Goals For MCB 5068

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3. Be able to critically read and evaluate the scientificliterature.

1. Obtain a solid foundation of knowledge in cell biology

2. Obtain a working knowledge of available techniques.

4. Be able to define and investigate a biological problem.

Goals For MCB 5068

Visit website: www.mcb5068.wustl.eduSign up for course.

Check out Self Assessment homework under Mercer-Introduction

Visit Discussion Sections: Read “Official” Instructions

To Do:

Molecular Cell Biology 5068

TA’s:

1st Session:

Nana Owusu-Boaitey

[email protected]

2nd Session:

Shankar Parajuli

[email protected]

3rd Session:

Jeff Kremer

[email protected]

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biochemistry genetics cytology

What is Cell Biology?

physiology

Molecular Cell Biology

CELL BIOLOGY/MICROSCOPE

Microscope first built in 1595 by Hans and Zacharias Jensen in Holland

Zacharias Jensen

CELL BIOLOGY/MICROSCOPERobert Hooke accomplished in physics, astronomy, chemistry, biology,geology, and architecture. Invented universal joint, iris diaphragm, anchorescapement & balance spring, devised equation describing elasticity(“Hooke’s Law”). In 1665 publishes Micrographia

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Robert Hooke

. . . “I could exceedingly plainlyperceive it to be all perforatedand porous, much like a Honey-comb, but that the pores of itwere not regular. . . . these pores,or cells, . . . were indeed the firstmicroscopical pores I ever saw, andperhaps, that were ever seen, for Ihad not met with any Writer orPerson, that had made anymention of them before this. . .”


Antony van Leeuwenhoek (1632-1723)


Antony van Leeuwenhoek (1632-1723)

a tradesman of Delft, Holland, in1673, with no formal training,makes some of the mostimportant discoveries in biology.He discovered bacteria, free-living and parasitic microscopicprotists, sperm cells, blood cellsand more. All of this from a verysimple device that could magnifyup to 300X.

Red blood cells

Spiral bacteria

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THE CELL THEORYMatthias Jakob Schleiden 1804-1881Theodor Schwann 1810-1882

Schleiden Schwann

THE CELL THEORY

First coined by Theodore Schwann in 1839, and formed fromthe ideas of Matthias Schleiden, Schwann, and RudolfVirchow. 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 GENERATIONFrom ancient time, through the Middle Ages, and until the latenineteenth century, it was generally accepted that some lifeforms arose spontaneously from non-living organic matter.

Jan Baptista van Helmont (1577-1644) Flemish physican,chemist and physiologist. Invented the word “gas”. Recipe formice:

Place a dirty shirt or some rags in an open pot or barrelcontaining a few grains of wheat or some wheat bran, and in21 days, mice will appear

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SPONTANEOUS GENERATION(1668-1859)

Although the belief in the spontaneous generationof large organisms wanes after 1668, the inventionof the microscope serves to enhance the belief inspontaneous generation. Microscopy revealed awhole new class of organisms (animalcules) thatappeared to arise spontaneously. It was quicklylearned that you needed only to place hay in waterand wait a few days before examining your newcreations under the microscope. This belief persistedfor nearly two centuries.

SPONTANEOUS GENERATION(1668-1859)

In 1859, after years of debate The French Academy ofSciences sponsors a contest for the best experiment eitherproving or disproving spontaneous generation. The Frenchchemist, Louis Pasteur (1822-1895) uses a variation of themethods of Needham and Spallanzani. He boils meat brothin a flask, heats the neck of the flask in a flame until itbecame pliable, and bent it into the shape of an S. Air couldenter the flask, but airborne microorganisms could not -they would settle by gravity in the neck. As Pasteur hadexpected, no microorganisms grew. When Pasteur tilted theflask so that the broth reached the lowest point in the neck,where any airborne particles would have settled, the brothrapidly became cloudy with life. Pasteur had both refutedthe theory of spontaneous generation and convincinglydemonstrated that microorganisms are everywhere - evenin the air.

CELL BIOLOGY/MICROSCOPECamillo Golgi (1843-1926)

In 1898, Golgi develops a stainingtechnique (silver nitrate) that allowsthe identification of an "internalreticular apparatus" that now bearshis name: the "Golgi complex” or the“Golgi”.

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By the late 1800’s to the early 1900’s the limits to the lightmicroscope had been reached.

Resolving ability roughly 1/2 λ of light used: ≈ 0.2 µm

In 1930 A.A. Lebedeff designs and builds the firstinterference microscope.

In 1932 Frits Zernike (1888-1966) invents the phase-contrastmicroscope. It is first brought to market in 1941 in Germany.

Both microscopes aid in elucidating the details in unstainedliving cells.


In 1932 Zernike traveling from Amsterdam,visits the Zeiss factory in Germany topresent his method of phase contrastmicroscopy. After reviewing Zernike'smethod an older scientist said:

"If this really had any practical value, thenwe would have invented it a long timeago."

In 1953 Zernike was awarded the NobelPrize for his phase contrast work.

Wavelength sets limitson what one can see

Light behaves as a Wave

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Resolution = 0.61 x wavelength of light NA (numerical aperture)

The effect of NAon the image ofa point.

The need forsepara6on toallow resolu6on

θθ

θ

Lower limits on spatial resolution aredefined by the Rayleigh Criterion

NA = nsinθn = refrac6ve index of the mediumθ = semi-‐angle of an objec6ve lens

Contrast in the Image is Necessary:Types of Optical Microscopy GenerateContrast in Different Ways

•Bright field - a conventional lightmicroscope

•DIC (Differential Interference Contrast -Nomarski)

•Phase contrast•Fluorescence•Polarization•Dark field

Bright-field Optics: Light PassingStraight Through the Sample

•Most living cells are optically clear, so stainsare essential to get bright field contrast

•Preserving cell structure during staining andsubsequent observation is essential, so cellsmust be treated with “fixatives” that makethem stable

•Fixing and staining is an art

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Generating Contrast

StainingCoefficients of absorption among different

materials differ by >10,000, so contrast canbe big

Without stainingEverything is brightMost biological macromolecules do not

absorb visible lightContrast depends on small differences

between big numbersNeed an optical trick

Mammalian Cell:Bright-field and Phase-contrast Optics

Principles of bright fieldand phase contrast optics

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Differential Interference Contrast(DIC)

• Optical trick to visualize the interferencebetween two parts of a light beam that passthrough adjacent regions of the specimen

• Small amounts of contrast can be expandedelectronically

• Lots of light: Video camera with low brightness& high gain

Brightfield vs DIC

FluorescenceMicroscopy

•Absorption of high-energy(low wavelength) photon

•Loss of electronic energy(vibration)

•Emission of lower-energy(higher wavelength) photon

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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

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Immunofluorescence

• Primary Abs recognize the antigen (Ag)• Secondary Abs recognize the primary Ab• Secondary Abs are labeled

Immunofluorescence Example

•Ab to tubulin

•Ab tokinetochoreproteins

•DNA stain(DAPI)

Biological microscopy problem: Cells are3D objects, and pictures are 2D images.

• Single cells are thicker than the wavelength ofvisible light, so they must be visualized withmany “optical sections”

• In an image of one section, one must removelight from other sections

• Achieving a narrow “depth-of-field”

• A “confocal light microscope”

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Laser-ScanningConfocal Light

Microscopy

•Laser thru pinhole

•Illuminates samplewith tiny spot oflight

•Scan the spot overthe sample

•Pinhole in front ofdetector: Receiveonly light emittedfrom the spot

Light from points thatare in focus versus outof focus

Spinning-disk confocal microscopy:Higher speed and sensitivity

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Example: Confocal imaging lessensblur from out-of-focus light

Optically Sectioning a Thick Sample:Pollen Grain

Multiple optical sections assembled toform a 3D image

Fluorescence can Measure Concentration of Ca2+ Ions in Cells:Sea Urchin Egg Fertilization

Phase Contrast Fluorescence

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Total Internal Reflection Fluorescence (TIRF)Microscopy

www.leica-microsystems.com

The penetrationdepth of thefield typicallyranges from 60to 100 nm

Total Internal Reflection Fluorescence (TIRF)Microscopy

www.leica-microsystems.com

Summary

• Light microscopy provides sufficient resolution toobserve events that occur inside cells

• Since light passes though water, it can be used tolook at live as well as fixed material

• Phase contrast and DIC optics: Good contrast

• Fluorescence optics: Defined molecules can belocalized within cells

• “Vital” fluorescent stains: Watch particularmolecular species in live cells

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In 1924 at the Faculty of Sciences at ParisUniversity he delivers a thesis Recherches surla Théorie des Quanta (Researches on thequantum theory), which earned him hisdoctorate. This thesis contained a series ofimportant findings that he had obtained inthe course of about two years. This researchculminated in the de Broglie hypothesisstating that any moving particle or object hadan associated wave. Therefore a movingelectron has wavelike properties.

In 1929 he received the Nobel Prize for thisobservation.

Louis de Broglie (1892-1987)



Light Microscope TransmissionElectron

Microscope

ScanningElectron

Microscope

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The Cell

1. Compartmentalizedchemical reactions

2. Modify intra- and extra-cellular environment

3. Different properties andfunctions.

The Cell

Surface Area to Volume Ratio Limits Cell Size

In general, the surface area increases in proportionto the square of the width and volume as thecube of the width.

Xenopus oocyte

Electron micrograph of a thin section of a hormone-secreting cell from the rat pituitary,showing the subcellular features typical of many animal cells.

Membranes Define the Cell

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CELL BIOLOGY/MEMBRANES

In the late 1890’s Charles Ernest Overton was working ona doctoral degree in botany at the University of Zurich. Hisresearch was related to heredity in plants and in order tocomplete his studies he needed to find substances thatwould be readily absorbed into plant cells. He found thatthe ability of a substance to pass through the membranewas 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 atthe time that the membrane was impermeable to almostanything but water.


Based on his observations of what substances pass throughthe membrane, Overton proposes:

1. There are some similarities between cell membranesand lipids such as olive oil.

2. Certain molecules (i.e., lipids) pass through themembrane by "dissolving" in the lipid interior of themembrane.


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CELL BIOLOGY/MEMBRANESIrving Langmuir (1881-1957)

Trained in physical chemistry under Nobellaureate Walther Nernst, Langmuir workedin the laboratories of General Electric doingresearch on molecular monolayers. Hisresearch eventually turned to lipids and theinteraction of oil films with water. Byimproving an existing apparatus for thestudy of lipids (referred to today as aLangmuir trough), he was able to makecareful measurements of surface areasoccupied by known quantities of oil.

CELL BIOLOGY/MEMBRANESIrving Langmuir (1881-1957)

Based on his studies he proposed that the fatty acid moleculesform a monolayer by orienting themselves vertically with thehydrocarbon chains away from the water and the carboxylgroups in contact with the surface of the water.

In 1932 he received the Nobel Prize for Chemistry “for hisdiscoveries and investigations in surface chemistry.”

His improvement of vacuum techniques led to the invention of thehigh-vacuum tube. He and colleague Lewi Tonks discovered thatthe lifetime of a tungsten filament was greatly lengthened by fillingthe bulb with an inert gas, such as argon. He also discoveredatomic hydrogen, which he put to use by inventing the atomichydrogen welding process. During WWII Langmuir worked todevelop protective smoke screens and methods for de-icing aircraftwings. This research led him to discover that the introduction ofdry ice and iodide into a sufficiently moist cloud of lowtemperature could induce precipitation (cloud seeding).Time Magazine

August 28, 1950

CELL BIOLOGY/MEMBRANESIn 1925 Evert Gorter and his research assistant, J.Grendel extracted the lipidsfrom red blood cells with acetone and other organic solvents. Using amodified trough, similar to Langmuir, they were able to demonstrate thatlipid 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 extractedfrom the red blood cells was about twice the surface area of the cellsthemselves.

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 isapproximately equal to twice the surface area of the cells) and repeatedstudies 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 thatis “two molecules thick”

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Lipid Monolayer

Lipid Bilayer


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CELL BIOLOGY/MEMBRANE MODELS

In 1935 James F. Danielli and Hugh Davson propose the firstwidely accepted membrane model. The model proposed byDanielli 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" andprotein lined pores.



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 lighterinner region. According to the unit membrane model, the two outer, darkerlines are the protein layers and the inner region the lipid bilayer.

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CELL BIOLOGY/MEMBRANE MODELSIn the early 1970’s the unit membrane model was replaced bythe fluid mosaic model. This model was first proposed bybiochemists S.J. Singer and Garth L. Nicolson. The modelretains the basic lipid bilayer structure, however, proteins arethought to be globular and to float within the lipid bilayer.

As in the other models, the hydrophobic tails of thephospholipids face inward, away from the water. Thehydrophilic heads of the phospholipids are on the outsidewhere they interact with water molecules in the fluidenvironment of the cell. Floating within this bilayer are theproteins, some of which span the entire bilayer and maycontain channels, or pores, to allow passage of moleculesthrough the membrane. The entire membrane is fluid—the lipidmolecules move within the layers of the bilayer while the"floating" proteins also freely move within the bilayer.


The fluid-mosaic model of membrane structure as initially proposed bySinger and Nicolson in 1972.


Left. Image of the upper surface of a lipid bilayer containing phosphatidylcholine (blackbackground), and sphingomyelin molecules, which organize themselves spontaneously into theorange-colored rafts. The yellow peaks represent a GPI-anchored protein, which is almostexclusively raft-associated. This image is provided by an atomic force microscope,which measuresthe height of various parts of the specimen at the molecular level. Right. Schematic model of a lipidraft 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 fattyacids also tend to concentrate in this region. A GPI-anchored protein is localized in the raft. Thelipids in the outer leaflet of the raft have an organizing effect on the lipids of the inner leaflet. As aresult, the inner leaflet raft lipids consist primarily of cholesterol and glycerophospholipids withsaturated fatty acyl tails. The inner leaflet tends to concentrate lipid-anchored proteins, such as srckinase, that are involved in cell signaling.

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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 bothlipids and proteins

Fluidity of membranes is determinedby both temperature and composition.

Temperature:

Left. Above the transition temperature, the lipid molecules and theirhydrophobic tails, although ordered are free to move in certain directions.Right. Below the transition temperature, the movement of the lipidmolecules is greatly restricted and the bilayer takes on properties of acrystalline gel.

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Fluidity of membranes is determined byboth temperature and composition.

Unsaturated vs. saturated fatty acids.Concentration of cholesterol.

The cholesterol molecules (green) inthe lipid bilayer, interfere with thetight packing of the phospholipids,making the bilayer more fluid.

Crooked, unsaturated fatty acidsinterfere with tight packing of thephospholipids, making the bilayermore fluid.

The Cell

1. Compartmentalizedchemical reactions

2. Modify intra- and extra-cellular environment

3. Different properties andfunctions.

Individual cells will direct the function of tissues and organs

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

Reductionism Science

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Approaches to Cell Biology ResearchGenetics

• Screen for mutants with a phenotype.

• Crosses to define complementationgroups.

• Details of the phenotypes. Divide intoclasses.

• Order the classes by epistasis.

• Clone the genes.

Approaches to Cell Biology Research

Anatomy

• Structure of cells and tissues.

• Ultrastructure (EM), to detect finestructures, such as filaments ormembranes.

• Correlate structures with function.

• Identify molecules if possible.

Approaches to Cell Biology ResearchBiochemistry

• Purify molecules, such as metabolites,proteins, or even membranes.

• Study their chemical properties in vitro.

• Attempt to re-create in vitro aphenomenon observed in vivo.

• Reconstitution as an ultimate test for“sufficiency.”

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Physiology

• Observe the phenomena exhibited byliving cells or organisms, such asmovement.

• Quantify parameters such as rate ofmovement and ask how they correlatewith each other factors.

• Decrease or increase the activity of acomponent.


Pharmacology

• Find drugs (chemicals) that inhibit orenhance a phenomenon, such asmovement.

• Identify their molecular targets, such asproteins.

• Use in physiology studies to inhibit aprocess acutely.

Example of How the Techniques Interact

Find a cell that moves, like Dictyostelium.

• Study its movement up a chemotacticgradient, and quantify variousparameters.

• Find drugs that inhibit this movement.

Study the fine structure of the cell, especiallythe areas that seem to be moving.

• Are there small structures, such asfilaments and crosslinkers, and are theyin an arrangement that suggests howmovement can be generated?

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Purify proteins that make up those fine structures,such as filaments.

• Purify proteins that bind to those proteins.

• Look for how the different proteins regulatethe relevant activity (which you have to guessat).

• Determine whether the drugs above affectthis in vitro activity.


Localize the proteins

• Ab staining of cells to show that theproteins really are associated withthose fine structures.

• GFP fusions once cDNA is obtained(later)

Microinject Abs or fragments of proteinslooking for an effect on cell movement(inhibition or enhancement).

Example of How the Techniques InteractReverse genetics.

• Use the protein to clone cDNA’sand/or genes encoding it. Modernequivalent - database search.

• Correlate expression with cellmovement.

• Use the cDNA to inhibit the protein(antisense or knockout)

• Overexpress the protein• Express fragments or mutated versions

of the protein (dominant effects).

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Example of How the Techniques InteractForward genetics.

• Start by making mutants.• Study phenotype and classify.

Information about different steps at whichone can stall the process. Use the physiologyand anatomy to classify.

• Epistasis to order the genes.• Clone and sequence the genes.• Make protein, make Abs and cDNAs, and do

the experiments above.


Reconstitution as an ultimate goal.

• Genetics defines a set ofgenes/proteins important formovement.

• Make and mix together pureproteins to create the movement.

Hypothesis-Driven Experiments

•State the hypothesisNot a “straw man” or trivial

•State the experiment

•Possible outcomes

•Interpretation of each outcome

•Controls - positive, negative

•Limitations and Alternative Interpretations

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“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 thealternatives?

• Obligation to raise and test crediblealternatives

• Or the ones that others find compelling

Revolutions and Paradigms

•Thomas Kuhn, The Structure ofScientific Revolutions, 1962

•Evidence against the current paradigmis 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 afterphotobleaching (FRAP), etc.)

• Kinetics of molecular interactions, enzyme reactions(msec - min)

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Catalyst (enzyme): increases rate of a reactionSubstrate: molecule on which enzyme acts to form product

S ------> P enzyme

Free energy of reactionnot changed by enzyme.For a favored reaction(ΔG negative), enzymeaccelerates reaction.

Graph:ΔG* = activation energyΔG negative overall for forward reaction

Enzymes are catalysts for chemical reactions in cells

Active Site: Region of the enzyme that does the work. Aminoacid 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, facilitatingtransition 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

Enzymes as Catalysts

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

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What Can You Learn from WhatHappens 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-steadystate) kinetic analysis

Need an assay that measures the product of the chemicalreaction. 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 productthat absorbs light (creates color). Measure absorbance.

How to Measure Enzyme Activity atSteady State

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

Color-Producing Substrates for β-galactosidase

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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

One Single Experiment at One Substrate Concentration

•Plot product vs time•Determine rate during initial linear phase

Equilibrium?

Steady-state?

Measure Velocity of Reaction

Run the Assay at DifferentSubstrate Concentrations

Plot initial rate (v0)vs

Concentration ofSubstrate [S]

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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

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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 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)

Competitive Inhibition

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Example of Competitive Inhibition

• EtOH Rx for MeOH poisoning

• Methanol (ingested from solid alcohol, paint strippers,windshield washer fluid, etc.) is metabolized by alcoholdehydrogenase to formaldehyde and formic acid. Leads tometabolic acidosis and optic neuritis (from formate) that cancause blindness.

• Treatment: Infuse EtOH to keep blood concentration at 100-200 mg/dL (legally intoxicated) for long enough to excretethe MeOH.

• EtOH serves as a competitive inhibitor. Ethylene glycolpoisoning is treated in the same way.

Noncompetitive Inhibition

Noncompetitive inhibitor :

• Binds to a site on the enzyme (E or ES) thatinactivates the enzyme

• Decreases total amount of enzyme availablefor catalysis, decreasing Vmax

• Remaining active enzyme molecules areunaffected, so Km is unchanged

Uncompetitive Inhibition

Uncompetitive inhibitor:

• Binds specifically to the [ES] complex (andinactivates it

• Fraction of enzyme inhibited increases as [S]increases

• So both Km and Vmax are affected

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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

• Uncompetitive

• Noncompetitive

No inhibitor

No inhibitor

No inhibitor

Lineweaver-BurkePlots show curveswith no inhibitorvs. presence oftwo differentconcentrations ofinhibitor

Reading and Homework for Kinetics

• Alberts (5th edition) pp. 159-166• Lodish (6th edition) pp. 79-85• See handout or website for homework

Documents

Goals For MCB 5068