the presence of oxygen
✓ Coccis (pl. cocci): spherical cells
▪ tetanus
✓ Bacillus (pl. bacilli): rod-shaped cells
➢ Facultative: growth is possible
✓ The smallest cells: hard to identify the
either aerobically or anaerobically
shape; has a separate category
▪ it can shift to metabolism
✓ Spirillum (pl. spirilla): spiral cells
that makes use of oxygen if
there is no oxygen; then it
✓ The shape is important because one has to
shifts to another kind of
know that morphology, its requirements
metabolism, that does not
(anaerobic or aerobic), pH for growth,
require oxygen
✓ Energy & Carbon Source: temperature, etc.

Primary Cell Types

✓ Despite the diversity of cells, all cells are
classified as one of two primary cell types:
➢ eukaryotic and prokaryotic.

➢ Living organisms obtain their free energy

and carbon in different ways.
➢ On Energy Sources:

▪ Phototrophs: when energy

is from sunlight; they feed
on light as energy source
▪ Chemotrophs: when
energy is derived from the
consumption of chemical
compounds.
✓ Not only do microorganisms occupy a
wide range of habitats and have different
growth requirements, but they also come
in a wide range of sizes and shapes.
✓ Some cells may even change shape in
response to changes in their local
environment.
✓ Main differences between eukaryotic
and prokaryotic cells:
1. Compartmentalization of DNA (DNA in
organelles):
In prokaryotes, DNA is exposed and
floating in the cytoplasm; called “naked
DNA”
2. Number of DNA molecules:
 Prokaryotes usually have only one circular
DNA while eukaryotes have many.
3. Membrane bound organelles:
There are many membrane bound
organelles present in eukaryotes and not
in prokaryotes such as the mitochondria,
ER, golgi apparatus, etc.
Prokaryotes
✓ Above is the bacterium Vibrio cholerae
and its simple organization:
➢ Flagellum: a helical appendage at
one end; rotates as a propeller to
drive the cell forward; it can infect
human small intestine to cause
cholera
➢ DNA is floating inside the
cytoplasm and there are a lot of
ribosomes scattered around.
➢ Plasma membrane: surrounding
your microorganism.
✓ Bacteria Gram Strains:
✓ Bacteria gram strains differentiate
bacteria in two classes: gram positive
cells and gram negative cells.
✓ Procedure:
➢ We use crystal violet and initially,
all material will be stained with
crystal violet, and we add iodine as
a fixating agent, then we wash it
with alcohol. Upon washing it with
alcohol, some cells will retain that
crystal violet stain; some will be
clear again.
➢ We will then add the second dye.
✓ Gram positive microorganisms: have a
thick peptidoglycan layer; peptidoglycan
means you have macromolecules that are
combinations of carbohydrates and
proteins.
➢ Underneath this, there is just one
plasma membrane.
➢ E.coli is one of the most famous
examples of this type of cell.
✓ Gram negative microorganisms:
peptidoglycan layer is thinner, although
there is an additional outer membrane.
➢ there is an additional outer
membrane, hence, sandwiching the
peptidoglycan layer.
✓ Genome analyses have made it clear that
prokaryotes comprise two distinct groups:
bacteria (or eubacteria) and the
archaea (or archaebacteria).
 ✓ Tree of Life:
✓ The green areas are called the “missing
links”
✓ Eukaryotes came from archaea, and not
bacteria
✓ Three main domains of life:
➢ Bacteria (prokaryotes)
➢ Acraea (prokaryotes)
➢ Eukaryotes (where humans
evolved from)
✓ Archaea
➢ Archaea are often found inhabiting
environments that humans avoid,
such as bogs, sewage treatment
plants, ocean abysses, salt brines,
and hot acid springs—most of
theme are extremophiles (those
who inhabit extreme
environments)
Eukaryotes
✓ Fungi, algae, protozoa, and animal and
plant cells constitute the eukaryotes.
✓ Eukaryotes are 5-10 times larger than
prokaryotes in diameter and have a true
nucleus, in addition to a number of cellular
organelles inside the cytoplasm.
✓ In cell wall and cell membrane structure,
eukaryotes are similar to prokaryotes.
✓ Plants have cell walls, but animal cells
don’t—for this treason, animal cells are
very shear-sensitive and fragile. This
factor significantly complicates the design
of large-scale bioreactors for animal cells.
✓ Mixing does not kill the cells: plant cells
can withstand mixing, but it is a limited
case for animal cells.
✓ This is an animal cell because it does not
have a cell wall.
✓ Part of the cytoskeleton (gives
mechanical support, strength and
structure to the cell):
➢ Microtubule
➢ Intermediate filaments
➢ Actin filaments
✓ Main parts:
➢ Nucleus
➢ Nuclear membrane or nuclear
envelope: it has force because
information has to come out of this
set of this component
➢ Nucleolus: this is the site for
ribosome synthesis
➢ Ribosomes: scattered all around
the cytoplasm (the black dots), but
they can be attached to the ER.
▪ The ribosomes are the
“protein factories”
➢ Rough endoplasmic reticulum: ER
with attached ribosome; involved
in protein synthesis and
medication (attachment of sugars
to proteins to produce
glycoproteins; also include the
attachment of lipids to proteins to
produce lipoproteins).
➢ Smooth endoplasmic reticulum:
does not have attached ribosomes;
involved in lipid synthesis
 ➢ Centrosomes: involved in the
reproduction of the cell;
attachment of microtubules; also
involved in locating the center of
the cell (it does that by extending
the microtubules until it reaches
the periphery of the cells;
important in cell division)
➢ Golgi apparatus or bodies: involved
in packaging and release of
materials that has to be secreted by
the cell; packaging of protein that 2. Aspergillus niger: used in the
has to be sent outside the cell; also production of citric acid.
involved in modification. 3. Penicillium chrysogenum: production of
➢ Plasma membrane: serves as a penicillin
semipermeable barrier. 4. Algae: used in wastewater treatment as
➢ Mitochondrion: powerhouse of the well as in simultaneous production of
cell; provide energy by single cell protein (commonly used as a
synthesizing ATP (energy currency feed for livestock or food for humans;
of the cell) considered as “food of the future”); also
➢ Lysosome: responsible for the used in producing biofuels and biooils
degradation of whatever needs to
be degraded; degradation sites

✓ The most common eukaryotic cells used in

bioprocesses are:
1. Saccharomyces cerevisiae: used in both
winemaking or simply alcohol production
from as well as in baking for yeast
- One strain produced ethanol
in an anaerobic process
- Other strain produced CO2
(for baking) in aerobic
process
1.2. CELL COMPOSITION
Chemical Components
✓ Living organisms are made of only a small
selection of the 92 naturally occurring
elements, four of which—carbon (C),
nitrogen (N), hydrogen (H), and oxygen
(O)—make up 96.5% of an organism’s
weight.
 ✓ By weight, macromolecules are the most
abundant carbon-containing molecules in
a living cell.

✓ The four elements in red constitute 99%

of the total number of atoms present in the
human body.
✓ We have 3 volumetric macromolecules:
✓ An additional of seven elements,
o Only lipids are not polymeric while
highlighted in blue, together represent
polysaccharide, protein, and
about 0.9% of the total.
nucleic acids are.
✓ The elements in green are required in
trace amounts by humans; needed in small Proteins
amounts; if they exceed in this amount,
✓ Proteins are the most abundant organic
they become toxic.
molecules in living cells. They are built
✓ The elements in yellow are those unclear
from amino acid monomers, and have
if they are essential in humans.
diverse biological functions:
➢ structural (glycoproteins,
✓ The small organic molecules of the cell are
collagen, keratin, microtubules,
carbon-based compounds that have
etc.)
molecular weights in the range of 100-
➢ catalytic (enzymes)
1000 and can contain up to 30 or so carbon
➢ transport (hemoglobin, serum
atoms.
albumin, membrane transport
✓ Some of these are used as monomers to
proteins, etc.)
construct giant polymeric
➢ regulatory (hormones)
biomacromolecules. Others act as energy
➢ protective (antibodies, thrombin,
sources to be broken down via metabolic
etc.)
pathways.
✓ We have 20 amino acids (make up proteins
✓ Broadly speaking, cells contain four major
in living cells) and some of these have
families of small organic molecules:
hydrophobic residues, charged residues,
sugars, fatty acids, nucleotides, and
and can either be acidic or basic, some
amino acids. These four families of small
have aromatic groups.
organic molecules, together with their
✓ The general structure (R is the residue):
polymers, account for a large fraction of
the cell mass.
 ✓ In terms of structure, cellulose is a
✓ Amino acids require nitrogen. carbohydrate and cellulose is responsible
✓ Some of the residues also contain sulfur. for the rigidity of plant cells.
✓ The sequence of proteins when attached ✓ Storage in plants – starch
together make up your primary structure; ✓ Storage in animals or humans – glycogen
that primary structure dictates how the ✓ Most common carbohydrates:
protein will look like in 3D space, so that
will give rise to your secondary structure.
✓ Combinations of different secondary
structures will make up the final tertiary
structure.
✓ When different units combine to form a
much larger structure, it is called the
quaternary structure.

✓ Cellulose has an upward pointing

connection
✓ Starch and glycogen have downward
pointed connections
✓ We cannot degrade cellulose, but we
can degrade starch; this is due to their
connection differences

✓ Ex. Hemoglobin, a transport protein found Lipids

in blood, is responsible for transporting ✓ Lipids are hydrophobic biological
oxygen and carbon dioxide; it is consisting compounds that are insoluble in water, but
of 4 subunits. Hence, it is a quaternary soluble in nonpolar solvents like benzene,
structure, and those subunits are tertiary chloroform, and ether. They are usually
structures. present in plasma membranes.
Carbohydrates ✓ Cells can alter the mix of lipids in their
membranes to compensate, at least
✓ Carbohydrates are the most abundant partially, for changes in temperature or to
organic molecules in nature. These increase their tolerance to the presence of
molecules play key roles as structural and chemical agents such as ethanol.
storage compounds in cells, as well as in
some aspects of chemical signaling in
animals and plants.
✓ Carbohydrates are represented by the
general “hydrated carbon” formula,
(𝐶𝐻2 𝑂)𝑛 , 𝑛 ≥ 3.
✓ Another function of carbohydrates is that
when they are clasped with a protein, they
can shield that protein from recognition by
the immune system.
✓ Lipids are actually in general class of Nucleic Acids
molecules that can be extracted by
✓ Nucleic acids play the central role in the
organics solvents; anything that can be
reproduction of living cells.
extracted from organic solvents are lipids.
Deoxyribonucleic acid (DNA) stores and
✓ Cholesterol is a good structural support.
preserves the genetic information, while
✓ Other molecules also serve as the OH
ribonucleic acid (RNA) converts
source for esterification; in this case, fatty
segments of DNA into short “manuals” for
acids react with a phosphate group to form
use by ribosomes in protein synthesis,
a phospholipid.
among other functions like regulation and
✓ Lipids make up out cell membrane.
structure.
➢ Not all cell membranes are made up
✓ There are also other classes aside from
of phospholipids, but others are
DNA or RNA like SNRNA, MI RNA, etc.
made up of special lipids like
✓ 4 nucleotides make up the nucleic acids
sphingolipids.
o These nucleotides have
➢ Lipids are important for protection
nitrogenous base
and cell membrane structure.
o Contains sugar; this sugar is the
➢ Cell membrane is made up of lipids
deoxyribose for ribose in RNA
and some protein (it can span
o It also has at least one phosphate
either the whole membrane of be
groups
present in just one layer or only in
o To give rise nucleic acids,
the surface); carbohydrates can
phosphate and sugar groups must
also be found on these proteins
have a reaction
➢ The small orange circles in the
figure are cholesterol; they assist in
making the cell membrane more
rigid; prevent freezing of
membrane at low temperatures
✓ If your product is intracellular, it means
inside the cell, you must be able to destruct
the lipid membrane to be able to get the
product from the interior of the cell.
➢ To do that, we must know what the
membrane is composed of.

✓ 4 base base pairs: A, T, G, C

➢ However, in RNA, thymine is
replaced with uracil

CELL COMPOSITION
✓ A cell’s composition differs greatly from its
environment, and it must selectively
remove desirable compounds from its
extracellular environment and retain
other compounds within itself (not spill).
✓ Since the cell differs greatly in composition
from its environment, active
concentration differences are present, and
the cell must expend energy to maintain
itself away from thermodynamic
equilibrium, or practically, death.
✓ Allowing equilibrium in living systems
means death.
✓ The intracellular composition of cells
varies depending on the type and age of
the cells, and the composition of nutrient
media in which the cells are growing.
✓ Different compositions between
organisms:
1.3. CELL NUTRIENTS
✓ Most of the products formed by organisms
are produced as a result of their response
to environmental conditions, such as
nutrients, growth hormones, and ions.
✓ The qualitative and quantitative
nutritional requirements of cells need to
be determined experimentally to optimize
growth and product formation.
✓ Cell nutrients can be classified into two
categories:
➢ Macronutrients: needed in
concentrations larger than 10-4M;
carbon, oxygen, nitrogen,
hydrogen, sulfur, phosphorus,
Mg2+, and K+ are major
macronutrients
➢ Micronutrients: needed in
concentrations less than 10-4M;
trace elements such as Mo2+, Zn2+,
Cu2+, Mn2+, Ca2+, Na+, vitamins,
growth hormones, and metabolic
precursors are micronutrients
Macronutrients
✓ Carbon compounds are major sources of
cellular carbon and energy. The most
common carbon sources in industrial
fermentations are molasses (sucrose),
starch (glucose, dextrin), corn syrup, and
waste sulfite liquor (glucose).
➢ In lab fermentations, glucose, sucrose,
and fructose are the most common
carbon sources. Methanol, ethanol, and
methane also constitute cheap carbon
sources for some fermentations.
➢ In aerobic fermentations, about 50% of
substrate carbon is converted into cell
mass, and 50% is used as energy
source. In anaerobic fermentations,
more carbon is converted to products,
and a smaller fraction is converted to
cell mass (<30%).
 ➢ In anaerobic fermentations, there is a Micronutrients
higher yield in product compared to
✓ Trace elements are essential to microbial
aerobic fermentations.
nutrition—lack of essential trace elements
increases the time lag from inoculation to
✓ Nitrogen constitutes about 10-14% of cell
growth in culture and may decrease the
dry weight. The most widely used sources
growth rate.
are ammonia or the ammonium salts
(chloride, sulfate, or nitrate), proteins,
✓ Micronutrients are grouped into three:
peptides, and amino acids.
1. those that are most widely needed
➢ Urea may also be used as a nitrogen
(Fe, Zn, Mn)
source by some organisms. Organic
2. those that are needed under
nitrogen sources such as yeast extract
specific growth conditions (Cu, Co,
and peptone are expensive compared
Mo, Ca, Na, Cl, Ni, Se)
to ammonium salts but are
3. Those that are rarely required (B,
nevertheless common in lab
Al, Si, Cr, V, Sn, Be, F, Ti, Ga, Ge, Br,
fermentations.
Zr, W, Li, I)

✓ In addition, chelating agents such as

EDTA, citric acid, polyphosphates,
histidine, tyrosine, and cysteine, are
needed to form soluble compounds with
ions that are prone to precipitation in the
culture medium.

✓ Growth factors may also be added to

stimulate the growth and synthesis of
some metabolites. Vitamins, hormones,
and amino acids are major growth factors.
➢ We never use pure sources because it
Growth Media
is expensive.
Other macronutrients and their function: ✓ Two major types of growth media are
defined and complex media:

➢ Defined media are those whose

components are present at specific
amounts. Although more expensive,
defined media have the advantage of
reproducibility and better control over
fermentation. Product recovery and
purification are often easier and
cheaper.
➢ Complex media are those containing
natural compounds whose chemical
compositions are not exactly known,
such as yeast extracts, peptone,
molasses, and corn steep liquor.
 Complex media are usually able to
provide the necessary growth factors,
vitamins, hormones, and trace
elements, resulting in higher cell
yields, compared to defined media.
✓ Growth media can either be solid or liquid
➢ In solid form, usually dissolved in
agarose; agarose is a powder but if you
melt it with hot water, it will start on
polymerizing and form the ager gel.
➢ Medias also have different colors;
yellow for agarose.
➢ Some microorganisms prefer to grow
on surfaces, some prefer to grow in
solutions.
Enzymes
✓ Enzymes are biological catalysts (of
nature) that mediate the transformation of
molecules or energy from one form into
another.
✓ The catalytic activity of enzymes
accelerates typical reactions to usually 103
to 1017 times faster than the uncatalyzed
rate.
✓ Although nearly all enzymes are proteins,
some enzymes are also RNA molecules—
called ribozymes.
✓ By utilizing the full repertoire of
intermolecular forces (e.g. hydrophobic
reactions, hydrostatic reactions, van der
Waals reactions), enzymes bring
substrates together in an optimal
orientation, the requirement for a reaction
to occur.
✓ In the illustration, the coils are alpha
helices; sheets are beta sheets;
everything else are random coils.
✓ Rate enhancement by selected
enzymes:
 ✓ Like any catalyst, enzymes provide an
alternate pathway for the reaction to
occur. This pathway requires a lower
activation energy.
✓ Because enzymatic pathways have
lowered EA, enhancements in reaction
rates can be enormous.
✓ Activation energy and rate of reaction
are inversely related; this
independence is exponential.
𝐸𝐴
✓ Arrhenius equation (𝑘 = 𝐴𝑒 −𝑅𝑇 ) is
exponential but also an inverse one.
✓ Enzymes lower the activation energy
by facilitating the formation of, and
stabilizing, transition states.
➢ proximity effects: bringing
substances closer together; for
✓ In addition, enzymes act on specific
a biomolecular reaction, a
collision of two substrates
should occur
➢ orientation effects: everything
✓ We can think of it as if the enzyme having
must be oriented for there to
have a reaction
✓ Enzymes usually work under mild
✓ For example:
➢ Proteases: only catalyze the
hydrolysis of peptide bonds
between specific amino acids in
proteins; e.g. papain (from
papaya and can hydrolize any
peptide bonds), trypsin (can
cleave peptide bonds but only
those involving amino acids like
arginine or lysine),
chymotrypsin
➢ Amylases: work only on
glycosidic bonds between
glucose molecules in starch
➢ Lipases: only attack fats,
degrading them into fatty acids
and glycerol
substrates or they can only bind to a
specific enantiomer—they are
stereospecific.
insertion sites or “active sites” that can
only bind complimentary substrates
➢ E.g. ibuprofen: we have R and S
ibuprofen enantiomers, and the
body can only metabolize one
conditions: pH 4 to 9 and temperatures
between 20oC and 70oC.

✓ An important property of enzymes is that

they are highly specific: one enzyme
usually catalyzes a single chemical
reaction or a set of closely related
reactions.
✓ The Enzyme Commission number (EC
number) is a numerical classification ✓ Top-level EC Numbers:
scheme for enzymes based on the chemical
reactions they catalyze. Class Reaction Catalyzed Typical Enzyme
Reaction example(s)
✓ Every EC number is associated with a with trivial
recommended name for the respective name
enzyme.
✓ The current (6th) edition of the
EC 1 Oxidation/reduction AH + B → A Dehydrogenase,
nomenclature scheme is published by the Oxidoreductases reactions; transfer + BH oxidase
of H and O atoms or (reduced)
International Union of Biochemistry electrons from one A + O → AO
and Molecular Biology. substance to (oxidized)
another
EC 2 Transfer of a AB + C → A Transaminase,
Transferases functional group + BC kinase
from one substance
to another. The
group may be
methyl-, acyl-,
amino- or phosphate
group
EC 3 Formation of two AB + H2O Lipase, amylase,
✓ Every enzyme code consists of the letters Hydrolases products from a → AOH + peptidase,
substrate by BH phosphatase
“EC” followed by four numbers hydrolysis
EC 4 Non-hydrolytic RCOCOOH Decarboxylase
separated by periods, representing Lyases addition or removal → RCOH +
progressively finer classification of the of groups from CO2 or [X-
substrates. C-C, C-N, A+B-Y] →
enzyme. C-O or C-S bonds [A=B + X-
✓ For example, the tripeptide may be cleaved Y]
EC 5 Intramolecule ABC → BCA Isomerase,
aminopeptidases are EC 3.4.11.4: Isomerases rearrangement, i.e. mutase
isomerization
➢ EC 3 –hydrolase enzymes changes within a
➢ EC 3.4 –hydrolases acting on single molecule
EC 6 Join together two X + Y + ATP Synthetase
peptide bonds Ligases molecules by → XY +
➢ EC 3.4.11 –hydrolases cleaving synthesis of new C- ADP + Pi
O, C-S, C-N or C-C
off amino-terminal amino acids bonds with
simultaneous
from a polypeptide breakdown of ATP
➢ EC 3.4.11.4 –hydrolases EC 7 Catalyse the Transporter
Translocases movement of ions or
cleaving off amino-terminal molecules across
amino acids from a tripeptide membranes or their
separation within
membranes
✓ Expasy (database for enzymes)

✓ The structure above is the human carbonic

anhydrase II and its zinc site.
➢ (Left) Notice that the zinc ion is
bound to the imidazole rings of
three histidine residues as well
as to a water molecule.
➢ (Right) Notice the location of
the zinc site in a cleft near the
center of the enzyme.

✓ Many enzymes require small molecules

called cofactors for their catalytic activity.
This is where the micro or macronutrients
come in. Generally, these cofactors are able
to execute chemical reactions that cannot
be performed by the standard set of
twenty amino acids.

✓ An enzyme bound to its cofactor is called a

holoenzyme; without the cofactor, the
enzyme is known as an apoenzyme.

Apoenzyme + cofactor = holoenzyme

✓ Cofactors can be either metals or small

organic molecules called coenzymes.
These coenzymes are often derived from
vitamins.

✓ Tightly bound coenzymes (like covalently

attached) called prosthetic groups;
loosely associated coenzymes are
sometimes considered “cosubstrates.”
✓ Above is a quaternary structure of
deoxyhemoglobin.
 ➢ Hemoglobin, which is a similar manner to the fitting of a key
composed of two 𝛼 chains and into a lock.
two 𝛽 chains, functions as a ➢ Here, both enzyme and substrate are
pair of 𝛼𝛽 dimers, rigid. For a long time, this was the
➢ (A) a ribbon diagram; (B) a preferred model for E·S complex
space-filling model formation.
➢ For hemoglobin the cofactors
are both metal ion and organic
molecule.
The Enzyme-Substrate Complex
✓ The catalyzed reaction pathway proceeds
through an active intermediate (a
“transition state”), called the enzyme-
substrate complex (E·S complex).
✓ The substrate binds with a specific active site
of the enzyme to form the E·S complex.
✓ There are also sits called the allosteric sites.
✓ Much of the catalytic power is attributed to
the binding energy of the substrate to the
enzyme through multiple noncovalent bonds ✓ In the induced fit model (actually the
with specific functional groups on the enzyme. more useful model), both the enzyme and
All noncovalent bonds: substrate are distorted.
➢ electrostatic (ionic) interactions ➢ These changes in conformation distort
(represented by the + and – signs) one or more of the substrate bonds,
➢ hydrogen bonds making these bonds weaker and easier
➢ van der Waals forces to break.
➢ π-πstacking interactions ➢ Thus, the mechanism of enzymatic
catalysis is dynamic, involving
structural changes with multiple
intermediates of both substrates and
the enzyme.

✓ There are two models for enzyme-

substrate interactions:
➢ In the lock-and-key model, proposed
by Emil Fischer in 1890, the E·S
complex is pictured as being formed in

Mechanisms
✓ For a constant amount of enzyme, the rate
of catalysis increases with substrate
concentration, but begins to level off and
approach a maximum at higher substrate
concentrations—a phenomenon
recognized as saturation.
➢ This means that we have an upper limit
to the catalysis that can be done by
your enzymes.
✓ In 1913, Leonor Michaelis and Maud
Menten proposed a simple model to
account for these kinetic characteristics.
✓ The critical feature in their treatment is
that a specific ES complex is a necessary
intermediate in catalysis:
➢ E and S formed ES complex in a
rapid equilibrium.
➢ Once the ES complex is formed, it
will follow one or two things:
1. Either the substrate dissociates
to reform E and S.
2. Or the substrate becomes
processed to form the product,
P.
Note: The formation of the product from the ES is
irreversible with rate constant k2.
Michaelis-Menten Kinetics: Derivation
✓ It is assumed that the ES complex is
established rather rapidly, and the rate of
the reverse reaction of the second step is
negligible (when product accumulation is
negligible, i.e. at the beginning of the
reaction).
✓ We want an expression (rate law) that
relates the rate of catalysis to the
concentrations of substrate and enzyme,
and the rates of the individual steps.
✓ The rate of product formation is:
𝑑 [𝑃 ]
−𝑟𝑠 = 𝑟𝑝 = 𝑣 = ( ) = 𝑘2 [𝐸𝑆]
𝑑𝑡
Where
➢ 𝑟𝑝 = 𝑟𝑎𝑡𝑒 𝑜𝑓 𝑝𝑟𝑜𝑑𝑢𝑐𝑡𝑖𝑜𝑛 𝑓𝑜𝑟𝑚𝑎𝑡𝑖𝑜𝑛
➢ 𝑟𝑠 = 𝑟𝑎𝑡𝑒 𝑜𝑓 𝑠𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒 𝑐𝑜𝑛𝑠𝑢𝑚𝑝𝑡𝑖𝑜𝑛
➢ 𝑣 = 𝑣𝑜𝑙𝑙𝑢𝑚𝑒𝑡𝑟𝑖𝑐 𝑟𝑒𝑎𝑐𝑡𝑖𝑜𝑛 𝑟𝑎𝑡𝑒
Notes
➢ ES is a transition state and cannot be
isolated. Hence, it cannot be measured or
quantified.
➢ We convert ES to actually measurable
quantities: 𝑘2 [𝐸𝑆(𝑆, 𝐸𝑜 )] or the initial
substrate concentration and initial
enzyme concentration
THE RATE OF PRODUCTION FORMATION:
𝒅[𝑷]
∴ −𝒓𝒔 = 𝒓𝒑 = 𝒗 = ( ) = 𝒌𝟐 [𝑬𝑺]
𝒅𝒕
𝒘𝒉𝒆𝒓𝒆 𝒌𝟐 [𝑬𝑺] → 𝒌𝟐 [𝑬𝑺(𝑺, 𝑬𝒐 )]
➢ The goal is to derive ES and let it have
measurable properties.
THE TIME VARIATION OF [ES]:
𝒅[𝑬𝑺]
= 𝒌𝟏 [𝑬][𝑺] − 𝒌−𝟏 [𝑬𝑺] − 𝒌𝟐 [𝑬𝑺]
𝒅𝒕
➢ The time variation allows us to relate ES to
S; E is the amount of enzyme that will not
be bound to any substrate (also
immeasurable).
 ✓ However, [E] cannot be quantified.
Instead, we want [E] in terms of the initial
ENZYME CONSERVATION EQUATION
amount of enzyme. Hence, using Enzyme
[𝑬]𝟎 = [𝑬] + [𝑬𝑺] Conservation Equation:

[𝐸]0 = [𝐸] + [𝐸𝑆] → (𝒆𝒒. 𝟐)

➢ It means that whatever amount of enzyme
we add to the reaction will be split into
✓ Rearranging (eq.1) we know that:
two. 𝐾𝑚 ′ [
𝐸𝑆]
➢ Parts of the enzyme added will be bound to [𝐸 ] = → (𝒆𝒒. 𝟑)
[𝑆]
the substrate while the other will remain
✓ Plugging (eq.3) to (eq.2)
unbonded.
𝐾 ′ [𝐸𝑆]
➢ In the equations so far, the only known [𝐸]0 = 𝑚 + [𝐸𝑆] → (𝑬𝒒. 𝟒)
[𝑆 ]
parameters are [𝑺] and [𝑬]𝟎; we want to
✓ Factoring out [ES]
replace this with ES in the final equation.
𝐾′
➢ Two ways to derive the equation from the [𝐸]0 = [𝐸𝑆] ( 𝑚 + 1) → (𝒆𝒒. 𝟓)
[𝑆]
starting equations:
1. Rapid Equilibrium Assumption ✓ Expressing [ES] in terms of [𝐸]0
2. Pseudo-Steady State Hypothesis [𝐸 ] 0
[𝐸𝑆] = ′ → (𝒆𝒒. 𝟔)
(PSSH) 𝐾𝑚
( + 1)
[𝑆 ]
✓ Simplifying by multiplying both the
1. RAPID EQUILIBRIUM ASSUMPTION
numerator and the denomination with [S]
✓ Michaelis and Menten, along with
[𝐸]0 [𝑆 ]
V.C.R. Henri, used the assumption that [𝐸𝑆] = ( )
𝐾′ [ ]
equilibrium between the enzyme ( 𝑚 + 1) 𝑆
[𝑆]
and substrate is rapid, forming the
[𝐸]0 [𝑆]
ES complex. An equilibrium constant ∴ [𝐸𝑆] = ′ → (𝒆𝒒. 𝟕)
𝐾𝑚 + [𝑆]
can therefore be used to express [ES] in
✓ Since we now have a measurable quantity
terms of [S].
for [ES], we can use the rate of product
✓ Because this is rapid, the 𝑘2 can be
formation equation:
neglected.
𝑑[𝑃]
✓ The equilibrium constant, written in 𝑟𝑠 = 𝑟𝑝 = 𝑣 = ( ) = 𝑘2 [𝐸𝑆]
the form of a dissociation constant 𝑑𝑡
(reciprocal of 𝑲𝑪 ), is 𝑑[𝑃] 𝑑 [𝑆]
𝑣=( )=− = 𝑘2 [𝐸𝑆]
𝑑𝑡 𝑑𝑡
′ (=
1 𝑘−1 [𝐸][𝑆] ✓ Simplifying:
𝐾𝑚 )=( )=
𝐾𝐶 𝑘1 [𝐸𝑆]
RATE LAW FOR RAPID EQUILIBRIUM
Note:
For a reversible reaction 𝐸 + 𝑆 ⇌ 𝐸𝑆, [𝑬]𝟎 [𝑺]
𝒗 = (𝒌𝟐 )
𝑲′𝒎 + [𝑺]
[𝐸𝑆] 𝑘1
𝐾𝑐 = =
[𝐸][𝑆] 𝑘−1
Derivation:
✓ Write 𝐾𝑚
′
as the ratio of the rate constants,
′
𝑘−1 [𝐸][𝑆]
𝐾𝑚 = = → (𝒆𝒒. 𝟏)
𝑘1 [𝐸𝑆]
 2. PSEUDO-STEADY STATE HYPOTHESIS ✓ The PSSH assumes (eq. 1) to be 0.
(PSSH) or the QUASI-STEADY STATE
𝑑 [𝐸𝑆]
ASSUMPTION = 𝑘1 [𝐸][𝑆] − 𝑘−1 [𝐸𝑆] − 𝑘2 [𝐸𝑆] = 0
𝑑𝑡
→ (𝒆𝒒. 𝟐)
✓ In many cases, the rapid equilibrium
assumption is not valid, although the ✓ Factor-out the [ES]
enzyme-substrate reaction will still show
𝑘1 [𝐸][𝑆] = (𝑘−1 + 𝑘2 )[𝐸𝑆] → (𝒆𝒒. 𝟑)
saturation-type kinetics.
✓ Hence, this assumption is the most ✓ Isolate [ES]
acceptable one. 𝑘1
[𝐸𝑆] = [𝐸][𝑆] → (𝒆𝒒. 𝟒)
✓ The PSSH was first proposed by G.E. Briggs 𝑘−1 + 𝑘2
and J.B.S. Haldane. In a closed system, ✓ Recall the enzyme conservation equation:
after a brief transient, and if [𝑺]𝟎 ≫ [𝐸]0 = [𝐸] + [𝐸𝑆] → (𝒆𝒒. 𝟓)
[𝑬]𝟎 ,
𝒅[𝑬𝑺]
≈ 𝟎. (there will be an instance ✓ Isolate [E]
𝒅𝒕
[𝐸] = [𝐸]0 − [𝐸𝑆] → (𝒆𝒒. 𝟔)
wherein the [ES] complex will be constant)
✓ Substitute (eq. 6) to (eq.4)
✓ This is as long as the substrate remains at
𝑘1
a high concentration relative to th4 [𝐸𝑆] = ([𝐸]0 − [𝐸𝑆])[𝑆]
enzyme concentration. 𝑘−1 + 𝑘2
→ (𝒆𝒒. 𝟕)
✓ Factor the introduced [ES]
𝑘 [𝐸] [𝑆] 𝑘1 [𝐸𝑆][𝑆]
[𝐸𝑆] = 1 0 − → (𝒆𝒒. 𝟖)
𝑘−1 + 𝑘2 𝑘−1 + 𝑘2
✓ Transfer the second term to the left
𝑘 [𝐸𝑆][𝑆] 𝑘1 [𝐸]0 [𝑆]
[𝐸𝑆] + 1 = → (𝒆𝒒. 𝟖)
𝑘−1 + 𝑘2 𝑘−1 + 𝑘2
𝑘1 [𝑆] 𝑘1 [𝐸]0 [𝑆]
∴ [𝐸𝑆] (1 + )=
𝑘−1 + 𝑘2 𝑘−1 + 𝑘2
→ (𝒆𝒒. 𝟗)
✓ Introduce a new 𝐾𝑚 (without the prime)
𝑘−1 + 𝑘2
𝐾𝑚 = → (𝒆𝒒. 𝟏𝟎)
𝑘1
✓ Using (eq. 10) to (eq. 9) for simplification
[𝑆] [𝐸]0 [𝑆]
[𝐸𝑆] (1 + )= → (𝒆𝒒. 𝟏𝟏)
𝐾𝑚 𝐾𝑚
✓ PSSH is at steady state since it is a batch
reaction: ✓ Simplify by multiplying entire equation
𝑑[𝐸𝑆] with 𝐾𝑚
𝑃𝑆𝑆𝐻: =0
𝑑𝑡
✓ Also known as the Briggs-Haldane [𝑆] [𝐸]0 [𝑆]
{[𝐸𝑆] (1 + )= } (𝐾𝑚 ) → (𝒆𝒒. 𝟏𝟐)
assumption. 𝐾𝑚 𝐾𝑚

Derivation: ∴ [𝐸𝑆](𝐾𝑚 + [𝑆]) = [𝐸]0 [𝑆] → (𝒆𝒒. 𝟏𝟑)

✓ Using the time variation of [ES] equation: ✓ Rearranging to isolate [ES]

𝑑[𝐸𝑆] [𝐸]0 [𝑆]
= 𝑘1 [𝐸][𝑆] − 𝑘−1 [𝐸𝑆] − 𝑘2 [𝐸𝑆] [𝐸𝑆] = → (𝒆𝒒. 𝟏𝟒)
𝑑𝑡 (𝐾𝑚 + [𝑆])
→ (𝒆𝒒. 𝟏)
 ✓ Accounting the rate of production APPLYING THE FINAL MICHAELIS-MENTEN
formation equation: EQUATION
𝑑 [𝑃 ]
𝑣=( ) = 𝑘2 [𝐸𝑆] → (𝒆𝒒. 𝟏𝟓) ✓ For a given total enzyme concentration, a
𝑑𝑡
sketch of the rate of disappearance of the
✓ Substituting (eq.14) to (eq. 15) substrate (=rate of formation of product)
is shown:
RATE LAW FOR PSSH ✓ This is called a Michaelis-Menten plot:

[𝑬]𝟎 [𝑺]
𝒗 = (𝒌𝟐 )
(𝑲𝒎 + [𝑺])

✓ We will use PSSH because it is more

general; the same result as the rapid
equilibrium except the dissociation
constant. Hence, PSSH accounts the third
reaction or k2.
✓ 𝑘2 is also called 𝑘𝑐𝑎𝑡
Significant Parameters
✓ The parameter 𝒌𝒄𝒂𝒕 (unit 𝟏/𝒔) is also ✓ The blue line is the plot for Michaelis-
called the turnover number and Menten equation for the given data points
represents the number of substrate ✓ We are satisfying the saturation constraint
molecules converted to product in a given because there is an upper limit.
time, on a single-enzyme molecule when
the enzyme is saturated with substrate. ✓ We have two extremes and 1 condition:

✓ Ideal for enzymes: 𝒉𝒊𝒈𝒉 𝒌𝒄𝒂𝒕 , 𝒍𝒐𝒘 𝑲𝑴

1. At low substrate concentrations ([𝑺] ≪
✓ The constant 𝑲𝑴 is called the Michaelis 𝑲𝑴)
constant and, for simple systems, is a
measure of the attraction of the enzyme 𝑖𝑓 [𝑆] ≪ 𝐾𝑀 , [𝑆] + 𝐾𝑀 ≈ 𝐾𝑀
for its substrate. It is also called the 𝒗𝒎𝒂𝒙[𝑺] 𝑽𝒎𝒂𝒙
affinity constant. 𝒗≈ ≈( ) [𝑺]
𝑲𝑴 𝑲𝑴
✓ Since [𝐸]0 will be constant for a given
reaction, we can group together 𝑘𝑐𝑎𝑡 and Note: This is approximately 1𝑠𝑡 order,
[𝐸]0 into one parameter, which we call the when substrate concentration is very low.
maximum velocity 𝒗𝒎𝒂𝒙:

MAXIMUM VELOCITY FORMULA

𝒗𝒎𝒂𝒙 = 𝒌𝒄𝒂𝒕 [𝑬]𝟎

MICHAELIS-MENTEN EQUATION
𝒗𝒎𝒂𝒙 [𝑺]
𝒗=
𝑲𝑴 + [𝑺]
 2. At high substrate concentrations ([𝑺] ≫ One way to compare the catalytic efficiencies of
𝑲𝑴) 𝒌𝒄𝒂𝒕
two enzymes is to compare their ratios .
𝑲𝑴

𝑖𝑓 [𝑆] ≫ 𝐾𝑀 , [𝑆] + 𝐾𝑀 ≈ [𝑆] ✓ We can get 𝑘𝑐𝑎𝑡 once we know 𝑣𝑚𝑎𝑥

𝒗𝒎𝒂𝒙[𝑺] 𝑣𝑚𝑎𝑥 = 𝑘𝑐𝑎𝑡 [𝐸]0
𝒗≈ ≈ 𝒗𝒎𝒂𝒙 𝑣𝑚𝑎𝑥
[𝑺] 𝑘𝑐𝑎𝑡 =
[𝐸]0
Protein Engineering
Note: This is approximately 0𝑡ℎ order; at high
concnetration, it becomes constant until it is
asymptotic to vmax.

3. When [𝑺] = 𝑲𝑴,

✓ This implies that 𝐾𝑀 also has units
of concentration.

𝑤ℎ𝑒𝑛 [𝑆] = 𝐾𝑚
𝒗𝒎𝒂𝒙[𝑺] 𝒗𝒎𝒂𝒙 [𝑺] 𝒗𝒎𝒂𝒙[𝑺]
𝒗= = =
𝑲𝑴 + [𝑺] [𝑺] + [𝑺] 𝟐[𝑺]
𝒗𝒎𝒂𝒙 ✓ We start with a natural form of enzyme
∴𝒗= with a goal of designing an ezyme that
𝟐
starts from the same native enzyme but is
more efficient.
✓ This means that when the susbtrate ✓ For one to imprve enzymes, we must do
concentration is equal to 𝐾𝑀 , the interventions immediately in DNA; they
reaction will be running at half the sometimes use mutageneis.
maximum velocity. ✓ The overall goal of protein engineerring is
✓ the Michaelis constant is equal to catalytic efficiency together with
the substrate concentration at catalytic stability.
which the rate of reaction is equal
to one-half the maximum rate.
In summary
For reactions described by Michaelis-
Mentenkinetics, two parameters are needed:
✓ 𝒗𝒎𝒂𝒙 (𝒌𝒄𝒂𝒕 [𝑬]𝟎 ) which is a function of the
total enzyme concentration, and
✓ 𝑲𝑴, which is not a function of total enzyme
concentration; it is only a function of rate
parameters.
Two enzymes may have the same values for
𝑘𝑐𝑎𝑡 but different affinities for the substrate
(represented by 𝐾𝑀 ).
Linearizing Michaelis-Menten Equations Exercise 1.4.1
✓ Given rate versus substrate concentration Determine the Michaelis-Menten parameters
data, the parameters 𝑣𝑚𝑎𝑥 and 𝐾𝑀 may be 𝑣𝑚𝑎𝑥 and 𝐾𝑀 for the reaction:
obtained by curve-fitting or linear
regression.
✓ There are several ways of linearizing the
Michaelis-Mentenequation.

The rate of reaction is given as a function of urea

concentration (𝐶𝑆 ):

✓ Lineweaver-Burk is also called the “double Solution (Exercise 1.4.1):

reciprocal” method and the most common: ✓ Using the Lineweaver-Burk Equation
✓ Initial Assumptions:
➢ We invert Michaelis-Menten on both sides:
𝑣𝑚𝑎𝑥 [𝑆]
𝑓𝑟𝑜𝑚 𝑣 =
𝐾𝑀 + [𝑆]

1 𝐾𝑀 + [𝑆]
𝑡𝑜 𝑡ℎ𝑖𝑠 =
𝑣 𝑣𝑚𝑎𝑥 [𝑆]

𝑡ℎ𝑒 𝑛𝑢𝑚𝑒𝑟𝑎𝑡𝑜𝑟 𝑐𝑎𝑛 𝑏𝑒 𝑓𝑎𝑐𝑡𝑜𝑟𝑒𝑑 𝑡𝑜 2:

1 𝐾𝑀 + [𝑆]
=
𝑣 𝑣𝑚𝑎𝑥 [𝑆]
1 𝐾𝑀 1 1
= ∙ +
𝑣 𝑣𝑚𝑎𝑥 [𝑆] 𝑣𝑚𝑎𝑥

✓ The Michaelis-Menten Equation:

𝑣𝑚𝑎𝑥 [𝑆]
𝑣= → (𝒆𝒒. 𝟏)
𝐾𝑀 + [𝑆]

✓ Lineweaver-Burk equation:
1 𝐾𝑀 + [𝑆]
= → (𝒆𝒒. 𝟐)
𝑣 𝑣𝑚𝑎𝑥 [𝑆]

✓ Separating the numerators:

1 𝐾𝑀 1 1
= ∙ +
𝑣 𝑣𝑚𝑎𝑥 [𝑆] 𝑣𝑚𝑎𝑥
1 1
✓ Plot 𝑉 as y, [𝑆] as x
 1 [𝑆] ([𝑆]0 −[𝑆])
✓ Therefore, a plot of 𝑡 ln ( [𝑆]0 ) 𝑣𝑠. 𝑡

1 𝑣𝑚𝑎𝑥
Results in a line of slope − 𝐾 and intercept .
𝑀 𝐾𝑀

𝑲𝑴 𝒂𝒏𝒅 𝒗𝒎𝒂𝒙
✓ While 𝐾𝑀 is an intrinsic parameter,
𝑣𝑚𝑎𝑥 is not.
o 𝐾𝑀 is a function of rate parameters
and is expected to change with
temperature or pH.
o 𝑣𝑚𝑎𝑥 is a function of both the rate
parameter 𝑘𝑐𝑎𝑡 (𝑘2 ) and the initial
enzyme concentration.

✓ For highly purified enzyme preparations,

it may be possible to express [𝐸]0 in terms
of M or g/L.

✓ From the Excel plot, ✓ When the enzyme is part of a crude

preparation, its concentration is in terms
𝑦 = 0.02073𝑥 + 0.75308
of units. One enzyme unit (U or IU) is
Where 0.02073 is the 𝑣
𝐾𝑀
; 0.75308 is the 𝑣
1 defined as the amount of enzyme that
𝑚𝑎𝑥 𝑚𝑎𝑥 catalyzes the conversion of one micromole
✓ Solving for 𝑣𝑚𝑎𝑥 of substrate to product per minute, under
1 𝒌𝒎𝒐𝒍 specific reaction (usually optimal)
𝑣𝑚𝑎𝑥 = = 𝟏. 𝟑𝟑 𝟑 conditions.
0.75308 𝒎 ∙𝒔
✓ Solving for 𝐾𝑀 ✓ The SI unit is the katal, which is defined as
𝐾𝑀 = (𝑚)(𝑣𝑚𝑎𝑥 ) the amount of enzyme that catalyzes the
𝒌𝒎𝒐𝒍 conversion of one mole of substrate to
𝐾𝑀 = (0.02073)(1.33) = 𝟎. 𝟎𝟐𝟕𝟓
𝒎𝟑 product per second.

Use of Batch Reactor

✓ A batch reactor can also be used to study
time course variation of [S].

✓ Integration of
𝑑 [𝑆] 𝑣𝑚𝑎𝑥 [𝑆]
𝑣=− =
𝑑𝑡 𝐾𝑀 + [𝑆]
✓ Yields

𝐾𝑀
[𝑆]0 − [𝑆] ln[𝑆]0
𝑣𝑚𝑎𝑥 − = 𝑡
𝑡 [𝑆]
1.1. ALLOSTERY
Kinetics of Allosteric Binding
✓ Some enzymes have multiple binding sites,
and the binding of one substrate can
facilitate the binding of the subsequent
substrates. This is known as cooperative
binding or allostery.
o The affinity of the second substrate
increases because of the binding of
the first substrate; this is true for all
subsequent binding substrates.
✓ The activity of allosteric enzymes can be
altered by regulatory molecules binding
on allosteric sites; their properties can
thus be adjusted to meet the immediate
needs of a cell.
o Binding of substrates in allosteric
sites helps enzyme regulation; ✓ When one oxygen molecule binds
there is cooperative binding, thus, (refer to the hemoglobin), the binding
affinity will increase. of that first oxygen molecule causes a
✓ Michaelis-Menten kinetics fails to describe structural change in the hemoglobin. It
the kinetics of cooperative binding; enhances the binding of other oxygen
allosteric enzymes often display molecules.
sigmoidal plots. ✓ The rate expression in this case of
allosteric enzymes becomes a three-
parameter rate law:
✓ The dark yellow color: one pair or 𝛼
and 𝛽 monomers.

RATE EXPRRESION (ALLOSTERIC

ENZYMES)
𝒅[𝑺] 𝒗𝒎𝒂𝒙 [𝑺]𝒏
𝒗=− = ′′
𝒅𝒕 𝑲𝒎 + [𝑺]𝒏

where n is the Hill coefficient, and

𝑛 > 1 indicates cooperativity.

For n=1, the expression reduces to the

Michaelis-Menten expression.
 ✓ The Hill coefficient can be determined Enzyme Inhibition: Competitive
by linear regression:
✓ In competitive inhibition, the enzyme
𝑣 binds either substrate or inhibitor.
′′
ln ( ) = 𝑛 ln[𝑆] − 𝑙𝑛𝐾𝑚 Competitive inhibitors are usually
𝑣𝑚𝑎𝑥 − 𝑣
substrate analogs and compete with
𝑣 substrate for the active site of the enzyme.
✓ A plot of ln 𝑣 versus ln[𝑆] gives a
𝑚𝑎𝑥 −𝑣
straight line whose slope is n.

1.2. ENZYME INHIBITION

✓ Enzyme inhibition is the opposite of

allostery because you are decreasing
the catalytic activity of the enzyme.
✓ It is a way of regulating enzyme activity
as well.
✓ The effect of inhibitors is seen as a
✓ Enzyme activity can be inhibited by the
decrease in 𝐾𝑀 .
binding of specific small molecules and
✓ The inhibition scheme can be described as:
ions.
✓ The ions responsible are called
inhibitors.
✓ Like allostery, inhibition also serves as
a major control mechanism in
biological systems. In addition, many
drugs and toxic agents act by inhibiting
enzymes.
✓ Enzyme inhibition can be irreversible
Enzyme Inhibition (Competitive) Derivation:
or reversible:
✓ Our goal is to replace [ES] with a
1. Irreversible inhibitors dissociate measurable quantity
very slowly from its target enzyme; 𝑣 = 𝑘2 [𝐸𝑆] → (𝒆𝒒. 𝟏)
they are either covalently or non- ✓ Note that the only measurable quantities
covalently bound to it. Examples are are:
heavy metals (Pb, Cd, Hg, etc.) (toxic). [𝑆], [𝐸]0 , [𝐼 ]
✓ Begin with the balance on the transition
2. Reversible inhibitors rapidly states
dissociate from the enzyme-inhibitor 𝑑 [𝐸𝑆]
= 𝑘1 [𝐸][𝑆] − (𝑘−1 + 𝑘2 )[𝐸𝑆]
complex, and can be competitive, 𝑑𝑡
uncompetitive, or noncompetitive. → (𝒆𝒒. 𝟐)
In some cases, the substrate can also be ✓ Using the PSSH approach, we can see to it
inhibitory if accumulated at high that,
concentrations. 𝑑 [𝐸𝑆]
= 𝑘1 [𝐸][𝑆] − (𝑘−1 + 𝑘2 )[𝐸𝑆] = 0
𝑑𝑡
→ (𝒆𝒒. 𝟑)
✓ Rewriting in terms of [E]
𝑘 + 𝑘2 [𝐸𝑆]
[𝐸] = −1 ∙ → (𝒆𝒒. 𝟒)
𝑘1 [𝑆]
 ✓ Isolating [ES] to have [ES] as a function of
all measurable properties
✓ Recall that [𝐸]0
[𝐸𝑆] = → (𝒆𝒒. 𝟏𝟏)
𝑘−1 + 𝑘2 𝐾𝑀 𝐾𝑀 [𝐼 ]
(1 + )
𝑘1
= 𝐾𝑀 [𝑆] + 𝑘𝐼 [𝑆]
∴ 𝑢𝑠𝑖𝑛𝑔 (𝒆𝒒. 𝟒), ✓ Now that [ES] is now a function of
[𝐸𝑆] measurable properties, we circle back to
𝐾𝑀 ∙ →→ (𝒆𝒒. 𝟓) (𝒆𝒒. 𝟏). then substitute (eq. 11)
[𝑆]
𝑣 = 𝑘2 [𝐸𝑆] → (𝒆𝒒. 𝟏)
✓ Performing balance on the inhibitor’s side.
At steady state, which occurs in the middle 𝑘2 [𝐸]0
𝑣= → (𝒆𝒒. 𝟏𝟐)
of the reaction, 𝐾𝑀 𝐾𝑀 [𝐼 ]
(1 + )
𝑑 [𝐸𝐼 ] [𝑆] + 𝑘𝐼 [𝑆]
= 𝑘3 [𝐸][𝐼 ] − 𝑘−3 [𝐸𝐼 ] = 0 ✓ Multiply the numerator and denominator
𝑑𝑡
Note that: with [S]
𝑘2 [𝐸]0 [𝑆]
𝑣= ∙
𝐾 𝐾 [𝐼 ] [𝑆]
(1 + 𝑀 + 𝑀 )
[𝑆] 𝑘𝐼 [𝑆]
𝑘2 [𝐸]0 [𝑆]
∴ → (𝒆𝒒. 𝟏𝟑)
𝐾𝑀 [𝐼 ]
([𝑆] + 𝐾𝑀 + )
𝑘𝐼
✓ Rearranging the denominator in the form
of 𝐾𝑀
✓ Isolating [EI] 𝑘2 [𝐸]0 [𝑆]
𝑘 [𝐸][𝐼 ] → (𝒆𝒒. 𝟏𝟒)
[𝐸𝐼 ] = 3 [𝐸][𝐼 ] = → (𝒆𝒒. 𝟔) [𝐼 ]
𝑘−3 𝑘𝐼 𝐾𝑀 (1 + 𝐾 ) + [𝑆]
𝐼
✓ Recall that,
✓ Substitute (eq.6) to (eq.5) 𝑣𝑚𝑎𝑥 = 𝑘2 [𝐸]0
𝐾 [𝐸𝑆][𝐼 ] And
[𝐸] = 𝑀 → (𝒆𝒒. 𝟕)
𝑘𝐼 [𝑆] [𝐼 ]
𝐾𝑀,𝑎𝑝𝑝 = 𝐾𝑀 (1 + )
𝐾𝐼
✓ Using the enzyme conservation equation ✓ Therefore, using the recalled equations
and the third form of [EI]: and (eq. 14),
[𝐸]0 = [𝐸] + [𝐸𝑆] + [𝐸𝐼] → (𝒆𝒒. 𝟖) 𝒗𝒎𝒂𝒙[𝑺]
𝒗= → (𝒆𝒒. 𝟏𝟓)
𝑲𝑴,𝒂𝒑𝒑 + [𝑺]
✓ Write everything in terms of [ES] from
(eq.7)
THE RATE LAW FOR COMPETETIVE
𝐾 𝐾 [𝐼]
[𝐸]0 = 𝑀 [𝐸𝑆] + [𝐸𝑆] + 𝑀 [𝐸𝑆] INHIBITION IS:
[𝑆 ] 𝑘𝐼 [𝑆]
→ (𝒆𝒒. 𝟗)
𝒗𝒎𝒂𝒙[𝑺] 𝒗𝒎𝒂𝒙 [𝑺]
✓ Factor out [ES] 𝒗= =
[𝑰] 𝑲𝑴,𝒂𝒑𝒑 + [𝑺]
𝐾 𝐾 [𝐼 ] 𝑲𝑴 (𝟏 + 𝑲 ) + [𝑺]
[𝐸]0 = [𝐸𝑆] (1 + 𝑀 + 𝑀 ) → (𝒆𝒒. 𝟏𝟎) 𝑰
[𝑆] 𝑘𝐼 [𝑆] where

[𝑰]
𝑲𝑴,𝒂𝒑𝒑 = 𝑲𝑴 (𝟏 + )
𝑲𝑰
 ✓ The net effect of competitive inhibition is
an apparently higher value of𝐾𝑚 . Since
𝑣𝑚𝑎𝑥 is unaffected, high concentrations of
substrate can overcome the inhibition to
reach 𝑣𝑚𝑎𝑥.

✓ A competitive inhibitor reduces the

rate of catalysis by reducing the
proportion of enzyme molecules bound
to a substrate.
o If there is an inhibitor in the
enzyme, you can kick that out; only Derivation
when substrate concentration is
high.
✓ Only competitive inhibition can overcome
high substrate concentration.
Enzyme Inhibition: Noncompetitive
✓ In noncompetitive inhibition, the
inhibitors are not substrate analogs.
Inhibitors bind sites other than the active
site and reduce enzyme affinity to the
substrate. Noncompetitive inhibitors can
bind free enzyme or the ES complex.
✓ Assumes the dissociation constant for ESI
and EI are the same (not functional version
of the proteins because ES is the only
functional one)

✓ The inhibition scheme can be described as:

 THE RATE LAW FOR
NONCOMPETETIVE INHIBITION IS:

𝒗𝒎𝒂𝒙[𝑺] 𝒗𝒎𝒂𝒙,𝒂𝒑𝒑 [𝑺]

𝒗= =
[𝑰] 𝑲𝑴 + [𝑺]
(𝟏 + ) (𝑲𝒎 + [𝑺])
𝑲𝑰

where

𝒗𝒎𝒂𝒙
𝒗𝒎𝒂𝒙,𝒂𝒑𝒑 =
[𝑰]
𝟏+𝑲
𝑰
✓ The inhibition scheme can be described as:

✓ This means that 𝑣𝑚𝑎𝑥 decreases at higher

concentrations.
✓ The maximum possible rate decreases if
𝑣𝑚𝑎𝑥 is affected.
✓ The net effect of noncompetitive inhibition
is an apparently lower value of 𝑣𝑚𝑎𝑥.
Therefore, substrate concentrations
cannot overcome noncompetitive
inhibition; the initial 𝑣𝑚𝑎𝑥cannot be
restored.

✓ A noncompetitive inhibitor lowers the

concentration of functional enzyme.
The resulting solution behaves as a
more dilute solution of the enzyme
does (same amount of enzyme but k2
decreases).

Enzyme Inhibition: Uncompetitive

✓ In uncompetitive inhibition, the inhibitors
bind to the ES complex only; this implies
that substrate must bind first before
uncompetitive inhibitors can take effect.
 Comparing and Contrast Enzyme Inhibitions
✓ Measurements of the rates of catalysis at
different concentrations of substrate and
inhibitor can serve to distinguish the three
types of reversible inhibition.

✓ For Competitive Inhibition

THE RATE LAW FOR UNCOMPETETIVE

INHIBITION IS:

Where
✓ For Noncompetitive Inhibition

✓ The net effect of uncompetitive inhibition

is a decrease in both 𝐾𝑚 and 𝑣𝑚𝑎𝑥. The
rate is more sensitive to changes in 𝑣𝑚𝑎𝑥,
so the net result is a decrease in reaction
rate. As in uncompetitive inhibition, high
substrate concentrations cannot
overcome uncompetitive inhibition.

✓ Because some unproductive ESI

complex will always be present,
[𝑬]𝟎 will be lower and so will 𝒗𝒎𝒂𝒙. Also,
because ES is consumed to form ESI, the
equilibrium shifts to more binding of S,
lowering the apparent value of 𝑲𝒎.
 ✓ For Uncompetitive Inhibition
✓ The following scheme may be used to
describe the pH dependence of enzymatic
reaction rates for ionizing enzymes:

THE RATE LAW (ACCOUNTING

EFFECTS OF Ph)
✓ SUMMARY:
➢ 𝑣𝑚𝑎𝑥 𝑐ℎ𝑎𝑛𝑔𝑒𝑠: noncompetitive
➢ 𝐾𝑚 𝑐ℎ𝑎𝑛𝑔𝑒𝑠: competitive
➢ 𝑣𝑚𝑎𝑥 𝑎𝑛𝑑 𝐾𝑚 𝑐ℎ𝑎𝑛𝑔𝑒: uncompetitive

1.3. EFFECTS OF pH AND TEMPERATURE

Effects of pH
✓ Recall that some amino acid residues have
side chains that are ionizable; certain
enzymes have these groups on their active ✓ As a result of this behavior, the pH
sites, and their extents of protonation optimum of the enzyme is between pK1
depend on the prevailing pH of the solution. and pK2.
✓ Changes in solution pH result in changes in ✓ Theoretical prediction of the pH optimum
enzyme activity due to different of enzymes requires a knowledge of the
ionizations, which may also result in active site characteristics of enzymes,
changes in the three-dimensional shape of which are very difficult to obtain. It is
the enzyme. In some cases, the pH of the usually determined experimentally.
medium can also affect the ionization state
of the substrate, and hence its affinity to
the enzyme.
✓ For these reasons, enzymes are only
active over a certain pH range.
 substitution of the previous expression
gives:
Effects of Temperature
✓ The rate of enzyme-catalyzed reactions
increases with temperature up to a
certain limit only, above which, activity Where
𝑬𝒂
decreases with temperature due to 𝒌𝟐 = 𝑨𝒆−𝑹𝑻
denaturation (there will be no 3D [𝑬] = [𝑬]𝟎 𝒆−𝒌𝒅 𝒕
structure anymore). 𝑬𝒂
𝒌𝒅 𝜶 𝑨𝒅 𝒆−𝑹𝑻

✓ The activation energies of enzyme-

catalyzed reactions are within the 4-20
kcal/mol range, whereas deactivation
energies vary between 40-130 kcal/mol
range.
✓ Higher activation energy means it is more
sensitive to temperature changes and vice-
versa; denaturation is more dominant at
high temperatures.
✓ There is a point wherein two rates are
✓ The initial increase in the activity with equal called the optimal point.
temperature is called temperature ✓ As a result, enzyme denaturation is more
activation, and this follows the Arrhenius sensitive to temperature changes; a rise in
equation: temperature from 30o to 40oC results in a
1.8-fold increase in enzyme activity, but a
𝑬𝒂 41-fold increase in enzyme denaturation.
𝒌𝟐 = 𝑨𝒆−𝑹𝑻
✓ The descending part is called the
temperature deactivation or thermal
denaturation stage. Denaturation
kinetics follows a first-order rate law:

✓ The rate of enzyme-catalyzed reactions

increases with temperature up to a
certain limit only, above which, activity
decreases with temperature due to
denaturation.
✓ The denaturation constant also follows the
Arrhenius equation:

✓ Since 𝑣𝑚𝑎𝑥 = 𝑘2 [𝐸] (not [𝐸]0 if it is

changing) (exponentially decreasing),
 ✓ Isolate [ES],
[𝐸]𝑂
[𝐸𝑆] = → (𝒆𝒒. 𝟕)
𝐾𝑀′ 𝐾𝑀′ [𝐼 ]
[𝑆] + 1 + [𝑆] ∙ 𝐾𝐼
1. ENZYME INHIBITION: COMPETETIVE ✓ Multiply the numerator and denominator
DERIVATION (RAPID EQUILIBRIUIM with [S],
APPROACH) [𝐸]𝑂 [𝑆]
[𝐸𝑆] = ′ ′ [ ]∙
𝐾𝑀 𝐾𝑀 𝐼 [𝑆]
[𝑆] + 1 + [𝑆] ∙ 𝐾𝐼
[𝐸]𝑂 [𝑆]
∴ [𝐸𝑆] = → (𝒆𝒒. 𝟖)
′ ′ [𝐼 ]
𝐾𝑀 + [𝑆] + 𝐾𝑀 ∙ 𝐾
𝐼
✓ Factor 𝐾𝑀′
[𝐸]𝑂 [𝑆]
[𝐸𝑆] = → (𝒆𝒒. 𝟗)
′ [𝐼 ]
𝐾𝑀 + (1 + 𝐾 ) + [𝑆]
𝐼
✓ We can now express everything in terms of
✓ In the Rapid Equilibrium Approach, we
𝑣
assume that 𝑘2 is extremely small. 𝑑 [𝑃] 𝑑[𝑆]
Defining the constants, 𝑣= =− = 𝑘2 [𝐸𝑆] → (𝒆𝒒. 𝟏𝟎)
𝑑𝑡 𝑑𝑡
[𝐸][𝑆]
𝐾𝑀′ = → (𝒆𝒒. 𝟏) ✓ Substituting (eq.9) to (eq.10)
[𝐸𝑆] [𝐸]𝑂 [𝑆]
[𝐸][𝐼 ] 𝑣=
𝐾𝐼 = → (𝒆𝒒. 𝟐) [𝐼 ]
[𝐸𝐼 ] 𝐾𝑀′ + (1 + 𝐾 ) + [𝑆]
𝐼
✓ We then invoke the enzyme conservation, ✓ Note that,
and remember that for competitive [𝐼 ]
inhibition, enzyme exists in three forms: 𝐾𝑀′ ,𝑎𝑝𝑝 = 𝐾𝑀′ + (1 + ) → (𝒆𝒒. 𝟏𝟏)
𝐾𝐼
[𝐸]𝑂 = [𝐸] + [𝐸𝑆] + [𝐸𝐼 ] → (𝒆𝒒. 𝟑) ✓ Thus, the final equation is:

✓ The objective is to express [ES] as a 𝒗𝒎𝒂𝒙 [𝑺]

function of measurable properties: 𝒗=
(𝑲𝒎,𝒂𝒑𝒑 + [𝑺])
[𝐸]𝑂 , [𝑆], [𝐼]
✓ Express (eq.3) as a function of [ES] from
(eq.1) and (eq.2)

′ [
𝐾𝑚 𝐸𝑆] [𝐸][𝐼 ]
[𝐸]𝑂 = + [𝐸𝑆] + → (𝒆𝒒. 𝟒)
[𝑆 ] 𝐾𝐼
✓ Note that,
𝐾 ′ [𝐸𝑆]
[𝐸] = 𝑚
[𝑆 ]
′ [
𝐾𝑚 𝐸𝑆] [𝐼 ] 𝐾𝑀′ [𝐸𝑆]
[ ]
∴ 𝐸𝑂= [
+ 𝐸𝑆 +] ∙( )
[𝑆] 𝐾𝐼 [𝑆]
→ (𝒆𝒒. 𝟓)
✓ Factor [ES] out,
𝐾𝑀′ 𝐾𝑀′ [𝐼 ]
[𝐸]𝑂 = [𝐸𝑆]( +1+ ∙ ) → (𝒆𝒒. 𝟔)
[𝑆] [𝑆] [𝐾𝐼 ]
2. ENZYME INHIBITION: NON [𝐸]𝑂 [𝑆]
[𝐸𝑆] = ∙
COMPETETIVE DERIVATION (RAPID 𝐾𝑀′ ′ [ ]
𝐾𝑀 𝐼 [𝐼 ] [𝑆]
+ 1 + +
EQUILIBRIUIM APPROACH) [𝑆 ] 𝐾𝐼 [𝑆] 𝐾𝐼
→ (𝒆𝒒. 𝟕)
[𝐸]𝑂 [𝑆]
∴ [𝐸𝑆] =
𝐾 ′ [𝐼 ] [𝐼 ]
𝐾𝑀′ + [𝑆] + 𝑀 + [𝑆 ]
𝐾𝐼 𝐾𝐼
(
→ 𝒆𝒒. 𝟖 )
✓ Factor 𝐾𝑀′

[𝐸]𝑂 [𝑆]
[𝐸𝑆] =
[𝐼 ] [𝐼 ]
✓ Defining the constants, 𝐾𝑀′ (1 + 𝐾 ) + [𝑆] (1 + 𝐾 )
𝐼 𝐼
[𝐸][𝑆] [𝐸𝐼 ][𝑆] → (𝒆𝒒. 𝟗)
𝐾𝑀′ = = → (𝒆𝒒. 𝟏)
[𝐸𝑆] [𝐸𝑆𝐼 ] ✓ Group the denominator
✓ Note that in this model, we assume that [𝐸]𝑂 [𝑆]
[𝐸𝑆] = → (𝒆𝒒. 𝟏𝟎)
Km for the formation of the ES complex is [𝐼 ] ′ + [𝑆])
(1 + ) (𝐾𝑚
the same as the formation of the ESI 𝐾𝐼
complex from EI + S. Same goes for I. ✓ Expressing in terms of v
[𝐸][𝐼 ] [𝐸𝑆][𝐼 ] 𝑣 = 𝑘2 [𝐸𝑆] → (𝒆𝒒. 𝟏𝟏)
𝐾𝐼 = = → (𝒆𝒒. 𝟐) ✓ Substituting (eq.10) to (eq.11)
[𝐸𝐼 ] [𝐸𝑆𝐼 ]
✓ For the total enzyme balance, [𝐸]𝑂 [𝑆]
𝑣 = 𝑘2
[𝐸]𝑂 = [𝐸] + [𝐸𝑆] + [𝐸𝐼 ] + [𝐸𝑆𝐼 ] [𝐼 ] ′ + [𝑆 ])
(1 + ) (𝐾𝑚
→ (𝒆𝒒. 𝟑) 𝐾𝐼
✓ The objective is to express [ES] as a [𝐸]𝑂 [𝑆]
𝑣= ∙ ′ → (𝒆𝒒. 𝟏𝟐)
function of measurable properties: [𝐼 ] (𝐾𝑚 + [𝑆])
(1 + )
[𝐸]𝑂 , [𝑆], [𝐼] 𝐾𝐼
✓ Express (eq.3) as a function of [ES] from ✓ Note that
[𝐸 ]𝑂
(eq.1) and (eq.2) 𝑣𝑚𝑎𝑥,𝑎𝑝𝑝 = → (𝒆𝒒. 𝟏𝟑)
[𝐼 ]
(1 + )
𝐾𝐼
𝐾𝑀′ [𝐸][𝐼 ] [𝐸𝑆][𝐼 ]
[𝐸]𝑂 = [𝐸𝑆] + [𝐸𝑆] + + ✓ Thus, the final equation is,
[𝑆 ] 𝐾𝐼 𝐾𝐼
→ (𝒆𝒒. 𝟒)
𝒗𝒎𝒂𝒙,𝒂𝒑𝒑 [𝑺]
✓ Note that: 𝒗=
(𝑲′𝒎 + [𝑺])
𝐾′
[𝐸] = 𝑀 [𝐸𝑆]
[𝑆]
′
𝐾𝑀 𝐾 ′ [𝐼 ]
∴ [𝐸]𝑂 = [𝐸𝑆] + [𝐸𝑆] + 𝑀 [𝐸𝑆]
[𝑆] 𝐾𝐼 [𝑆]
[𝐼 ]
+ [𝐸𝑆] → (𝒆𝒒. 𝟓)
𝐾𝐼
✓ Factor out the [ES]
𝐾′ 𝐾 ′ [𝐼 ] [𝐼 ]
[𝐸]𝑂 = [𝐸𝑆] ( 𝑀 + 1 + 𝑀 + )
[𝑆 ] 𝐾𝐼 [𝑆] 𝐾𝐼
→ (𝒆𝒒. 𝟔)
✓ Isolate [ES] and multiply the numerator
and denominator with [S]
3. ENZYME INHIBITION: UNCOMPETETIVE ✓ Factor out [S]
DERIVATION (RAPID EQUILIBRIUIM [𝐸]𝑂 [𝑆]
[𝐸𝑆] = → (𝒆𝒒. 𝟖)
APPROACH) [𝐼 ]
𝐾𝑀′
+ [𝑆] (1 + 𝐾 )
𝐼
✓ Multiply numerator and denominator by
[𝐼]
(1 + 𝐾 )
𝐼
[𝐸]𝑂 [𝑆]
[𝐼 ]
(1 + )
𝐾𝐼
[𝐸𝑆] = → (𝒆𝒒. 𝟗)
𝐾𝑀′
+ [𝑆]
[𝐼 ]
(1 + )
𝐾𝐼
✓ we can now express the equation in terms
of v,
𝑣 = 𝑘2 [𝐸𝑆] → (𝒆𝒒. 𝟏𝟎)

✓ Defining the constants, ✓ Substituting (eq.9) to (eq.10)

[𝐸][𝑆] 𝑘2 [𝐸]𝑂
𝐾𝑀′ = → (𝒆𝒒. 𝟏)
[𝐸𝑆] [𝐼 ] ∙ [𝑆]
(1 + )
✓ Note that in this model, we assume that 𝐾𝐼
𝑣= → (𝒆𝒒. 𝟏𝟏)
Km for the formation of the ES complex is 𝐾𝑀′
+ [𝑆]
the same as the formation of the ESI [𝐼 ]
(1 + )
complex from EI + S. Same goes for I. 𝐾𝐼
[𝐸𝑆][𝐼 ] ✓ Take note that,
𝐾𝐼 = → (𝒆𝒒. 𝟐) 𝑣𝑚𝑎𝑥 = 𝑘2 [𝐸]𝑂 → (𝒆𝒒. 𝟏𝟐)
[𝐸𝑆𝐼 ]
✓ For the total enzyme balance,
[𝐸]𝑂 = [𝐸] + [𝐸𝑆] + [𝐸𝑆𝐼 ] → (𝒆𝒒. 𝟑) 𝑘2 [𝐸]𝑂
𝑣𝑚𝑎𝑥,𝑎𝑝𝑝 = → (𝒆𝒒. 𝟏𝟑)
✓ The objective is to express [ES] as a [𝐼 ]
(1 + )
𝐾𝐼
function of measurable properties:
[𝐸]𝑂 , [𝑆], [𝐼]
𝐾𝑀′
✓ Express (eq.3) as a function of [ES] from 𝐾𝑚,𝑎𝑝𝑝 = → (𝒆𝒒. 𝟏𝟒)
[𝐼 ]
(eq.1) and (eq.2) (1 + )
𝐾𝐼
𝐾𝑀′ [𝐼 ]
[𝐸]𝑂 = [𝐸𝑆] + [𝐸𝑆] + [𝐸𝑆] ✓ Hence, the final equation is,
[𝑆 ] 𝐾𝐼
→ (𝒆𝒒. 𝟒) 𝒗𝒎𝒂𝒙,𝒂𝒑𝒑 [𝑺]
✓ Factor out the [ES] 𝒗=
𝑲𝑴,𝒂𝒑𝒑[𝑺]
𝐾′ [𝐼 ]
[𝐸]𝑂 = [𝐸𝑆] ( 𝑀 + 1 + + ) → (𝒆𝒒. 𝟓)
[𝑆 ] 𝐾𝐼
✓ Isolate [ES] and multiply the numerator
and denominator with [S]
[𝐸]𝑂 [𝑆]
[𝐸𝑆] = ′ ∙ → (𝒆𝒒. 𝟔)
𝐾𝑀 [𝐼 ] [𝑆]
+ 1 +
[𝑆] 𝐾𝐼
[𝐸]𝑂 [𝑆]
∴ [𝐸𝑆] = → (𝒆𝒒. 𝟕)
′ [𝐼 ]
[ ]
𝐾𝑀 + 𝑆 + 𝐾 𝑆 [ ]
𝐼
Exercise 1.5.1 ✓ Solve for x-intercept:
𝑥 − 𝑖𝑛𝑡𝑒𝑟𝑐𝑒𝑝𝑡:
The hydrolysis of urea by urease shows 1 1 𝐾𝑚 1
inhibition. Hydrolysis data are given: 𝑦 − 𝑖𝑛𝑡 = = 0 = ∙ +
𝑣 [𝑆] 𝑣𝑚𝑎𝑥 𝑣𝑚𝑎𝑥
✓ Rearranging,

1 1 1 1
= 𝐾𝑚 + 1 → =−
𝑣 [𝑆 ] [𝑆] 𝐾𝑚
✓ Plot in excel only the chosen points

What type of inhibition do the data show?

Based on the type of inhibition, determine Km and
KI.
Solution to Exercise 1.5.1
✓ We have two substrate concentrations,
plotting those data: ✓ The resulting graph is:

✓ We are going to select specific points:

o We need inhibitions points that
would be very close to each other
or exactly the same like as follows,
✓ Calculate Km represented by each line
𝑠𝑙𝑜𝑝𝑒
𝐾𝑚 =
𝑦 − 𝑖𝑛𝑡𝑒𝑟𝑐𝑒𝑝𝑡
a. [𝐼 ] = 0
0.0102
𝐾𝑀𝐼 = = 𝟎. 𝟎𝟔𝟎𝟒
0.1689
b. [𝐼 ] = 0.0012
0.0153
𝐾𝑀2 = = 𝟎. 𝟎𝟔𝟎𝟒
0.2533
✓ Recall Lineweaver-Burk equation:
c. [𝐼 ] = 0.0044
1 1 𝐾𝑚 1
= ∙ + 0.0436
𝑣 [𝑆] 𝑣𝑚𝑎𝑥 𝑣𝑚𝑎𝑥 𝐾𝑀3 = = 𝟎. 𝟎𝟖𝟎𝟒
0.5422
d. [𝐼 ] = 0060
0.0573
𝐾𝑀4 = = 𝟎. 𝟎𝟗𝟔𝟔
0.5433
From the accumulated data, the inhibitor changes
vmax only since Km is not statistically different from
one another. Henceforth, the inhibitor is
NONCOMPETETIVE.

✓ Solve for 𝐾𝑚 average (obtained data)

𝑲𝒎,𝒂𝒗𝒈 = 𝟎. 𝟕𝟒𝟒𝟓 𝑴
✓ Solve for 𝐾𝐼
✓ Recall that for noncompetitive inhibition,
𝑣𝑚𝑎𝑥 [𝑆 ]
𝑣= ∙
[𝐼 ] 𝐾𝑚 + [𝑆]
1+ 𝐾
𝐼
𝑣𝑚𝑎𝑥
𝑣𝑚𝑎𝑥,𝑎𝑝𝑝 =
[𝐼 ]
1+
𝑘2
✓ We can use any point, for our case, we used
0.33

→ 𝑓𝑟𝑜𝑚 𝑡ℎ𝑒 𝑝𝑙𝑜𝑡

1
𝑣𝑚𝑎𝑥 = = 𝟑. 𝟗𝟓
0.2533
✓ Choose inhibitor point where 1/v=0.33
and [I]=0.0012
∴ 𝑣 = 0.33; 𝑣𝑚𝑎𝑥 = 3.95; [𝐼 ] = 0.0012;
[𝑆] = 0.2; 𝐾𝑚 𝑎𝑣𝑔 = 0.7445
3.95 0.2
0.33 = ∙
0.0012 0.7445
1+ 𝐾
𝐼
✓ Solve for 𝐾𝐼
𝑲𝑰 = 𝟔𝒙𝟏𝟎−𝟑 𝑴