Goals For MCB 5068
1. Obtain a solid foundation of knowledge in cell biology
2. Obtain a working knowledge of available techniques.
3. Be able to critically read and evaluate the scientific
literature.
4. Be able to define and investigate a biological problem.
Molecular Cell Biology 5068
To Do:
Visit website: www.mcb5068.wustl.edu
Sign up for course.
Check out Self Assessment homework under MercerIntroduction
Visit Discussion Sections: Read “Official” Instructions
TA’s:
Ji Woong Park
park.jiwoong@wustl.edu
Tatenda Mahlokozera
mahlokozerat@wusm.wustl.edu
What is Cell Biology?
biochemistry
genetics
cytology
Molecular Cell Biology
physiology
CELL BIOLOGY/MICROSCOPE
Microscope first built in 1595 by Hans and Zacharias Jensen in Holland
Zacharias Jensen
CELL BIOLOGY/MICROSCOPE
Robert Hooke accomplished in physics, astronomy, chemistry, biology,
geology, and architecture. Invented universal joint, iris diaphragm, anchor
escapement & balance spring, devised equation describing elasticity
(“Hooke’s Law”). In 1665 publishes Micrographia
CELL BIOLOGY/MICROSCOPE
Robert Hooke
. . . “I could exceedingly plainly
perceive it to be all perforated and
porous, much like a Honey-comb,
but that the pores of it were not
regular. . . . these pores, or cells, .
. . were indeed the first
microscopical pores I ever saw,
and perhaps, that were ever
seen, for I had not met with any
Writer or Person, that had made
any mention of them before this. .
.”
CELL BIOLOGY/MICROSCOPE
Antony van Leeuwenhoek (1632-1723)
CELL BIOLOGY/MICROSCOPE
Antony van Leeuwenhoek (1632-1723)
a tradesman of Delft, Holland, in
1673, with no formal training,
makes some of the most
important discoveries in biology.
He discovered bacteria, free-living
and parasitic microscopic protists,
sperm cells, blood cells and more.
All of this from a very simple
device that could magnify up to
300X.
Red blood cells
Spiral bacteria
THE CELL THEORY
Matthias Jakob Schleiden 1804-1881
Theodor Schwann 1810-1882
Schleiden
Schwann
THE CELL THEORY
First coined by Theodore Schwann in 1839, and formed from
the ideas of Matthias Schleiden, Schwann, and Rudolf
Virchow. The theory proposes that:
1. Anything that is alive is made up of cells.
2. The chemical reactions that occur in
organisms occur in cells.
3. All cells come from preexisting cells.
SPONTANEOUS GENERATION
From ancient time, through the Middle Ages, and until the late
nineteenth century, it was generally accepted that some life
forms arose spontaneously from non-living organic matter.
Jan Baptista van Helmont (1577-1644) Flemish physican,
chemist and physiologist. Invented the word “gas”. Recipe for
mice:
Place a dirty shirt or some rags in an open pot or barrel
containing a few grains of wheat or some wheat bran, and in
21 days, mice will appear
SPONTANEOUS GENERATION
(1668-1859)
Although the belief in the spontaneous generation of
large organisms wanes after 1668, the invention of
the microscope serves to enhance the belief in
spontaneous generation. Microscopy revealed a
whole new class of organisms (animalcules) that
appeared to arise spontaneously. It was quickly
learned that you needed only to place hay in water
and wait a few days before examining your new
creations under the microscope. This belief
persisted for nearly two centuries.
SPONTANEOUS GENERATION
(1668-1859)
In 1859, after years of debate The French Academy of
Sciences sponsors a contest for the best experiment either
proving or disproving spontaneous generation. The French
chemist, Louis Pasteur (1822-1895) uses a variation of the
methods of Needham and Spallanzani. He boils meat broth
in a flask, heats the neck of the flask in a flame until it
became pliable, and bent it into the shape of an S. Air could
enter the flask, but airborne microorganisms could not - they
would settle by gravity in the neck. As Pasteur had
expected, no microorganisms grew. When Pasteur tilted the
flask so that the broth reached the lowest point in the neck,
where any airborne particles would have settled, the broth
rapidly became cloudy with life. Pasteur had both refuted
the theory of spontaneous generation and convincingly
demonstrated that microorganisms are everywhere - even in
the air.
CELL BIOLOGY/MICROSCOPE
Camillo Golgi (1843-1926)
In 1898, Golgi develops a staining
technique (silver nitrate) that allows
the identification of an "internal
reticular apparatus" that now bears
his name: the "Golgi complex” or the
“Golgi”.
CELL BIOLOGY/MICROSCOPE
By the late 1800’s to the early 1900’s the limits to the light
microscope had been reached.
Resolving ability roughly 1/2 l of light used: ≈ 0.2 µm
In 1930 A.A. Lebedeff designs and builds the first interference
microscope.
In 1932 Frits Zernike (1888-1966) invents the phase-contrast
microscope. It is first brought to market in 1941 in Germany.
Both microscopes aid in elucidating the details in unstained
living cells.
CELL BIOLOGY/MICROSCOPE
In 1932 Zernike traveling from Amsterdam,
visits the Zeiss factory in Germany to
present his method of phase contrast
microscopy. After reviewing Zernike's
method an older scientist said:
"If this really had any practical value, then
we would have invented it a long time ago."
In 1953 Zernike was awarded the Nobel
Prize for his phase contrast work.
Light behaves as a Wave
Wavelength sets limits
on what one can see
Lower limits on spatial resolution are
defined by the Rayleigh Criterion
Resolution = 0.61 x wavelength of light
NA (numerical aperture)
NA = nsinθ
n = refractive index of the medium
θ = semi-angle of an objective lens
θ
θ
The effect of NA
on the image of
a point.
θ
The need for
separation to
allow resolution
Contrast in the Image is Necessary:
Types of Optical Microscopy Generate
Contrast in Different Ways
• Bright field - a conventional light
microscope
• DIC (Differential Interference Contrast Nomarski)
• Phase contrast
• Fluorescence
• Polarization
• Dark field
Bright-field Optics: Light Passing
Straight Through the Sample
• Most living cells are optically clear, so stains
are essential to get bright field contrast
• Preserving cell structure during staining and
subsequent observation is essential, so cells
must be treated with “fixatives” that make
them stable
• Fixing and staining is an art
Generating Contrast
Staining
Coefficients of absorption among different
materials differ by >10,000, so contrast
can be big
Without staining
Everything is bright
Most biological macromolecules do not
absorb visible light
Contrast depends on small differences
between big numbers
Need an optical trick
Mammalian Cell:
Bright-field and Phase-contrast Optics
Principles of bright field
and phase contrast optics
Differential Interference Contrast
(DIC)
• Optical trick to visualize the interference between
two parts of a light beam that pass through
adjacent regions of the specimen
• Small amounts of contrast can be expanded
electronically
• Lots of light: Video camera with low brightness &
high gain
Brightfield vs DIC
Fluorescence
Microscopy
• Absorption of high-energy
(low wavelength) photon
• Loss of electronic energy
(vibration)
• Emission of lower-energy
(higher wavelength) photon
Design of a Fluorescence Microscope
Green Fluorescent Protein - Considerations
• Color - Not just green
• Brightness
• Size/Location 26.9 kDa
• Time for folding
• Time to bleaching
GFP-Cadherin in cultured epithelial cells
Immunofluorescence
• Primary Abs recognize the antigen (Ag)
• Secondary Abs recognize the primary Ab
• Secondary Abs are labeled
Immunofluorescence Example
• Ab to tubulin
• Ab to
kinetochore
proteins
• DNA stain
(DAPI)
Biological microscopy problem: Cells are
3D objects, and pictures are 2D images.
• Single cells are thicker than the wavelength of
visible light, so they must be visualized with
many “optical sections”
• In an image of one section, one must remove
light from other sections
• Achieving a narrow “depth-of-field”
• A “confocal light microscope”
Laser-Scanning
Confocal Light
Microscopy
• Laser thru pinhole
• Illuminates sample
with tiny spot of light
• Scan the spot over
the sample
• Pinhole in front of
detector: Receive
only light emitted
from the spot
Light from points that
are in focus versus out
of focus
Spinning-disk confocal microscopy:
Higher speed and sensitivity
Example: Confocal imaging lessens
blur from out-of-focus light
Optically Sectioning a Thick Sample:
Pollen Grain
Multiple optical sections assembled to
form a 3D image
Fluorescence can Measure Concentration of Ca2+ Ions in
Cells:
Sea Urchin Egg Fertilization
Phase Contrast
Fluorescence
Total Internal Reflection Fluorescence (TIRF)
Microscopy
The penetration
depth of the field
typically ranges
from 60 to
100 nm
www.leica-microsystems.com
Total Internal Reflection Fluorescence (TIRF)
Microscopy
www.leica-microsystems.com
Summary
• Light microscopy provides sufficient resolution to
observe events that occur inside cells
• Since light passes though water, it can be used to
look at live as well as fixed material
• Phase contrast and DIC optics: Good contrast
• Fluorescence optics: Defined molecules can be
localized within cells
• “Vital” fluorescent stains: Watch particular
molecular species in live cells
CELL BIOLOGY/MICROSCOPE
Louis de Broglie (1892-1987)
In 1924 at the Faculty of Sciences at Paris
University he delivers a thesis Recherches
sur la Théorie des Quanta (Researches on
the quantum theory), which earned him his
doctorate. This thesis contained a series of
important findings that he had obtained in the
course of about two years. This research
culminated in the de Broglie hypothesis
stating that any moving particle or object had
an associated wave. Therefore a moving
electron has wavelike properties.
In 1929 he received the Nobel Prize for this
observation.
CELL BIOLOGY/MICROSCOPE
CELL BIOLOGY/MICROSCOPE
Light Microscope
Transmission
Electron
Microscope
Scanning
Electron
Microscope
The Cell
1. Compartmentalized
chemical reactions
2. Modify intra- and extracellular environment
3. Different properties and
functions.
The Cell
Surface Area to Volume Ratio Limits Cell Size
In general, the surface area increases in proportion
to the square of the width and volume as the
cube of the width.
Xenopus oocyte
Membranes Define the Cell
Electron micrograph of a thin section of a hormone-secreting cell from the rat pituitary,
showing the subcellular features typical of many animal cells.
CELL BIOLOGY/MEMBRANES
In the late 1890’s Charles Ernest Overton was working
on a doctoral degree in botany at the University of Zurich.
His research was related to heredity in plants and in order
to complete his studies he needed to find substances that
would be readily absorbed into plant cells. He found that
the ability of a substance to pass through the membrane
was related to its chemical nature. Nonpolar substances,
would pass quickly through the membrane into the cell.
This discovery was quite contrary to the prevalent view at
the time that the membrane was impermeable to almost
anything but water.
CELL BIOLOGY/MEMBRANES
Based on his observations of what substances pass
through the membrane, Overton proposes:
1. There are some similarities between cell membranes
and lipids such as olive oil.
2. Certain molecules (i.e., lipids) pass through the
membrane by "dissolving" in the lipid interior of the
membrane.
CELL BIOLOGY/MEMBRANES
CELL BIOLOGY/MEMBRANES
Irving Langmuir (1881-1957)
Trained in physical chemistry under Nobel
laureate Walther Nernst, Langmuir worked in
the laboratories of General Electric doing
research on molecular monolayers. His
research eventually turned to lipids and the
interaction of oil films with water. By
improving an existing apparatus for the
study of lipids (referred to today as a
Langmuir trough), he was able to make
careful measurements of surface areas
occupied by known quantities of oil.
CELL BIOLOGY/MEMBRANES
Irving Langmuir (1881-1957)
Based on his studies he proposed that the fatty acid
molecules form a monolayer by orienting themselves vertically
with the hydrocarbon chains away from the water and the
carboxyl groups in contact with the surface of the water.
In 1932 he received the Nobel Prize for Chemistry “for his
discoveries and investigations in surface chemistry.”
Time Magazine
August 28, 1950
His improvement of vacuum techniques led to the invention of the
high-vacuum tube. He and colleague Lewi Tonks discovered that
the lifetime of a tungsten filament was greatly lengthened by filling
the bulb with an inert gas, such as argon. He also discovered
atomic hydrogen, which he put to use by inventing the atomic
hydrogen welding process. During WWII Langmuir worked to
develop protective smoke screens and methods for de-icing aircraft
wings. This research led him to discover that the introduction of dry
ice and iodide into a sufficiently moist cloud of low temperature
could induce precipitation (cloud seeding).
CELL BIOLOGY/MEMBRANES
In 1925 Evert Gorter and his research assistant, J.Grendel extracted the
lipids from red blood cells with acetone and other organic solvents. Using a
modified trough, similar to Langmuir, they were able to demonstrate that lipid
molecules could form a double layer, or bilayer as well as a monolayer.
Further, they were able to show that the surface area of the lipids extracted
from the red blood cells was about twice the surface area of the cells
themselves.
Based on these two observations (i.e., that lipid molecules can form
bilayers, and that the surface area of the monolayer extracted from the cells
is approximately equal to twice the surface area of the cells) and repeated
studies with red blood cells from several animals (human, rabbit, dog, guinea
pig, sheep, and goat) Gorter and Grendel concluded that "chromocytes [red
blood cells] are covered by a layer of fatty substances that is “two molecules
thick”
CELL BIOLOGY/MEMBRANES
CELL BIOLOGY/MEMBRANES
Lipid Monolayer
Lipid Bilayer
CELL BIOLOGY/MEMBRANES
CELL BIOLOGY/MEMBRANE MODELS
In 1935 James F. Danielli and Hugh Davson propose the
first widely accepted membrane model. The model proposed
by Danielli and Davson was basically a "sandwich" of lipids
(arranged in a bilayer) covered on both sides with proteins.
Later versions of the model included "active patches" and
protein lined pores.
CELL BIOLOGY/MEMBRANES
CELL BIOLOGY/MEMBRANES
In 1957 J.D. Robertson proposed a modified version of the membrane
model, based primarily on EM studies, which he called the "unit membrane".
Under the high magnification of the TEM, membranes have a characteristic
"trilaminar" appearance consisting of two darker outer lines and a lighter
inner region. According to the unit membrane model, the two outer, darker
lines are the protein layers and the inner region the lipid bilayer.
CELL BIOLOGY/MEMBRANE MODELS
In the early 1970’s the unit membrane model was replaced by
the fluid mosaic model. This model was first proposed by
biochemists S.J. Singer and Garth L. Nicolson. The model
retains the basic lipid bilayer structure, however, proteins are
thought to be globular and to float within the lipid bilayer.
As in the other models, the hydrophobic tails of the
phospholipids face inward, away from the water. The
hydrophilic heads of the phospholipids are on the outside where
they interact with water molecules in the fluid environment of
the cell. Floating within this bilayer are the proteins, some of
which span the entire bilayer and may contain channels, or
pores, to allow passage of molecules through the membrane.
The entire membrane is fluid—the lipid molecules move within
the layers of the bilayer while the "floating" proteins also freely
move within the bilayer.
CELL BIOLOGY/MEMBRANE MODELS
The fluid-mosaic model of membrane structure as initially proposed by
Singer and Nicolson in 1972.
CELL BIOLOGY/MEMBRANE MODELS
Left. Image of the upper surface of a lipid bilayer containing phosphatidylcholine (black
background), and sphingomyelin molecules, which organize themselves spontaneously into the
orange-colored rafts. The yellow peaks represent a GPI-anchored protein, which is almost
exclusively raft-associated. This image is provided by an atomic force microscope,which measures
the height of various parts of the specimen at the molecular level. Right. Schematic model of a lipid
raft within a cell. The outer leaflet of the raft consists primarily of cholesterol and sphingolipids (red
head groups). Phosphatidylcholine molecules (blue head groups) with long saturated fatty acids
also tend to concentrate in this region. A GPI-anchored protein is localized in the raft. The lipids in
the outer leaflet of the raft have an organizing effect on the lipids of the inner leaflet. As a result, the
inner leaflet raft lipids consist primarily of cholesterol and glycerophospholipids with saturated fatty
acyl tails. The inner leaflet tends to concentrate lipid-anchored proteins, such as src kinase, that are
involved in cell signaling.
FUNCTIONS OF MEMBRANES
1. Compartmentalization
2. Permeability barrier - regulate what gets through
3. Selective pumps & gates - regulate & accelerate
molecular passage
4. Generate signals for cell communication
5. Flow of information between cells & between
environment & cells
6. Surfaces for ordered array of reactions
The properties of membranes derive from both
lipids and proteins
Fluidity of membranes is determined by
both temperature and composition.
Temperature:
Left. Above the transition temperature, the lipid molecules and their
hydrophobic tails, although ordered are free to move in certain directions.
Right. Below the transition temperature, the movement of the lipid molecules
is greatly restricted and the bilayer takes on properties of a crystalline gel.
Fluidity of membranes is determined by both
temperature and composition.
Concentration of cholesterol.
Unsaturated vs. saturated fatty acids.
The cholesterol molecules (green) in
the lipid bilayer, interfere with the tight
packing of the phospholipids, making
the bilayer more fluid.
Crooked, unsaturated fatty acids
interfere with tight packing of the
phospholipids, making the bilayer
more fluid.
The Cell
1. Compartmentalized
chemical reactions
2. Modify intra- and extracellular environment
3. Different properties and
functions.
Individual cells will direct the function of tissues and organs
Reductionism Science
1. Define a biological problem
Genetics, physiology, medicine
2. Inventory of parts
Biochemistry, genetics, genomics
3. Concentrations
Biochemistry, microscopy
4. Molecular structures
X-ray crystallography, NMR
5. Partners
Biochemistry, genetics
6. Rate & equilibrium constants
Biophysics, microscopy
7. Biochemical reconstitution
Biochemistry, microscopy
8. Mathematical model
Analytical or numerical
9. Physiological tests
Drugs, genetics, RNAi
Approaches to Cell Biology Research
Genetics
• Screen for mutants with a phenotype.
• Crosses to define complementation
groups.
• Details of the phenotypes. Divide into
classes.
• Order the classes by epistasis.
• Clone the genes.
Approaches to Cell Biology Research
Anatomy
• Structure of cells and tissues.
• Ultrastructure (EM), to detect fine
structures, such as filaments or
membranes.
• Correlate structures with function.
• Identify molecules if possible.
Approaches to Cell Biology Research
Biochemistry
• Purify molecules, such as metabolites,
proteins, or even membranes.
• Study their chemical properties in vitro.
• Attempt to re-create in vitro a
phenomenon observed in vivo.
• Reconstitution as an ultimate test for
“sufficiency.”
Approaches to Cell Biology Research
Physiology
• Observe the phenomena exhibited by
living cells or organisms, such as
movement.
• Quantify parameters such as rate of
movement and ask how they correlate
with each other factors.
• Decrease or increase the activity of a
component.
Approaches to Cell Biology Research
Pharmacology
• Find drugs (chemicals) that inhibit or
enhance a phenomenon, such as
movement.
• Identify their molecular targets, such
as proteins.
• Use in physiology studies to inhibit a
process acutely.
Example of How the Techniques Interact
Find a cell that moves, like Dictyostelium.
• Study its movement up a chemotactic
gradient, and quantify various
parameters.
• Find drugs that inhibit this movement.
Study the fine structure of the cell, especially
the areas that seem to be moving.
• Are there small structures, such as
filaments and crosslinkers, and are they
in an arrangement that suggests how
movement can be generated?
Example of How the Techniques Interact
Purify proteins that make up those fine
structures, such as filaments.
• Purify proteins that bind to those
proteins.
• Look for how the different proteins
regulate the relevant activity (which you
have to guess at).
• Determine whether the drugs above
affect this in vitro activity.
Example of How the Techniques Interact
Localize the proteins
• Ab staining of cells to show that the
proteins really are associated with
those fine structures.
• GFP fusions once cDNA is obtained
(later)
Microinject Abs or fragments of proteins
looking for an effect on cell movement
(inhibition or enhancement).
Example of How the Techniques Interact
Reverse genetics.
• Use the protein to clone cDNA’s and/or
genes encoding it. Modern equivalent database search.
• Correlate expression with cell
movement.
• Use the cDNA to inhibit the protein
(antisense or knockout)
• Overexpress the protein
• Express fragments or mutated versions
of the protein (dominant effects).
Example of How the Techniques Interact
Forward genetics.
• Start by making mutants.
• Study phenotype and classify.
Information about different steps at
which one can stall the process. Use
the physiology and anatomy to
classify.
• Epistasis to order the genes.
• Clone and sequence the genes.
• Make protein, make Abs and cDNAs,
and do the experiments above.
Example of How the Techniques Interact
Reconstitution as an ultimate goal.
• Genetics defines a set of
genes/proteins important for
movement.
• Make and mix together pure proteins
to create the movement.
Hypothesis-Driven Experiments
•State the hypothesis
Not a “straw man” or trivial
•State the experiment
•Possible outcomes
•Interpretation of each outcome
•Controls - positive, negative
•Limitations and Alternative Interpretations
“Proof” of a Hypothesis or Model
Observed Results as Predicted
What Alternatives are Excluded?
• Karl Popper, The Logic of Scientific Discovery,
1934
• How strong is the evidence against the
alternatives?
• Obligation to raise and test credible
alternatives
• Or the ones that others find compelling
Revolutions and Paradigms
•Thomas Kuhn, The Structure of
Scientific Revolutions, 1962
•Evidence against the current paradigm
is the most interesting and important
Kinetic analysis
• How cells change over long time periods
(development, long term adaptive changes; hours years)
• Movement of proteins and membranes within cells dynamics of cellular events (sec - hrs)
– Pulse chase analyses
– Real time imaging: GFP and other fluorophores
allow measurement of trafficking, diffusion, etc.
(time-lapse, fluorescence recovery after
photobleaching (FRAP), etc.)
• Kinetics of molecular interactions, enzyme reactions
(msec - min)
Enzymes are catalysts for chemical reactions in cells
Catalyst (enzyme): increases rate of a reaction
Substrate: molecule on which enzyme acts to form product
S ------> P
enzyme
Free energy of reaction
not changed by enzyme.
For a favored reaction
(ΔG negative), enzyme
accelerates reaction.
Graph:
ΔG* = activation energy
ΔG negative overall for forward reaction
Enzymes as Catalysts
Active Site: Region of the enzyme that does the work. Amino
acid residues in this site assume certain 3D conformation,
which promotes the desired reaction.
What does the Enzyme do to cause catalysis?
• High affinity for substrate in its transition state, facilitating
transition to product
• Increased probability of proper orientation of substrates
• Increased local concentration of substrates
• Has atoms in places that push the reaction forward
• Change hydration sphere of substrates
Phases of Enzyme Reactions
• Transient phase
– Accelerating Velocity
– Short (<1s)
– Formation Enzyme-Substrate
Intermediates
• Steady-state phase
–
–
–
–
–
–
May Not Occur
Constant Velocity
Duration up to Several Minutes
Little Change Levels of Enzyme
Small Fraction Substrate Consumed
Small Levels Product Formed
• Exhaustion phase
–
–
–
–
Decreasing Velocity
Depletion of Substrate
Accumulation of Product
Inactivation of Unstable Enzyme
What Can You Learn from What
Happens at Steady State?
• Turnover number => catalytic efficiency of enzyme
• Affinity of enzyme for substrates
• Lower bounds for rate constants
• Inhibitors and pH variations to probe active site
• Details of mechanism require transient (pre-steady
state) kinetic analysis
How to Measure Enzyme Activity at
Steady State
Need an assay that measures the product of the chemical
reaction. For example...
Enzyme β-galactosidase catalyzes this reaction:
lactose --------------------> glucose + galactose
Measure the amount of glucose or galactose over time.
Trick - use a substrate that produces a reaction product
that absorbs light (creates color). Measure absorbance.
Color-Producing Substrates for β-galactosidase
ONPG = ONP-galactose (ONP = o-nitro-phenol)
ONPG --------------> galactose
+
ONP
(colorless)
(colorless)
(yellow)
X-gal = X-galactose (X = 4-chloro-3-bromo indole)
X-gal ---------------> galactose
+
4-Cl-3-Br-indigo
(colorless)
(colorless)
(deep blue)
Measure absorbance with a spectrophotometer
•Beer’s law - concentration proportional to absorbance
•96-well format instruments
Optimizing assay
• No Enzyme -> No Product
• Optimize pH, salt, other buffer
conditions
• Optimize temperature
• Choose set of conditions to be kept
constant
• Amount of enzyme
– Linear range of assay
– More is better
Measure Velocity of Reaction
One Single Experiment at One Substrate Concentration
•Plot product vs time
•Determine rate during initial linear phase
Equilibrium?
Steadystate?
Run the Assay at Different
Substrate Concentrations
Plot initial rate (v0)
vs
Concentration of
Substrate [S]
Michaelis-Menten Plot
• What’s interesting or useful about this plot?
• Can we use this plot to compare results for
different enzymes or conditions?
• Can we derive an equation for the curve?
How Km values affect metabolism
• Glucose + ATP --> glucose-6-P + ADP + H+
• Typical cell [glucose] = 5 mM
• Two enzymes catalyze above reaction
– Hexokinase
• Km (glucose) = 0.1 mM
• Km << [S], so velocity independent of [glucose]
• Reaction is inhibited by product--regulated by product
utilization
– Glucokinase
• Km (glucose) = 10 mM
• Km > [S], promotes glucose utilization only when [glucose] is
high
• Reaction not inhibited by product--regulated by substrate
availability
Determining Km and Vmax
• Estimate Vmax from asymptote, Km from conc. at Vmax/2
• Curve fitting w/ computer programs, inc Excel
• Visual inspection (Graph paper)
•
Lineweaver-Burke plot and others
Michaelis-Menten equation can be rearranged into
“Lineweaver-Burke” equation
From this graph, visually estimate Km and Vmax.
Regulating enzyme activity
• Allosteric regulation
• Reversible covalent modifications
• Enzyme availability (synthesis, degradation,
localization)
• Substrate availability (synthesis, degradation,
localization)
• Inhibition
– By specific metabolites within the cell
– By drugs, toxins, etc.
– By specific analogues in study of reaction mechanism
Competitive Inhibition
Competitive inhibitor:
• binds to free enzyme
• prevents simultaneous binding of substrate
-i.e. competes with substrate
• Apparent Km of the substrate is therefore increased
• High substrate concentration:
- substrate overcomes inhibition by mass action
- v0 approaches Vmax (which does not change)
Example of Competitive Inhibition
• EtOH Rx for MeOH poisoning
• Methanol (ingested from solid alcohol, paint strippers,
windshield washer fluid, etc.) is metabolized by alcohol
dehydrogenase to formaldehyde and formic acid. Leads to
metabolic acidosis and optic neuritis (from formate) that can
cause blindness.
• Treatment: Infuse EtOH to keep blood concentration at 100200 mg/dL (legally intoxicated) for long enough to excrete the
MeOH.
• EtOH serves as a competitive inhibitor. Ethylene glycol
poisoning is treated in the same way.
Noncompetitive Inhibition
Noncompetitive inhibitor :
• Binds to a site on the enzyme (E or ES) that
inactivates the enzyme
• Decreases total amount of enzyme available
for catalysis, decreasing Vmax
• Remaining active enzyme molecules are
unaffected, so Km is unchanged
Uncompetitive Inhibition
Uncompetitive inhibitor:
• Binds specifically to the [ES] complex (and
inactivates it
• Fraction of enzyme inhibited increases as
[S] increases
• So both Km and Vmax are affected
Summary: Types of Inhibitors
• Competitive
– Binds Free Enzyme Only
– Km Increased
• Noncompetitive
– Binds E and ES
– Vmax Decreased
• Uncompetitive
– Binds ES only
– Vmax Decreased
– Km Decreased
Plots to Distinguish Types of Inhibitors
• Competitive
No inhibitor
• Uncompetitive
No inhibitor
• Noncompetitive
No inhibitor
Lineweaver-Burke
Plots show curves
with no inhibitor
vs. presence of
two different
concentrations of
inhibitor
Reading and Homework for Kinetics
• Alberts (5th edition) pp. 159-166
• Lodish (6th edition) pp. 79-85
• See handout or website for homework