biochem handouts part 1 (eamor)

8/6/2019 BioChem Handouts Part 1 (EAmor)

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Introduction

On Life and Chemistry..

Living things are composed of lifelessmolecules (Albert Lehninger)

Chemistry is the logic of biologicalphenomena (Garrett and Grisham)

What on earth is notbiochemistry? (Anonymous)


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Scope of this Review

Proteins

Carbohydrates

Lipids

Nucleic Acids

Metabolism


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Polymer of L-amino acids

Have varied roles most important of which is catalysis and regulation

Polymer of monosaccharides

Main source of energy in most

organisms


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informational molecules of allliving organisms

structural and functional partsof units such as the ribosome

catalytic function (ribozymes)transport and providechemical energy in the form of

phosphate groups

important in cell regulationand signal transduction


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The language of nucleic acids

What makes us human

There are 46chromosomes,arranged in 23 pairs,in each cell in thebody

One chromosome ofeach pair iscontributed by thefather, and the otherby the mother


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Biochemistry for the MED Boards



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Distinctive Properties of Living Systems

Organisms are complicated and highlyorganized

Biological structures serve functionalpurposes

Living systems are actively engaged inenergy transformations

Living systems have a remarkablecapacity for self-replication


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IN008

The sequence of monomeric units in a biological polymer has the potential to contain

information if the diversity and order of the units are not overly simple or repetitive. Nucleic

acids and proteins are information-rich molecules; polysaccharides are not.


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Methods for Separating and Purifying

Biomolecules

Salt fractionation(precipitation of

proteins with

ammonium sulfate)

Chromatography paper, ion-exchange,

affinity, thin-layer,gas-liquid, high

pressure liquid, gel

filtration

Electrophoresis paper, high voltage,

agarose, cellulose

acetate, starch gel,

polyacrylamide gel,

SDS-PAGE

Ultracentrifugation

Methods for Determining Biomolecular

Structures

Elemental analysis UV-VIS, IR, NMR

spectroscopy

Acid/base hydrolysis Enzymatic

degradation

MS Specific sequencing

methods

X-ray crystallography


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Preparations for Studying Biochemical

Processes

Whole animal(transgenic and withgene knockout)

Isolated perfusedorgan

Tissue slice Whole cells Homogenate Isolated cell

organelles

Subfractionation oforganelles

Purified metabolitesand enzymes

Isolated genes (PCRand site-directed

mutagenesis)

Major Causes of Diseases

Physical agents mechanical trauma,extremes of T, suddenchanges inatmospheric P,radiation, electricshock

Chemical agents,including drugs:certain toxiccompounds,therapeutic drugs,etc.

Biologic agents:viruses, bacteria,fungi, higher forms ofparasites

All of the causes listed act by influencing the various biochemical mechanisms in

the cell or in the body.


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Major Causes of Diseases

Oxygen lack: loss ofblood supply,depletion of theoxygen carryingcapacity of the blood,poisoning of theoxidative enzymes

Genetic disorders congenital, molecular

Immunologicreactions anaphylaxis,autoimmune disease

Nutritional imbalances deficiencies,excesses

Endocrine imbalances hormonaldeficiencies,excesses

Amino Acids


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What Are the Structures and Properties of Amino

Acids, the Building Blocks of Proteins?

Amino acids contain a central tetrahedralcarbon atom

There are 20 common amino acids Amino acids can join via peptide bonds Several amino acids occur only rarely in

proteins Some amino acids are not found in

proteins


Anatomy of an amino acid. Except for proline and its derivatives, all of the

amino acids commonly found in proteins possess this type of structure.

Amino AcidsBuilding Blocks of Proteins


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20 Common Amino Acids

You should know names, structures, pKavalues, 3-letter and 1-letter codes

Non-polar amino acids Polar, uncharged amino acids Acidic amino acids Basic amino acids



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The ionic forms of the amino acids, shown without consideration of any ionizations onthe side chain. The cationic form is the low pH form, and the titration of the cationic

species with base yields the zwitterion and finally the anionic form.


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pKa Values of the Amino Acids

Alpha carboxyl group - pKa = 2 Alpha amino group - pKa = 9


Arginine, Arg, R: pKa(guanidinogroup) = 12.5

Aspartic Acid, Asp, D: pKa = 3.9 Cysteine, Cys, C: pKa = 8.3 Glutamic Acid, Glu, E: pKa = 4.3 Histidine, His, H: pKa = 6.0


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Lysine, Lys, K: pKa = 10.5 Serine, Ser, S: pKa = 13 Threonine, Thr, T: pKa = 13 Tyrosine, Tyr, Y: pKa = 10.1


Titration of glycine, a simple

amino acid. The isoelectric

point, pI, the pH where the

molecule has a net charge of

0, is defined as (pK1+ pK2)/2.


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Titration of glutamic acid.


Titration of lysine.


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A Sample Calculation

What is the pH of a glutamic acid solutionif the alpha carboxyl is 1/4 dissociated?

pH = 2 + log10 [1][3]

pH = 2 + (-0.477)pH = 1.523


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Reactions of Amino Acids

Carboxyl groups form amides & esters Amino groups form Schiff bases and

amides

Side chains show unique reactivitiesCys residues can form disulfides and

can be easily alkylatedFew reactions are specific to a single

kind of side chain



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The pathway of theninhydrin reaction,

which produces acolored product

called Ruhemanns

Purple that absorbslight at 570 nm. Note

that the reactioninvolves and

consumes twomolecules ofninhydrin.


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Stereochemistry of Amino Acids

All but glycine are chiral L-amino acids predominate in nature D,L-nomenclature is based on D- and L-

glyceraldehyde

R,S-nomenclature system is superior,since amino acids like isoleucine and

threonine (with two chiral centers) can be

named unambiguously


The configuration of thecommon L-amino acids

can be related to theconfiguration of L(-)-

glyceraldehyde as shown.These drawings areknown as Fischer

projections. The horizontallines of the Fischer

projections are meant toindicate bonds coming outof the page from the

central carbon, andvertical lines represent

bonds extending behindthe page from the centralcarbon atom.


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Enantiomeric molecules basedon a chiral carbon atom.

Enantiomers arenonsuperimposable mirror

images of each other.

Spectroscopic Properties

All amino acids absorb at infraredwavelengths

Only Phe, Tyr, and Trp absorb UV Absorbance at 280 nm is a good

diagnostic device for amino acids NMR spectra are characteristic of each

residue in a protein, and high resolutionNMR measurements can be used toelucidate three-dimensional structures ofproteins


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Cation (a) and anion (b)exchange resins commonly

used for biochemicalseparations.



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Proteins


Anatomy of an amino acid. Except for proline and its derivatives, all of the

amino acids commonly found in proteins possess this type of structure.

What Is the Fundamental Structural Pattern inProteins?


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The Peptide Bond

is usually found in the trans configurationhas partial (40%) double bond characteris about 0.133 nm long - shorter than a typicalsingle bond but longer than a double bond

Due to the double bond character, the six atomsof the peptide bond group are always planar!

N partially positive; O partially negative


Anatomy of an

amino acid.

Except for proline

and itsderivatives, all of

the amino acids

commonly found

in proteins

possess this type

of structure.

The Coplanar Nature of the Peptide Bond

Six atoms of the peptide group lie in a plane!


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The amide or peptide bond planes arejoined by the tetrahedral bonds of the

-carbon. The rotation parameters are and . The conformation shown

corresponds to = 180 and = 180.Note that positive values ofand correspond to clockwise rotation as

viewed from C. Starting from 0, arotation of 180 in the clockwisedirection (+180) is equivalent to arotation of 180 in the counterclockwisedirection (-180). (Illustration: Irving Geis.Rights owned by Howard Hughes MedicalInstitute. Not to be reproduced without

permission.)

The angles phi

and psi are

shown here


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Peptides

Short polymers of amino acids Each unit is called a residue 2 residues - dipeptide 3 residues - tripeptide 12-20 residues - oligopeptide many - polypeptide

Protein

One or more polypeptide chains

One polypeptide chain - a monomeric protein

More than one - multimeric protein Homomultimer - one kind of chain Heteromultimer - two or more different chains Hemoglobin, for example, is a heterotetramer It has two alpha chains and two beta chains


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What Architectural Arrangements

Characterize Protein Structure?

Proteins are classed according to shape andand solubility

Shape - globular or fibrous The four levels of protein structure

- Primary - sequence

- Secondary - local structures - H-bonds

- Tertiary - overall 3-dimensional shape

- Quaternary - subunit organization


(a) Proteins having structural roles in cells are typically fibrous and often water insoluble.

Collagen is a good example. Collagen is composed of three polypeptide chains that intertwine.

(b) Soluble proteins serving metabolic functions can be characterized as compactly folded

globular molecules, such as myoglobin. The folding pattern puts hydrophilic amino acid side

chains on the outside and buries hydrophobic side chains in the interior, making the protein

highly water soluble. (c) Membrane proteins fold so that hydrophobic amino acid side chains

are exposed in their membrane-associated regions. The portions of membrane proteins

extending into or exposed at the aqueous environments are hydrophilic in character, like

soluble proteins. Bacteriorhodopsin is a typical membrane protein; it binds the light-absorbing

pigment, cis-retinal, shown here in red.(a, b, Illustration: Irving Geis. Rights owned by Howard HughesMedical Institute. Not to be reproduced without permission.)


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Bovine pancreatic

ribonuclease A

contains 124 amino

acid residues, none of

which are tryptophan.

Four intrachain

disulfide bridges (S-

S) form crosslinks in

this polypeptide

between Cyc26 and

Cys84, Cys40 and

Cys95, Cys58 andCys110, and Cys65 and

Cys72. These

disulfides are

depicted by yellow

bars.

Classes of Secondary Structure

All these are local structures that arestabilized by hydrogen bonds

Alpha helix Other helices Beta sheet (composed of "beta strands") Tight turns (aka beta turns or beta bends) Beta bulge


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The arrangement of hydrogenbonds in (a) parallel and (b)

antiparallel -pleated sheets.

Biochemistry for the MED BoardsChemistry 40 (Summer 2007)


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The structures of two kinds of-turns (also called tight turns or-bends) (Irving Geis)


Three different kinds of-bulge structures involving a pair of adjacent polypeptidechains. (Adapted from Richardson, J. S., 1981. Advances in Protein Chemistry 34:167339.)


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Supersecondary Structures

1. 2. 3. -meander4. Greek key

Domain

Distinct compactunits within a protein

consisting of various

elements of

secondary structure

25 to 300 residues Combinations of

secondary

structures that form

the core of a domain


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Tertiary structure Disulfide bonds are

not generally foundin intracellularproteins but aresometimes found inextracellularprotein

May also holdtogether differentsubunits (i.e.,contribute toquaternarystructure)

Insulin A & B chains

Protein folding

dictated by primarystructure

Multiple intermediatesteps

Important drivingforces: Hydrophobic effect Hydrogen bonding Van der Waals Charge-charge


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Molecular chaperones

Increase the rate ofcorrect folding of

nascent polypeptide

chains

Aid in the assembly ofmultisubunit proteins

Protect proteins fromstress-induced damage

(eg. Heat shock)

Chaperonin

Molecular chaperones assist protein

folding


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Anfinsen experiment: Spontaneousrenaturation of Ribonuclease A

Primarystructurecontainssufficientinformationto allow

formation ofsecondaryand tertiarystructures

Predictive Algorithms

If the sequence holds the secrets of folding, can wefigure it out?

Many protein chemists have tried to predictstructure based on sequence

Chou-Fasman: each amino acid is assigned a"propensity" for forming helices or sheets

Chou-Fasman: is only modestly successfuland doesn't predict how sheets and helicesarrange


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Relative frequencies of occurrence of amino acid residues in a-helices, b-sheets, and b-turns in proteins of known structure. (Adapted from Bell , J. E., and Bell , E. T., 1988, Proteinsand Enzymes, Englewood Cliffs, NJ: Prentice-Hall.)



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Prion Misfolding Diseases

Prion- proteinaceous infectious particles(spongiform encelopathies)

Mad Cow Disease (cow) Creutzfeldt-Jacob disease (human) Scrapie (sheep)

Quaternary structure

Quaternary structure refers to theorganization and arrangement of subunits in

a protein with multiple subunits


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Quaternary structure

Can have more thantwo subunits

Subunits areindividual

polypeptides

Pyruvate dehydrogenase complex:60 subunits!

Levels of protein structure

Primary

Secondary

Tertiary Quaternary


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How Are Proteins Isolated and Purified fromCells?

The thousands of proteins in cells can beseparated and purified on the basis of sizeand electrical charge

Proteins tend to be least soluble at theirisoelectric point

Increasing ionic strength at first increasesthe solubility of proteins (salting-in), thendecreases it (salting-out)

1. If more than one polypeptide chain,separate.

2. Cleave (reduce) disulfide bridges 3. Determine composition of each chain 4. Determine N- and C-terminal

residues

Determining the SequenceAn Eight Step Strategy


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5. Cleave each chain into smallerfragments and determine the

sequence of each chain

6. Repeat step 5, using a differentcleavage procedure to generate a

different set of fragments.

Determining the SequenceAn Eight Step Strategy

Determining the Sequence

An Eight Step Strategy

7. Reconstruct the sequence of theprotein from the sequences of

overlapping fragments

8. Determine the positions of thedisulfide crosslinks


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Step 1:

Separation of chains

Subunit interactions depend on weakforces

Separation is achieved with:- extreme pH

- 8M urea

- 6M guanidine HCl

- high salt concentration (usuallyammonium sulfate)

Step 2:

Cleavage of Disulfide bridges

Performic acid oxidation Sulfhydryl reducing agents

- mercaptoethanol- dithiothreitol or dithioerythritol

- to prevent recombination, follow withan alkylating agent like iodoacetate


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Step 3A:

Identify N- and C-terminal residues

N-terminal analysis:Edman's reagentphenylisothiocyanatederivatives are phenylthiohydantoinsor PTH derivatives

Step 3B: :

Identify N- and C-terminal residues

C-terminal analysis

Enzymatic analysis (carboxypeptidase)Carboxypeptidase A cleaves any residueexcept Pro, Arg, and Lys

Carboxypeptidase B (hog pancreas) onlyworks on Arg and Lys


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Steps 4 and 5:

Fragmentation of the chains

Enzymatic fragmentationtrypsin, chymotrypsin, clostripain,

staphylococcal protease Chemical fragmentation

cyanogen bromide

Enzymatic Fragmentation

Trypsin - cleavage on the C-side of Lys, Arg Chymotrypsin - C-side of Phe, Tyr, Trp Clostripain - like trypsin, but attacks Arg

more than Lys Staphylococcal protease

C-side of Glu, Asp in phosphate bufferspecific for Glu in acetate or bicarbonate

buffer


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Reconstructing the Sequence

Compare cleavage by trypsin and staphylococcalprotease on a typical peptide:

Trypsin cleavage:A-E-F-S-G-I-T-P-K L-V-G-K

Staphylococcal protease:F-S-G-I-T-P-K L-V-G-K-A-E

Reconstructing the Sequence

L-V-G-K A-E-F-S-G-I-T-P-K

L-V-G-K-A-E F-S-G-I-T-P-K

Correct sequence:L-V-G-K-A-E-F-S-G-I-T-P-K


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Amino Acid Sequence Can Be Determined

by Mass Spectrometry

Mass spectrometry separates particles onthe basis of mass-to-charge ratio

Fragments of proteins can be generated invarious ways

MS can also separate these fragments

Do Proteins Have Chemical Groups Other

Than Amino Acids?

Proteins may be "conjugated" with otherchemical groups

If the non-amino acid part of the protein isimportant to its function, it is called aprosthetic group.

Be familiar with the terms: glycoprotein,lipoprotein, nucleoprotein, phosphoprotein,metalloprotein, hemoprotein, flavoprotein.


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What Are the Many Biological Functions of

Proteins?

Many proteins are enzymes Regulatory proteins control metabolism and

gene expression

Many DNA-binding proteins are gene-regulatoryproteins

Transport proteins carry substances from oneplace to another

Storage proteins serve as reservoirs of aminoacids or other nutrients

What Are the Many Biological Functions of

Proteins?

Movement is accomplished by contractileand motile proteins

Many proteins serve a structural role Proteins of signaling pathways include

scaffold proteins (adapter proteins)

Other proteins have protective andexploitive functions

A few proteins have exotic functions


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Death of a protein

In a typical day, a person who is in nitrogen

balance will consume 100 grams of protein, break down

400 grams of bodily protein, resynthesize 400 grams ofprotein, and excrete/catabolize100 grams. Individual

proteins exhibit tremendous variability in their metabolic

lifetimes, from a few minutes to a few months.

Proteins in extracellular environments,

such as digestive enzymes,

polypeptide hormones, and antibodies,

turn over quite rapidly, but proteins

with predominantly structural roles,

such as collagen of connective tissue,

are much more stable.

Protein Turnover


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Protein Turnover

Ubiquitination

Ubiquitin is a 76-amino acid residue

heat-stable protein found in all

eukaryotic cells. An ATP-dependent

reaction with proteins links ubiquitin'sC-terminal glycine to lysine amino

groups in the target protein.

PEST sequences

Virtually all short-lived proteins (i.e.,

half-lives less than 2 hours) contain

one or more regions rich in proline,

glutamate, serine, and threonine.

Insertion of these sequences into

long-lived proteins increases

their metabolic lability.

Protein Turnover


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N-terminal amino acid residue

An N-terminal protein residue

of Phe, Leu, Tyr, Trp, Lys, orArg is correlated with short

metabolic lifetimes.

Protein Turnover

Enzymes


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What Characteristic Features Define

Enzymes?

Enzymes endow cells with the remarkablecapacity to exert kinetic control over

thermodynamic potentiality

Enzymes are the agents of metabolicfunction

Catalytic power, specificity, regulation


Reaction profile showing large DG for glucose oxidation, free energy change of -2,870 kJ/

mol; catalysts lower DG, thereby accelerating rate.


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Enzyme Classification



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Vitamins

organic compounds essential in the diet insmall amounts

have little or no caloric value chemical composition is varied normally classified according to their

polarity

Classification of Vitamins

Fat-soluble vitamins(nonpolar)

Vitamin A

Vitamin D

Vitamin E

Vitamin K

Water-soluble vitamins(polar)

Vitamin C

Vitamin B Complex


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Fat-Soluble vitamins: A, D, E, K

Soluble in fatty tissues Stored in the body for long periods of time Not easily excreted Can be overconsumed (overdose)

Water-Soluble Vitamins:

C and B Complex

Soluble in water Excreted in the urine and pose little threat

of overdose

However, they must be consumed insufficient amounts on a daily basis


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Nutritional Minerals

elements,other than C, H, N, and O, neededfor good health.

many are present as ions rather than asneutral atoms

Major minerals (~4% of the bodys weight)Ca, P, Mg, Na, K, Cl, and S

Minor mineralsFe, Cu, Zn, I, Se, Mn, F, Cr, and Mo

Can the Rate of an Enzyme-Catalyzed

Reaction Be Defined in a Mathematical Way?

Enzymes can accelerate reactions as muchas 1016 over uncatalyzed rates!

Urease is a good example:Catalyzed rate: 3x104/secUncatalyzed rate: 3x10 -10/secRatio is 1x1014 !


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The pH activity profiles of four different enzymes. Trypsin, an intestinal protease, has slightlyalkaline pH optimum, whereaspepsin, a gastric protease, acts in the acidic confines of thestomach and has a pH optimum near 2. Papain, a protease found in papaya, is relativelyinsensitive to pHs between 4 and 8. Cholinesterase activity is pH sensitive below pH 7 but notbetween pH 7 and 10. The cholinesterase pH activity profile suggests that an ionizable group

with pK' near 6 is essential to its activity. Might it be a histidine residue within the active site?


The effect of temperature

on enzyme activity. The

relative activity of an

enzymatic reaction as a

function of temperature.

The decrease in the activity

above 50C is due to

thermal denaturation.


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Specificity

Enzymes selectively recognize propersubstrates over other molecules

Enzymes produce products in very highyields - often much greater than 95%

Specificity is controlled by structure - theunique fit of substrate with enzyme controlsthe selectivity for substrate and the productyield

How Can Enzymes Be So Specific?

Lock and key was the first explanation forspecificity

Induced fit provides a more accuratedescription

Induced fit favors formation of the transition-state intermediate


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Enzyme-Linked Immunosorbent Assay (ELISA)


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Several terms to remember

rate or velocity rate constant rate law order of a reaction molecularity of a reaction

The Michaelis-Menten Equation

Louis Michaelis and Maud Menten's theory It assumes the formation of an enzyme-

substrate complex

It assumes that the ES complex is in rapidequilibrium with free enzyme Breakdown of ES to form products is assumedto be slower than 1) formation of ES and 2)breakdown of ES to re-form E and S


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Understanding Km

The "kinetic activator constant" Km is a constant Km is a constant derived from rate

constants

Km is, under true Michaelis-Mentenconditions, an estimate of thedissociation constant of E from S

Small Km means tight binding; high Kmmeans weak binding


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Understanding Vmax

The theoretical maximal velocity Vmax is a constant Vmax is the theoretical maximal rate of the

reaction - but it is NEVER achieved inreality

To reach Vmax

would require that ALLenzyme molecules are tightly bound withsubstrate

Vmax is asymptotically approached assubstrate is increased

The dual nature of the Michaelis-Menten

equation

Combination of 0-order and 1st-order kinetics

When S is low, the equation for rate is 1storder in S

When S is high, the equation for rate is 0-order in S

The Michaelis-Menten equation describes arectangular hyperbolic dependence of v on S!


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The turnover number

A measure of catalytic activity

kcat, the turnover number, is the number ofsubstrate molecules converted to product per

enzyme molecule per unit of time, when E is

saturated with substrate. If the M-M model fits, k2 = kcat = Vmax/Et Values of kcat range from less than 1/sec to many

millions per sec


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The catalytic efficiency

Name for kcat/KmAn estimate of "how perfect" the enzyme is

kcat/Km is an apparent second-order rateconstant

It measures how the enzyme performswhen S is low

The upper limit for kcat/Km is the diffusionlimit - the rate at which E and S diffusetogether

Enzyme Units

IU (International Unit) amount of enzymethat catalyze the formation of 1 micromole

of product in 1 minute

Katal amount of enzyme that converts 1mole of substrate to product in 1 second

Specific activity enzyme unit per mg ofprotein


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Linear Plots of the Michaelis-Menten

Equation

Lineweaver-Burk Eadie-Hofstee Hanes-WoolfHanes-Woolf is best - why?

Smaller and more consistent errorsacross the plot


The Lineweaver-Burk double-reciprocal plot, depicting extrapolations that allow the

determination of thex- and y-intercepts and slope.


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Biochemistry for the MED BoardsA Hanes-Woolf plot of [S]/vversus [S], another straight-line rearrangement of the Michalelis-Menten equation.

Eadie Hofstee Linear Plot


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What Can Be Learned from the Inhibition of

Enzyme Activity?

Enzymes may be inhibited reversibly orirreversibly

Reversible inhibitors may bind at the activesite or at some other site

Enzymes may also be inhibited in anirreversible mannerPenicillin is an irreversible suicide inhibitor


Lineweaver-Burk plot of competitive inhibition, showing lines for no I, [I], and 2[I]. Note that

when [S] is infinitely large (1/[S] = 0), Vmax is the same, whether I is present of not. In the

presence of I, the negative


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Structures of succinate, the substrate of succinate dehydrogenase (SDH), and malonate,

the competitive inhibitor. Fumarate (the product of SDH action on succinate) is also shown.


Lineweaver-Burk plot of pure noncompetitive inhibition. Note that I does not alter Km but

that it decreases Vmax. In the presence of I, the y-intercept is equal to (1/Vmax)(1 + I/KI).


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Lineweaver-Burk plot of pure

uncompetitive inhibition. Note

that I decreases Km and Vmax. In

the presence of I, the y-interceptis equal to (1/Vmax)(1 + I/KI).


Penicillin is an irreversible inhibitor of

the enzyme glycoprotein peptidase,which catalyzes an essential step in

bacterial cell wall synthesis. Penicillin

consists of a thiazolidine ring fused to a

b-lactam ring to which a variable group

R is attached. A reactive peptide bond

in the b-lactam ring covalently attaches

to a serine residue in the active site of

the glycopeptide transpeptidase. (The

conformation of penicillin around its

reactive peptide bond resembles thetransition state of the normal

glycoprotein peptidase substrate.) The

penicilloyl-enzyme complex is

catalytically inactive. The bond

between the enzyme and penicillin is

indefinitely stable; that is, penicillin

binding is irreversible.


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Are All Enzymes Proteins?

Relatively new discoveries Ribozymes - segments of RNA that display

enzyme activity in the absence of protein

Examples: RNase P and peptidyl transferase Abzymes - antibodies raised to bind the

transition state of a reaction of interest

What Are the Mechanisms of Enzyme-

Induced Rated Accelerations?

Mechanisms of catalysis:

Entropy loss in ES formationDestabilization of ESCovalent catalysisGeneral acid/base catalysisMetal ion catalysisProximity and orientation


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What Factors Influence Enzymatic

Activity?

Six points: Rate slows as product accumulates Rate depends on substrate availability Genetic controls - induction and repression Enzymes can be modified covalently Allosteric effectors may be important Zymogens, isozymes and modulator

proteins may play a role


Enzymes regulated by covalent modification are called interconvertible enzymes. The

enzymes (protein kinase andprotein phosphatase, in the example shown here) catalyzingthe conversion of the interconvertible enzyme between its two forms are called converter

enzymes. In this example, the free enzyme form is catalytically active, whereas the

phosphoryl-enzyme form represents an inactive state. The -OH on the interconvertible

enzyme represents an -OH group on a specific amino acid side chain in the protein (for

example, a particular Ser residue) capable of accepting the phosphoryl group.


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Proinsulin is an 86-

residue precursor to

insulin (the sequence

shown here is human

proinsulin). Proteolytic

removal of residues 31

to 65 yields insulin.

Residues 1 through 30

(the B chain) remain

linked to residues 66

through 87 (the A chain)

by a pair of interchain

disulfide bridges.

Biochemistry for the MED BoardsThe proteolytic activation of chymotrypsinogen.


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The isozymes of lactate dehydrogenase (LDH). Active muscle tissue becomes anaerobic

and produces pyruvate from glucose via glycolysis. It needs LDH to regenerate NAD + from

NADH so glycolysis can continue. The lactate produced is released into the blood. The

muscle LDH isozyme (A4) works best in the NAD+-regenerating direction. Heart tissue is

aerobic and uses lactate as a fuel, converting it to pyruvate via LDH and using the pyruvate

to fuel the citric acid cycle to obtain energy. The heart LDH isozyme (B4) is inhibited by

excess pyruvate so the fuel wont be wasted.


Cyclic AMP- dependent protein kinase (also known as PKA) is a 150- to 170-kD R2C2tetramer in mammalian cells. The two R (regulatory) subunits bind cAMP (KD = 3 x 10

-8

M); cAMP binding releases the R subunits from the C (catalytic) subunits. C subunits areenzymatically active as monomers.


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Carbohydrates



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D-Fructose and L-fructose, an enantiomeric pair. Note that changing the configurationonly at C5 would change D-fructose to L-sorbose.


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the sweetest of all sugars (more than 50%sweeter than table sugar)

Which structure is a Haworth projection ofI?

Which structure is a carbon-2 (position 2) epimer ofI?

Which structure(s) has/have the "beta" configuration at the anomeric carbon?

Which structure(s) is/are a ketose?

Which structure is a pentose?


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D-Glucose can cyclize in two ways, forming either furanose or pyranose structures.


(a) Chair and boat conformations of a pyranose sugar. (b) Two possible chairconformations of-D-glucose.


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Monosaccharide Derivatives

Reducing sugars: sugars with free anomeric

carbons - they will reduce oxidizing agents, such

as peroxide, ferricyanide and some metals (Cu

and Ag)

These redox reactions convert the sugar to a

sugar acidGlucose is a reducing sugar - so these reactions

are the basis for diagnostic tests for blood sugar



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Structures of some sugar alcohols.

Biochemistry for the MED BoardsSeveral deoxy sugars and ouabain, which contains -L-rhamnose (Rha). Hydrogenatoms highlighted in red are deoxy positions.


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Several sugar esters important in metabolism.


Structures of D-glucosamine and D-galactosamine.


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Structures of muramicacid and neuraminic

acid and severaldepictions of sialic acid.


Acetals and ketals can be formed from hemiacetals and hemiketals, respectively.


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What is the Structure and Chemistry ofOligosaccharides?

Be able to identify anomeric carbons andreducing and nonreducing ends

Sucrose is NOT a reducing sugar Note carefully the nomenclature of links.Be able to recognize alpha(1,4), beta

(1,4), etc

Soy milk is good substitute forlactose intolerance


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The structures of several important disaccharides. Notethat the notation -HOH means that the configuration can

be either or. If the -OH group is above the ring, the

configuration is termed . The configuration is if the -

OH group is below the ring as shown. Also note thatsucrose has no free anomeric carbon atoms.


The structures of some interesting oligosaccharides.


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What is the Structure and Chemistry of

Polysaccharides?

Functions: storage, structure, recognition

Nomenclature: homopolysaccharide vs.heteropolysaccharide

Starch and glycogen are storage molecules Chitin and cellulose are structural molecules Cell surface polysaccharides are recognition

molecules

Starch

A plant storage polysaccharide

Two forms: amylose and amylopectin Most starch is 10-30% amylose and 70-90%

amylopectin

Branches in amylopectin every 12-30 residues Amylose has alpha(1,4) links, one reducing

end


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Amylose and amylopectin are the two forms of starch. Note that the linear linkages are

(1 4), but the branches in amylopectin are (1 6). Branches in polysaccharides caninvolve any of the hydroxyl groups on the monosaccharide components. Amylopectin is ahighly branched structure, with branches occurring every 12 to 30 residues.

Starch

A plant storage polysaccharide

Amylose is poorly soluble in water, but formsmicellar suspensions

In these suspensions, amylose is helicaliodine fits into the helices to produce a blue

color


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Glycogen

The glucose storage device in animals

Glycogen constitutes up to 10% of liver massand 1-2% of muscle mass

Glycogen is stored energy for the organism Only difference from starch: number of

branches Alpha(1,6) branches every 8-12 residues Like amylopectin, glycogen gives a red-violet

color with iodine

Structural Polysaccharides

Composition similar to storage polysaccharides,but small structural differences greatlyinfluence properties

Cellulose is the most abundant naturalpolymer on earth Cellulose is the principal strength and support

of trees and plants

Cellulose can also be soft and fuzzy - in cotton


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(a) Amylose, composed exclusively of the relatively bent (14) linkages, prefers toadopt a helical conformation, whereas (b) cellulose, with (14)-glycosidic linkages, can

adopt a fully extended conformation with alternating 180 flips of the glucose units. Thehydrogen bonding inherent in such extended structures is responsible for the great

strength of tree trunks and other cellulose-based materials.

Structural Polysaccharides

Composition similar to storage polysaccharides,but small structural differences greatlyinfluence properties

Beta(1,4) linkages make all the difference! Strands of cellulose form extended ribbons


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The structure ofcellulose, showing

the hydrogenbonds (blue)

between thesheets, whichstrengthen the

structure.Intrachain

hydrogen bonds

are in red andinterchain

hydrogen bondsare in green.

Other Structural Polysaccharides

Chitin - exoskeletons of crustaceans, insectsand spiders, and cell walls of fungi

similar to cellulose, but C-2s are N-acetylcellulose strands are parallel, chitins can be

parallel or antiparallel


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Like cellulose, chitin,mannan, and poly(D-

mannuronate) formextended ribbons and

pack together efficiently,taking advantage ofmultiple hydrogen

bonds.


Glycosaminoglycans areformed from repeating

disaccharide arrays.Glycosaminoglycans are

components of theproteoglycans.


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Characteristics of GAGsGAG Localization Comments

Hyaluronate

synovial fluid, vitreous

humor,ECM of loose connective

tissue

large polymers, shock

absorbing

Chondroitin sulfate cartilage, bone, heart valves most abundant GAG

Heparan sulfatebasement membranes,

components of cell surfaces

contains higher acetylated

glucosamine than heparin

Heparin

component of intracellular

granules of mast cellslining the arteries of the

lungs, liver and skin

more sulfated than heparan

sulfates

Dermatan sulfateskin, blood vessels, heart

valves

Keratan sulfate

cornea, bone,

cartilage aggregated withchondroitin sulfates

What Are Glycoproteins, and How Do They

Function in Cells?

Many structures and functions!

May be N-linked or O-linked N-linked saccharides are attached via the

amide nitrogens of asparagine residues O-linked saccharides are attached to

hydroxyl groups of serine, threonine orhydroxylysine


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The carbohydrate moieties ofglycoproteins may be linked to the

protein via (a) serine or threonine

residues (in the O-linked

saccharides) or(b) asparagine

residues (in the N-linked

saccharides). (c) N-Linked

glycoproteins are of three types:

high mannose, complex, and

hybrid, the latter of which

combines structures found in the

high mannose and complex

saccharides.

Blood Group Antigens


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Blood Group Antigens

Lipids


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Classes of Lipids

All biological lipids are amphipathic

Fatty acids Triacylglycerols Glycerophospholipids Sphingolipids Waxes Isoprene-based lipids (including steroids)



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The structures of some typical fatty acids. Note that most natural fatty acids contain aneven number of carbon atoms and that the double bonds are nearly always cis and

rarely conjugated.

Triacylglycerols

Also called triglycerides

A major energy source for many organisms Why?

Most reduced form of carbon in natureNo solvation neededEfficient packing


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Triacylglycerols are formed from glycerol and fatty acids.

Triacylglycerols - II

Other advantages accrue to users oftriacylglycerols

InsulationEnergy without nitrogenMetabolic water


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Glycerophospholipids

Glycerophospholipids are phospholipidsbut not necessarily vice versa


Phosphatidic acid, the parent compound for glycerophospholipids.


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Structures of several

glycerophospholipids andspace-filling models of

phosphatidylcholine,phosphatidylglycerol, andphosphatidylinositol.

Ether Glycerophospholipids

An ether instead of an acyl group at C-1

Plasmalogens are ether glycerophospholipids inwhich the alkyl chain is unsaturated


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A 1-alkyl 2-acyl-phosphatidylethanolamine (an ether glycerophospholipid).

Ether Glycerophospholipids

Platelet activating factor (PAF) is an etherglycerophospholipid

PAF is a potent biochemical signal moleculeNote the short (acetate) fatty acyl chain at theC-2 position in PAF


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The structure of1-alkyl 2-acetyl-phosphatidylcholine, also known as platelet activatingfactor or PAF.


The structure and a space-filling model of a choline

plasmalogen.


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Sphingolipids

Base structure is sphingosine

Sphingosine is an 18-carbon amino alcoholCeramides are amide linkages of fatty acids tothe nitrogen of sphingosine

Glycosphingolipids are ceramides with one ormore sugars in beta-glycosidic linkage at the 1-hydroxyl group

Biochemistry for the MED BoardsFormation of an amide linkage between a fatty acid and sphingosine produces aceramide.


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Sphingolipids

Glycosphingolipids with one sugar arecerebrosides

Gangliosides - ceramides with 3 or moresugars, one of which is a sialic acid


A structure and a space-

filling model of a cholinesphingomyelin formed from

stearic acid.


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The structure of a

cerebroside. Note thesphingosine backbone.

Biochemistry for the MED BoardsThe structures of several important gangliosides. Also shown is a space-filling model ofganglioside GM1.


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Waxes

Esters of long-chain alcohols with long-chainfatty acids

Highly insoluble Animal skin and fur are wax-coated Leaves of many plants Bird feathers


Figure 8.15 An exampleof a wax. Oleoyl alcohol

is esterified to stearicacid in this case.


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Terpenes

Based on the isoprene structure

Know nomenclature Understand linkage modes All sterols (including cholesterol) areterpene-based molecules Steroid hormones are terpene-based


The structure of isoprene (2-methyl-1,3-butadiene) and the structure of head-to-tail andtail-to-tail linkages. Isoprene itself can be formed by distillation of natural rubber, a linear

head-to-tail polymer of isoprene units.


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Many monoterpenes are readily recognized by their characteristic flavors or odors(limonene in lemons; citronellal in roses, geraniums, and some perfumes; pinene in

turpentine; and menthol from peppermint, used in cough drops and nasal inhalers). Thediterpenes, which are C20 terpenes, include retinal (the essential light-absorbing pigment in

rhodopsin, the photoreceptor protein of the eye), phytol (a constituent of chlorophyll), andthe gibberellins (potent plant hormones). The triterpene lanosterol is a constituent of woolfat. Lycopene is a carotenoid found in ripe fruit, especially tomatoes.


Dolichol phosphate is an initiation point for the synthesis of carbohydrate polymers in animals. The

analogous alcohol in bacterial systems, undecaprenol, also known as bactoprenol, consists of 11isoprene units. Undecaprenyl phosphate delivers sugars from the cytoplasm for the synthesis of cell

wall components such as peptidoglycans, lipopolysaccharides, and glycoproteins. Polyprenyl

compounds also serve as the side chains of vitamin K, the ubiquinones, plastoquinones, and

tocopherols (such as vitamin E).


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Steroids

Based on a core structure consisting of three6-membered rings and one 5-membered ring,all fused together

Cholesterol is the most common steroid inanimals and precursor for all other steroids inanimals

Steroid hormones serve many functions inanimals - including salt balance, metabolicfunction and sexual function


The structure of cholesterol, shown with steroid ring designations and carbon numbering.


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The structures of several important sterols derived from cholesterol.



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The Macronutrients and the Energy

They Provide to the Body

Fats and Oils Carbohydrates

1 Cal = 1000 cal

Membranes and Membrane

Transport


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What Are the Chemical and PhysicalProperties of Membranes?

Structures with many cell functions

Barrier to toxic molecules Help accumulate nutrients Carry out energy transductionFacilitate cell motion Assist in reproduction

Modulate signal transduction Mediate cell-cell interactions


Several spontaneously formed lipid structures.


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Lipids Form Ordered Structures

Spontaneously in Water

Hydrophobic interactions all!

Lipid bilayers can form in several waysunilamellar vesicles (liposomes)multilamellar vesicles (Alex Bangham)


Drawings of(a) a bilayer, (b) a unilamellar

vesicle, (c) a multilamellar vesicle, and (d) an

electron micrograph of a multilamellar Golgi

structure (X94,000).


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The Fluid Mosaic Model Describes MembraneDynamics

S. J. Singer and G. L. Nicolson

The phospholipid bilayer is a fluid matrix The bilayer is a two-dimensional solvent Lipids and proteins can undergo rotational and

lateral movement Two classes of proteins:

peripheral proteins (extrinsic proteins)integral proteins (intrinsic proteins)


The fluid mosaic model of membrane structure proposed by S. J. Singer and G. L. Nicolson. In

this model, the lipids and proteins are assumed to be mobile, so that they can move rapidly

and laterally in the plane of the membrane. Transverse motion may also occur, but it is much

slower.


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Phospholipids are arranged asymmetrically in most membranes, including the human

erythrocyte membrane, as shown here. Values are mole percentages. (After Rothman andLenard, 1977. Science194:1744.)


Phospholipids can be flipped across a bilayer membrane by the action of flippase proteins.

When, by normal diffusion through the bilayer, the lipid encounters a flippase, it can be moved

quickly to the other face of the bilayer.


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Membranes Undergo PhaseTransitionsThe "melting" of membrane lipids

Below a certain transition temperature,membrane lipids are rigid and tightly packed

Above the transition temperature, lipids aremore flexible and mobile

The transition temperature is characteristic ofthe lipids in the membraneOnly pure lipid systems give sharp, well-definedtransition temperatures


An illustration of the gel-to-liquid crystalline phase transition, which occurs when a membrane

is warmed through the transition temperature, Tm. Notice that the surface area must increaseand the thickness must decrease as the membrane goes through a phase transition. The

mobility of the lipid chains increases dramatically.


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What is Passive Diffusion?

No special proteins needed Transported species simply moves down its

concentration gradient - from high [c] to low[c]


Passive diffusion of an

uncharged species

across a membrane

depends only on the

concentrations (C1

and C2) on the two

sides of themembrane.


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The passive diffusion of

a charged species

across a membrane

depends upon the

concentration and also

on the charge of the

particle, Z, and the

electrical potential

difference across the

membrane, Dy.

How Does Facilitated Diffusion Occur?

G negative, but proteins assist Solutes only move in the

thermodynamically favored direction

But proteins may "facilitate" transport,increasing the rates of transport

Two important distinguishing features:solute flows only in the favored directiontransport displays saturation kinetics


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Passive diffusion and facilitated diffusion

may be distinguished graphically. The plots

for facilitated diffusion are similar to plots of

enzyme-catalyzed processes (Chapter 13)

and they display saturation behavior.

How Does Energy Input Drive Active

Transport Processes?

Energy input drives transport

Some transport must occur such that solutesflow against thermodynamic potential

Energy input drives transport Energy source and transport machinery are

"coupled"

Energy source may be ATP, light or aconcentration gradient


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The Sodium Pump

aka Na,K-ATPase

Large protein - 120 kD and 35 kD subunits Maintains intracellular Na low and K high Crucial for all organs, but especially for

neural tissue and the brain

ATP hydrolysis drives Na out and K in Alpha subunit has ten transmembrane

helices with large cytoplasmic domain

biochem handouts part 1 (eamor)

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