Biochemistry Section of Bio 41 (Fall 2007, Bob Simoni) 1. Lecture material most important 2. Reading...

Biochemistry Section of Bio 41(Fall 2007, Bob Simoni)

1. Lecture material most important

2. Reading in Berg, supplemental assignments for the 6th edition.

3. Problem sets, old exams as study guides. assume open book for homework

4. Don’t memorize structures

5. Don’t memorize equations

"Macromolecules" 1-1

• Proteins: most diverse, complex, responsible forall cell functions

• Lipids: Structural, cell membranes, energymetabolism

• Carbohydrates: Structure, energy metabolism

• Nucleic Acids: Genetic material

(Polymers of amino acids)

amino acid

(1-20)

found in proteins not found in proteinsbut are found in nature

Proteins

pp. 27

Nutritionally required in humans

The 20 amino acids present in proteins

pp. 33http://www.jbc.org/cgi/content/full/277/37/e25

(Polymers of amino acids)

amino acid

(1-20)

found in proteins not found in proteinsbut are found in nature

Proteins

pp. 27

Amino Acid R-groups: Very Diverse1. Polarity: hydrophobic, hydrophilic, charged

2. Charge: positive or negative

3. Size: big or small

4. Shape: flat, round

5. Reactivity: functional groups

6. Hydrogen bonding

R-group Polarity Types

Hydrophobic (non-polar)water hating

Hydrophilic (polar)water loving

Charged (polar)water lovingEnergetics Important

nitrogen oxygen

hydrogencarbon

Amino Acids

pp. 28

Hydrophobic R-groups

sulfur

pp. 29

Hydrophobic, aromatic amino acids

pp. 30

The Basic Amino Acids

pp. 32

Acidic Amino Acids

pp. 33

Amino acids are linked by the peptide bond

pp. 34

Every amino acid linked in the same way

The peptide backbone

pp. 35

The direction of the peptide chain

1 2 3 5……..…….

pp. 35N-terminal C-terminal

pH ~ 2 pH ~ 9.5

Dipolar or Zwitterion

pH ~ 7

Ionization of dibasic amino acids

fully protonated half protonated fully deprotonated

Ionization State Varies with pH(Dibasic amino acids)

pp. 27

Consider amino acids as acids or bases

What is pH? pH = log10(1/[H+]) = -log10[H+]

Consider a weak acid, HA <-> H+ + A-

The equilibrium constant, Ka, for this rxn is:Ka = [H+][A-]/[HA]

What is pK?pKa = -logKa = log(1/Ka)

pK is the pH at which a group is 50% ionized

Evaluating ionization state with pH and pK

The Henderson-Hasselbalch Equation

pH = pKa + log([A-]/[HA])

Dibasic

Titration of dibasic amino acid

What is a buffer?

Isoelectric Point/Isoelectric pH

pH at which an amino acid has no NET charge

isoelectric point of dibasic amino acids is the averageof the pK values of the carboxyl and amino groups

H pK (-COOH) = 2.4+NH3-C-COO- p K (-NH3) = 9.8

H isolectri cp = H 6.1

Glycine (gly, G)

Wh yd owe care about dibasic amino acids?

Except fo r N-termina l an dC-termina l amin o acid sall-amin o group san d -carboxy l groups areinpeptid elinkage

The amino and carboxyl are in peptide bond

Tribasicamino acids

Titration oftribasic amino acids

The Tribasic Amino AcidsAcidic Basicglutamic lysineaspartic arginine

histidine

COO-

Glutamic (glu,E) H3N+-C-CH2-C H2-COO-

H

pK1 (-COOH) = 2.2 Isoelectric pHpK2 (-COOH) = 4.3 (average 2 closest)pK3 (-NH3) = 9.7 =3.25

COO- +NH3 COO- +NH3

H3N+ COO-

Isoelectric point

Another type of covalent bond in proteins

disulfide bond

pp. 36

Proline: an imino acid

pp. 29

Protein Classification

1. Size: big-small

• peptides, a few amino acids• polypeptides, more amino acids• proteins, 50-5,000 amino acids

2. Composition

• simple: amino acids only• conjugated: other components

• lipoproteins• nucleoproteins• glycoproteins

3. Function

• Enzymes• Storage proteins• Structural proteins• Contractile/mechanical• Transport• Hormones• Defense

Protein Features

1. Diversity of function

2. Specificity of action

3. Complexity of structure

Specificity

galactose No reactionX

HO

How to explain?

1. Diversity of function

2. Specificity of action

3. Complexity of structure

Protein Structure Overview 2-31. Primary structure (1o)

composition and sequence

2. Secondary structure (2o)

helix, β-shee t formation

3. Tertiary structur e(3o)

Foldin gof polypepti deinto comple x 3-D structure

4. Quaternary Structure (4o)

interaction ofsevera l polypeptides

(Genetically determined)

R1 R2 R3 R4 R5

H3N+ COO-

N C

N C

Oligomeric proteins4 polypeptides or subunits or protomers

Primary Structure 3-1Composition

1. Purify protein2. Hydrolyze pure protein

N-----------------------------------------------C

6N HCl, 110o, 24 hrs

N--C, N--C, N--C, N--C, N--C, N--C, N--C, N--C, N--C,

3. Ion-exchange chromatography-separate andquantify amino acids

1. amino acids at low pH2. wash thru soln increasing pH3. collect fractions4. measure amino acids in eachfraction

…...….

.SO3

3-_

Fraction number1 100

Single amino acids

(Stein and Moore- Nobel Prize) www.jbc/org/cgi/content/full/280/9/e6

N-ala-gly-asp-phe-arg-gly-C

(ala,arg,asp,gly2,phe)pp. 78

Information from Composition 3-2

1. Not all proteins have all 20 amino acids

2. Composition highly variable

3. Protein properties reflect amino acid composition

• Proteins that are insoluble in water,membrane proteins, have high proportionof amino acids with hydrophobic R-groups

• Chromosomal proteins, histones, have highproportion of basic amino acids

(not much)

Primary Structure 3-3Amino Acid Sequence

The Edman Degradation

pp. 79

Break protein into small peptides to sequenceSpecific protein cleavage methods proteolytic enzymes

trypsin; cleaves after lysine, argininechymotrypsin; cleaves after phe, tyr,trp, leu, met

chemicalcyanogen bromide: cleaves after methionine

pp. 81

Trypsin Cleavage

pp. 80

Sequence determination

1. Determine amino acid composition

2. Generate peptide fragments using two or more different methods

3. Sequence peptides by Edman method

4. Align peptides to reconstruct complete sequence

General info from Sequence 3-5

1. Proteins with unique function have uniquesequence

2. Homologous proteins from different specieshave similar sequences

3. Sequence differences between homologousproteins from different species are not random

4. Within a species, amino acid substitutions as aresult of mutation can be harmful or not

5. Compare to other known sequences; learn functionall sequences in databases, easy to compare

6. Comparisons to similar proteins from other species;provide evolutionary insight

(Over 100,000 protein sequences are known)

Often easier to sequence gene and deduce protein sequence

Alternative to protein sequencing

pp. 83

protein sequence DNA sequence

inform each other

What can be learned from sequence?: Insulin

1. 1953 sequence determined by Fred Sanger and colleagues

2. Before Edman procedure, took 10 years and probably 100 person/years

3. Demonstrated proteins contained all L-amino acids

4. All linkages were peptide bonds *****

5. Sanger got Nobel Prize (1st of 2) pp. 36

Insulin:Comparative Sequences

1. Insulin is mammalian hormone

2. Sequences from over 12 species have identical hormone activity. (Use pig insulin to treat human diabetics,now use human recombinant insulin)

3. All 12 species have two polypeptide chains of 21 and 30 amino acids

4.Sequences nearly identical; only variations at 3 positions

5. When differences exist, not random

Amino acid differences in insulin

8 9

Bovine (cow) insulin

pp. 36

Position in A chain

8 9 10

Beef ala ser valPig thr ser ileSheep ala gly valHorse thr gly ileWhale thr ser ileHuman thr ser ileDog thr ser ileRabbit thr ser ile

Insulin Sequence Variation

Questions from insulin sequences.

1. Why are insulin molecules fromdifferent species so similar in structure?

2. How have sequence differences arisen?Survived?

3. What do the sequence differences andsimilarities tell us about the protein?

What can be learned from amino acid sequence? Cytochrome-c

1. Found in all species that use oxygen: bacteria-humans

2. Evolved >1.5 billion years ago, before divergence of plants and animals

3. Sequence known for over 80 species

4. Most have 104 amino acids, 26/104 invariant

5. # amino acids differences between 2 species proportional to time of evolutionary divergence

6. Amino acid differences are not random

7. Amino acid differences survived natural selection

Comparison of cytochrome c sequences

Cytochrome-c:

similar sequences- similar structures-same function

50 amino acid difference

pp. 520

bacteriabacteriafish

“Molecular Clock”

#amino acid Evolutionary divergencedifferences (millions of year)

Human-monkey 1 50-60Horse-cow 3 60-75Human-horse 12 70-75Human-dog 10 70-75Mammals-birds 10-15 280Mammals-fish 17-21 400Vertebrates-yeast 43-48 1,100

Molecular Evolution

Summarize Interspecies Sequence Information

1. Homologous proteins from different species have very similar sequences

2. Substitutions result from mutation

3. Substitutions we see have survived natural selection

4. # of differences correlate with evolutionary time

5. Surviving substitutions not random; in position, type

6. Conservation of function requires conservation of structure

Sequence differences within a species:Hemoglobin in humans

1. Conjugated protein heme + globin = hemoglobin2. Function to carry oxygen from lungs to tissues in red blood cells3. Oligomeric protein:

β

β4 polypeptides

or subunitsor protomers

Each -subunit: 141 amino acidsEach β-subunit: 146 amino acids1 heme (O2 carrier/subunit)

4. Many human hemoglobin mutations known, many benign5. Sickle cell disease, a molecular disease

Structure of Hemoglobin

Red blood cells sickle in low O2

low O2

Disease: cells get trapped in small blood vesselssevere anemia, organ damage, death

RBC Flow thru capillary

HbS forms filaments in absence of O2

HbS Filaments

Electrophoresis detects difference between HbA & HbS

migration

-

+

HbA/HbA HbA/HbS HbS/HbS

migration

+-

Electrophoresis of HbA and HbS

Electrophoresis shows HbA(normal) = HbS (sickle)

What is/are the difference(s) and how to determine?

1. -subunits HbA = HbS

2. β-subunits not identical

HbA val-his-leu-thr-pro-glu-glu-lys…………HbS val-his-leu-thr-pro-val-glu-lys………….

3. 1/146 amino acids change, harmful effect on structure and function

1 2 3 4 5 6 7 8

How has harmful mutation survived natural selection?

1. Sickle cell anemia, autosomal recessive genetic disease(first genetic disease with molecular explanation)

2. HbA/HbA = normal HbA/HbS = carrier, not symptomatic, 1% sickle cells HbS/HbS = sickle cell disease, 50% sickle cells

3. Incidence 4/1000 in black populations

4. Heterozygote is resistant to malaria. Malaria is caused by the malaria parasite that lives in red blood cells

Sickle cell disease frequency in Africa(correlates with high malaria frequency)

Summary of hemoglobin mutations

1. 1/146 amino acid changes can cause functional defect

2. Genetic disease depend on genes and environment

Primary structure determines three-dimensional structure

Ribonuclease: (enzyme digests RNA)

pp. 50

Mercaptoethanol breaks disulfide bonds

pp. 51

Unfolding and refolding of ribonuclease: primary structure is sufficient“Self assembly”

Most stable structure

pp. 51-52

“Renature”

“Denature”

Insulin violates principle of self-assembly?

ureamercaptoethanol

remove ureamercaptoethanol

Xnativematureinsulin

denaturedmature insulin

Insulin is made as precursor and processed

Assisted Protein Folding: Chaperones

1.While many proteins can fold like ribonuclease, for many the process is very inefficient.

2. Within the cell, special proteins called chaperones assist folding

Summary of Primary Structure

1. Every protein of unique function has unique sequence2. Homologous proteins from different species have

very similar sequences and structures: insulin & cytochrome-c

3. Sequence differences between homologous proteinsare not random

4. Within a species mutations can be deleterious: HbS

5. Amino acid sequence sufficient to dictate folding: self assembly

6. Proteins assume most stable structure

Higher Order Structure, 3-D2o, 3o, 4o

1. 3-D resolution

2. Atomic resolution requires 1-2 angstrom resolution

3. Nuclear magnetic resonance (structure in solution) Electron microscopy

4. X-Ray diffractionProtein crystalsSource of X-rays, 1.5 angstroms wavelengthDetector

Larger atom, higher electron density

pp. 96

X-rays

Secondary structure, 2o The -helix

Linus Pauling and Robert Corey(1939)

1. Studied structure of amino acids and small peptides by X-Ray diffraction

2. Determined bond angles and distances

3. Configuration of peptide bond is planar

OC - C - N - C

H

free rotation

4. Built models, CPK models, predicted -helix

The peptide bond is planar

pp. 37

-helix

3.6 amino acids/turnpp. 41

Why does -helix form?(Energetically favorable)

1. Pauling and Corey showed that hydrogen bonds stabilize the helix

What are hydrogen bonds? Water OH H

pp. 9

-

+

Hydrogen bonds are weak

H--------O ~1-3 kcal/mole

H O ~100 kcal/mole

pp. 8

Hydrogen bonds in -helix

pp. 41

Each amino acid hydrogen bonds to an amino acid 4 down the chain

pp. 41

2o brings some amino acids closer together

pp. 41

Amount of helix varies 0-100%

Ferritin

Keratin (Hair)Coiled coil

pp. 42

2o structure, β-sheets

1. Pauling and Corey also predicted β-sheets2. Hydrogen bonding between chains

pp. 43

pp.52

Effect of R-groups on helix formation

1. Most R-groups favor helix formation, helix is default structure

2. Bulky R-groups do not favor helix, steric effects

3. Adjacent like-charge R-groups destabilize helix

4. Proline destabilizes helix, cannot hydrogen bond

5. Destabilizing helix necessary for 3o structure

2o structure can be predicted?

1. Empirical data, which amino acids appear in certain structures

2. Theoretical, energy minimization, not so good

Tertiary structure, 3o

1. Myoglobin, first protein 3-D structure at atomic resolution

2. Oxygen carrier, found in muscle, deep diving animals

3. Contains heme group which is where O2 is boundheme is called “prosthetic” group, helper

4. Consists of 153 amino acids

5. Closely related to hemoglobin

6. Structure determined in 2 stages, 6 angstroms, backbone 2 angstroms, all atoms

Crystals of sperm whale myoglobin

Larger atom, higher electron density

pp. 96

X-rays

pp. 97

X-ray reflection pattern(intensities and positions)

pp. 97

Electron density map (fourier transform)

pp. 47

Backbone atoms(John Kendrew, 1957) 1. 8 regions of helix, 70% helix

2. Proline and other helix destablizing amino acids at bends3. Extremely compact, no room

for water inside4. Hydrophobic R-groups inside5. Hydrophilic R-groups outside6. Myoglobins from different species have similar sequences and similar structures7. Final structure is most stable

= charged

= hydrophobic

Polar amino acids outside; hydrophobic inside

intact molecule slice of molecule

pp. 47

Quaternary structure, 4o

1. Proteins with multiple subunits

2. Number of subunits, protomers, 2-1000s

3. Subunits same or different

4. Interactions between subunits, mostly surfacesalt, pH

oligomer protomers

5. Hemoglobin good example

Hemoglobin 3-D Structure

1. Hemoglobin comprised of ~10,000 atoms

2. Max Perutz determined structure/developed techniques

3. Took 23 years (1936-1959); a lifetimes work

4. Related to myoglobin, helped determine structure

Hemoglobin evolved from myoglobin

Striking structural similarity with only 24/141 identical amino acids between myoglobin, Hb , Hb β

heme

Hemoglobin, 2 β2

If myoglobin binds oxygen, why did hemoglobin evolve?

1. Oligomeric proteins have potential for cooperativity

2. Cooperative O2 binding makes hemoglobin very efficient for O2 transport and delivery

3. Myoglobin binds O2

3. Hemoglobin binds O2, CO2, H+ and BPG

4. Structures of oxy and deoxy hemoglobin differ

Physiology of respiration

1. Red cells circulate to lungs where O2 is high

2. Hemoglobin becomes saturated with O2; Hb-4O2

3. Red blood cells circulate to muscle, Hb releases O2

necessary for metabolism4. Hb picks up CO2 and H+ , products of metabolism,

and return to lungs5. Hb releases CO2 and H+ and picks up O2

How can Hb both bind and release O2?

Perutz noted structure of oxy and deoxy hemoglobin differ

β

β

+ O2

β

β

deoxy Hb oxy Hb

crystals crack

Not true for myoglobin!

O2

deoxy Hb oxy Hb

Structural change upon oxygenation

pp. 189

(tense) (relaxed)

Structural change on O2 bindingsubtle

sigmoid curve

hyperbolic curve

O2 binding by Hb is cooperative, allosteric(O2 binding regulated by O2)

(homotropic regulation)

pp187

The Concerted model for allosteric proteins Monod, Wyman and Changeux, MWC model

T-state, low O2 binding

R-state, high O2 binding

pp189

The sequential model for allosteric proteinsDaniel Koshland

Increasing O2 binding affinity

T-state R-state

pp190

Oxygen binding by Hb is a cooperative, allosteric process(homotropic regulation)

pp. 188

Hb O2 binding regulated by H+

(heterotropic regulation)lungs

muscle

pp.192

Hb O2 binding regulated by CO2

(heterotropic regulation)

pp. 193

Hb O2 binding regulated by BPG; 2,3 bisphosphoglycerate(heterotropic regulation)

tissues lungs

pp.191

How does fetus get O2?

1. Fetal Hb, Hb F, is comprised of 2 and 2 chains

2. is a separate gene product made by the fetus

Hb F binds O2 more tightly than HbA

HbF binds BPG less well than HbA

pp. 192

Summarize Hemoglobin

1. Single amino acid change in HbS changes structure/function

2. O2 homotropically activates O2 binding

3. H+, CO2 , BPG heterotropically inhibit O2 binding

4. HbF binds O2 more tightly than HbA

4. All effects tuned to physiology

5, Hb is one amazing molecule. All due to oligomeric structure

Summary of proteins

1. Primary structure, sequence, determines all higher order structures “self assembly”

2.Peptide backbone can form 2o structures, helix, β sheet

3. Higher order structures, 2o, 3o 4o, are energetically favored

4. Amino acids R-groups all important

5. Oligomeric proteins may exhibit cooperativity

6. Structural complexity explains diversity and specificity

Enzymes: The Dawn of Biochemistry

1. Pasteur, 1860s, recognized catalysis“vitalism” prevailed

2. Buchner, 1890s, cell free system

3. Sumner, enzymes were proteins!www.jbc.org/cgi/content/full/277/35/e23

4. Enzymology, 1st 50 years of biochemistry

Study of enzymes in vitro

1. find a source

2. prepare cell free extract

3. develop assaymeasure the reaction catalyzed

4. purify and study

Study of enzymes in vitro

1. find a source

2. prepare cell free extract

3. develop assaymeasure the reaction catalyzed

4. purify and study

The enzyme assay; an exampleThe enzyme, β-galactosidase

β-linkage

Nitrophenyl-glucose nitrophenol + glucose(colorless) yellow

Measure rate of appearance of yellow color

(substrate) (products)

Enzyme Classification1. Types of reactions catalyzed

2. Cofactor requirement-simple enzymes; amino acid R-groups -complex enzymes; protein + cofactor

VitaminsVit. B1RiboflavinNiacinVit. B6PantothenateBiotinVit B12folate

pp. 207

**

Examples of coenzymes for oxidation/reduction

(Reactions from the TCA Cycle)

enzyme

coenzyme

enzyme enzyme

coenzyme

oxidation oxidation

reduction reduction

Enzymes enhance rates of reactions

pp. 206

Enzymes are highly specific

Trypsin

Thrombin

proteolytic enzymes

pp. 207

How are enzymes such powerful and specific catalysts?

Intimate association of the substrate with the intricate,complex 3-D structure of the enzyme

Role of Free Energy, G, in reactions

Consider the reaction:

A + B C + D

G = Go + RT ln [C][D]/[A][B]

In biological systems use Go’, Go at pH 7

G = Go’ + RT ln [C][D]/[A][B] **

standard free energy gas constant temperature

(Review 1st and 2nd Laws and Entropy. pp 11-13)

G = Go’ + RT ln [C][D]/[A][B]Since at equilibrium, G = 0, rearrange:0 = Go’ + RT ln[C][D]/[A][B] or

Go’ = -RT ln [C][D]/[A][B] since

K’eq = [C][D]/[A][B]

Go’ = -RT ln K’eq **

G = Go’ + RT ln [C][D]/[A][B] **

Even if Go’ is positive, G can be negative(Problem Berg, pp. 210)

pp. 210

Thermodynamic considerations for enzyme reactions

1. A reaction can occur spontaneously if G is negative (exergonic)

2. A system is at equilibrium when G is zero

3. A reaction cannot occur spontaneously if G is positive (endergonic)

4. G is independent of pathway; only initial and final states

5. G provides no information about rate of reaction

How do enzymes accelerate reaction rates?

1. They do not alter equilibrium or change G values

2. They lower activation energy by formation/bindingof “transition state”

Enzymes lower activation energy

pp. 212

Consider a reaction:

ATP very slow ADP + Pi + Energy

ATP ATP* ADP + Pi + Energy

ATP ATP* ADP + Pi + Energy

very slow

very fast

Enzyme

ATP* = transition state, transition structure

I think that enzymes are molecules that are complementaryin structure to the activated complexes of the reactions they catalyze, that is, to the molecular configuration that is intermediate between the reacting substances and the the products of reaction for those catalyzed processes. The attraction of the enzyme molecule for the activated complex would thus lead to a decrease in its energy and hence to a decrease in the energy of activation of the reaction and to an increase in the rate of reaction.

- Linus Pauling- Nature 161, (1948): 707pp. 212

Linus Pauling strikes again!

Enzymes bind substrate transition statesFormation of the enzyme-substrate complex, [ES]

Enzyme kinetics

V

[S]

enzyme catalyzed rxn

non-catalyzed rxn

pp. 213

Saturationcurve,Saturationkinetics

“Active site”

1. Saturation kinetics implies an “active site” and [ES] complex:A discrete place in the enzyme where substrate binds

Evidence for “active site” and [ES] complex

Active sites are complementary to the substrate

The lock and key

pp. 215

1. Saturation kinetics implies an “active site” and [ES] complex:A discrete place in the enzyme where substrate binds

2. X-Ray crystallography demonstrates [ES]

Evidence for “active site” and [ES] complex

pp. 213Note active site residues

Induced Fit, Daniel Koshland ~1958“hand in glove”

pp. 215

Induced fit in carboxypeptidase

arg 145

tyr 248

glu 270

Induced fit in carboxypeptidase

Active site of carboxypeptidaseOH

enzyme

N Chis arg glu arg asn arg his tyr glu

1 69 71 72 127 144 145 196 248 270 307

Why do enzymes have such complex structures?

Active site, catalytic residues come from entire molecule(carboxypeptidase)

but only 9/307 so why are 307 necessary?

Michaelis-Menten (1913) Model Accounts for Enzyme Kinetics

Vo

[S]

E + S ES E + Pk1 k2

k-1 k-2 pp. 213

E + S ES E + Pk1 k2

k-1 k-2

The following assumptions allow M-M model to explain V vs S kinetics

1. Enzyme and substrate combine to form ES complex

2.Assume reverse rxn, k-2, is negligible

3.Assume [ES] is constant, steady state assumption: d[ES]/dt = 0

4. [E] <<<[S]. Does NOT mean enzyme is saturated with substrate

From these assumptions and simple rate equations derive M-M equationBerg, pp. 201-203

Vo = Vmax [S] Km + [S]

Steady State Kinetics

msec

E + S ES E + Pk1 k2

k-1 k-2

The following assumptions allow M-M model to explain V vs S kinetics

1. Enzyme and substrate combine to form ES complex

2.Assume reverse rxn, k-2, is negligible

3.Assume [ES] is constant, steady state assumption: d[ES]/dt = 0

4. [E] <<<[S]. Does NOT mean enzyme is saturated with substrate

From these assumptions and simple rate equations derive M-M equationBerg, pp. 217-219

v = Vo = Vmax [S] Km + [S]

v = Vo = Vmax [S] Km + [S]

v or Vo= d[P]/dt or -d[S]/dt initial velocity

Vmax = maximum rxn velocity; velocity limit as [S] infinity

Km = k-1 + k2 = Michaelis constant k1

when k2 <<<<< k-1 then Km ~ k-1 = [E][S] k1 [ES]

Km is measure of affinity of enzyme for substrateA low Km means high affinity.

An enzyme with a Km of 10-6 M binds substrate more tightly than one with a Km of 10-4 M

E + S ES E + Pk1 k2

k-1 k-2

Vo is measure of initial rates

low

high

pp. 217

Vo = Vmax [S] Km + [S]

Vo = d[P]/dt or -d[S]/dt initial velocity

Vmax = maximum rxn velocity; velocity limit as [S] infinity

Km = k-1 + k2 = Michaelis constant k1

when k2 <<<<< k-1 then Km ~ k-1 = [E][S] k1 [ES]

Km is measure of affinity of enzyme for substrateA low Km means high affinity.

An enzyme with a Km of 10-6 M binds substrate more tightly than one with a Km of 10-4 M

E + S ES E + Pk1 k2

k-1 k-2

E + S ES E + Pk1 k2

k-1 k-2

Vmax = Vo at [S] = infinity

Km = [S] at 1/2 Vmax

pp. 217

Factors that influence enzyme activity1. Substrate concentration2. Coenzyme concentration3. Temperature4. pH

pepsin urease trypsin % max activity

100

pH 1 2 3 4 5 6 7 8 9 10

Temp25 30 35 40 45 50 55 60 65

activity

enzyme rxn

chemical rxn

Interesting biology:thermophilic organismsacidophilic organisms

Km, Vmax: a better way

Vo = Vmax [S] Michaelis-Menten [S] + Km (hyperbola)

Instead, take reciprocal

1/Vo = 1/Vmax + Km/Vmax . 1/[S]

Lineweaver-Burk (straight line)

Lineweaver-Burk Plot for Km and Vmax

pp. 220

1/Vo = 1/Vmax + Km/Vmax . 1/[S]

The enzyme assay: How much enzyme is present?

Use optimal pH, temperature, Saturating substrate and coenzymeUnder saturating [S]

Vo

[E]

[S] >>>>> Km

[P]

Time

[E] = 2x

[E] = 1xOR

Final kinetic parameter: turnover numberMolecules substrate converted to product/per molecule of enzyme per second

pp.221

Kinetic parameters: who cares?

1. Important to understand catalytic mechanism

2. Km, Vmax characterize enzyme, physiology

3. Enzyme assay, practical considerations

4. Important for Bio 41 midterm

Not all enzymes obey Michaelis- Menten kinetics:allosteric, regulatory enzymes (more later)

Inhibition of enzyme activity

1. Reversible inhibition-competitive-non-competitive

2. Irreversible inhibition

Competitive vs non-competitive inhibition

pp. 225

Competitive inhibition

1. Inhibitor structurally similar to substrate

2. Can get formation of [ES] or [EI] but not [ESI]

3. “competition” for active site

Dihydrofolate reductase: purines, pyrimidines

(substrate)

competitive inhibitor

Kinetics of Competitive Inhibition

pp. 226Overcome inhibition with more substrate

Vo

Kinetics of competitive inhibition

Change inapparent Km

No change in apparent Vmax

o

pp.228

Competitive vs non-competitive inhibition

Kinetics of non-competitive inhibition

pp. 227

Kinetics of non-competitive inhibition

No change inKm Change in Vmax

pp. 228

Cannot overcome non-competitive inhibition with more substrate

Inhibition of enzyme activity

1. Reversible inhibition-competitive-non-competitive

2. Irreversible inhibition

Irreversible inhibitionPotent nerve gas, DIPF, blocks acetylcholinesterase, necessary for

transmission

active site serine covalentbond

Evidence for active site residues pp. 229

Summary of enzyme catalysis

1. Enzymes change rates not equlibria

2. Kinetic and structural evidence for active site

3. Enzymes lower activation energy, bind transition state

4. Enzymes can be self regulating **

5. Enzymes are wonderful

Regulation of enzyme activity:

1. Necessity for regulation, 1000s of biochemical reactions, all metabolism is interrelated

2. Control = efficiency

1000s of reactions, all interlinked

1. Control amount of enzyme: long term, hrs, days-enzyme synthesis: gene regulation-enzyme degradation

2. Control function of enzyme: short term, sec, min ** - allosteric regulation, non-covalent,

-covalent modification (phosphorylation)- proteolytic processing

Two major regulatory strategies

Regulatory, allosteric enzymes: some definitions

1. Allosteric = “other site” other than active site

2. Regulatory molecules called, effectors, modulators, regulatory molecules

3. Homotropic regulation: regulation by substrate at active site

4. Heterotropic regulation: regulation by molecule NOT substrate ( end products), at allosteric site

5. Few enzymes are allosteric

6. Allosteric enzymes DO NOT exhibit M-M kinetics

Physiology of allosteric enzymesConsider biochemical pathways:

-Homotropic regulation, substrate activation

activation E1 E2 E3 E4 E5

A B C D E F

-Heterotropic regulation, end product inhibition

E1 E2 E3 E4 E5

A B C D E Finhibition

D E FA B C

G H I

F inhibits C->Dpartially inhibitsA -> B

Which enzymes are allosteric?

E1 E2 E3 E4 E5

A B C D E F

1. 1st committed step in pathway

2. rate limiting step

Threonine Deaminase: Homotropic Activation

threonine deaminase

threonine B C D isoleucine

proteins proteins

d[B]/dt

[threonine]

90%

10%

M-M enzyme

threonine deaminase

It takes a smaller changein [S] to go from 10% to 90% activity

Vo

homotropic activation heterotropic inactivation

Substrate activation of threonine deaminase

active site

substrate substrate

T-state(less active)

R-state (more active)

structural transition

The Concerted model for allosteric proteins Monod, Wyman and Changeux, MWC model

T-state

R-state

Aspartate transcarbamoylase (ATCase): An allosteric enzyme(The physiological context)

ATCaseAspartate + carbamyl-P X UTP CTP

protein RNA DNA

ATCase makes sure that there is enough aspartate for protein synthesis and enough UTP and CTP for nucleic acid synthesis

homotropic activation heterotropic inhibition

ATCase: homotropic regulation, substrate activation

sigmoidal curve

The Concerted model for allosteric proteins Monod, Wyman and Changeux, MWC model

T-state

R-state

S-shaped curve is combo of R-state and T-stateA simulation

pp. 281

Aspartate transcarbamoylase (ATCase):Heterotropic regulation: feedback inhibition

ATCaseAspartate + carbamyl-P X UTP CTP

protein RNA DNA

heterotropic inhibition

Oligomeric structure of ATCase

R-state T-state

r r r r r r

C = catalyticR = regulatory

gentle heat

Kinetics of ATCase

inhibitor

activator

“C” subunits+/- CTP

Normal enzyme Normal + CTP

pp. 264

X-ray structure of ATCase

Structural Transition of ATCase

T = tenseR = relaxed pp.281

X-ray structure of ATCase- “side” view

CTP binding stabilizes the T-state

Inhibition of ATCase by CTP

pp. 282

ATP, heterotropic activator of ATCase

pp. 282

Summarize ATCase

Aspartate, substrate, is: homotropic activator substrate activator

CTP, end product inhibitor, is heterotropic inhibitor end product inhibitor

ATP, ?????? is heterotropic activator

Summary enzyme regulation1. Self regulation: allosteric enzymes

2. Control activity of existing enzymes

3. Short term regulation, min. sec.

4.Non-covalent regulation, reversible

5. Substrate activation, homotropic regulation

6. End product inhibition, heterotropic inhibition

7. Heterotropic activation

Summary of protein structure

1. Complex structure

2. Diverse, complex functions

3. High specificity

4. Enzymes most amazing, important

What do enzymes do?Metabolism

1. All biochemical reactions are interrelated, integrated

2. General discussion bacteria to humans

3. Strategies importantreactions in pathwaysenergetics importantregulation important

pp. 410

Light

Phototrophs HeterotrophsChemotrophs

Chemical oxidations

complex carbonglucose, amino acids, O2

CO2, H2O

Energy and material in the biosphere

Autotrophs

Heterotrophic requirements

E . coli Leuconostoc Humans(bacteria) (bacteria)

Carbon/Energy glucose glucose glucose

Nitrogen NH3 NH3 NH3

19 amino acids 9 aa4 nucleotides8 vitamins 15 vit.

Elements Na, K, Mg, Ca, Zn, Fe, PO4, SO4 etc.

Heterotrophic metabolismInterconversion of material and energy

Heterotrophic metabolism

Catabolism Anabolism(breakdown) (synthesis)yields energy, requiresprecursors energy,

precursors

How are catabolism and anabolism coupled?

coupled

Bioenergetics/Thermodynamics

catabolism/respiration

Go’ = -686 kcal/mol

C6H12O6 + 6O2 6CO2 + 6H2O (sugar) Go’ = 686 kcal/mol

anabolism/photosynthesis

G = Go’ + RT ln [C][D]/[A][B]

RememberFor the rxn: A + B C + D

Thermodynamically unfavorable rxns driven by favorable ones Gs are additive

Consider:

A B + C Go’ = +5 kcal/mol

B D Go’ = -8 kcal/mol

A C + D Go’ = -3 kcal/mol

Reaction coupling

pp. 411

Reaction Coupling

Go’

Glucose + PO4 glucose-6-PO4 4 kcal/mol

ATP + H2O ADP + Pi + H+ -7 kcal/mol

Glucose + ATP glucose-6-PO4 + ADP -3 kcal/mol

Hexokinase (couples the two reactions)

ATP couples energy between catabolism and anabolism

pp. 417

catabolism

anabolism

ATP: the universal currency of free energy“high energy” phosphate compound

ATP + H2O ADP + Pi + H+ Go’ = -7.3 kcal/mol

ADP + H2O AMP + Pi + H+ Go’ = -7.3 kcal/mol

phosphoanhydride

adenine

ribose

ATP is intermediate “high energy” compound

pp. 417

ATP is intermediate “high energy” compound

Go’

Coupling Oxidations/Reductions

catabolismReduced fuel Oxidized Fuel

NAD(ox) NADH(reduced)

Reduced Products Oxidized Precursors anabolism

NAD+(ox) NADH(reduced)Nicotinamide adenine dinucleotide

NADP NADPH(PO4)pp. 420

H: (hydride ion)

ATP/ADP couple energy of catabolism/anabolismNAD/NADH couple ox/red of catabolism/anabolism

Two coupling molecules

Catabolism/Energy Metabolism Overview

Glucose catabolismGlucose 6CO2 + 6H2O(C6H12O6) (requires O2)

Go’ = -686 kcal/mol

Occurs in 3 stages1. Glycolysis2. TCA cycle3. Electron transport/oxidative phosphorylation

no O2 required

1. Glycolysis: glucose lactate (muscle)ethanol (yeast)

What organisms use glycolysis?1. Anaerobes (grow without O2)2. Facultative organisms (grow with/without O2)3. Aerobes (grow only with O2)

History of Glycolysis (history of biochemistry)1. Buchner (1890)

sucrose ethanol

2. Meyerhof glucose lactic acid (lactate)

3. Harden and Young (1905)glucose + Pi fructose1,6 diphosphaterxn depends on

heat labile factors: zymase(enzymes)heat stable factors: cozymase (coenzymes)fluoride inhibits, causes intermediates to

accumulate

4. Embden-Meyerhof (1930s)worked out all steps, called Embden-Meyerhof pathwayGlycolysis

no O2

no O2

inhibitorX

yeast

muscle

What to know about glycolysis?

1. Relate structures to each other, don’t memorize2. Don’t memorize enzyme names, except a few3. Know general rxn sequences4. Follow: carbon, phosphates (ATP/ADP), electrons (NAD/NADH)

5. Understand rxn energetics6. Where in cell rxns take place7. How rxns are integrated8. How rxns are regulated

Stage 1 Stage 2 Stage 3

Glycolysis Overview

Stage 1 glycolysis

pp. 435

glycogen/starch

cell membrane

Glucose

many otherrxns

2 ATP 2ADP

regulatoryenzyme

Stage 1: Energy input, preparation

entry of many other sugars

Stage 2: 1 6-carbon sugar to 2 3-carbon compounds

pp. 438

Stage 2

pp. 438

Fructose 1, 6, bisphosphate

Stage 3: energy yield

pp. 441

Stage 3: NADH and ATP Produced

4 ADP 4 ATP

2 NAD 2 NADH

?

many other rxns

There is no NET oxidation in glycolysis

pp. 446

Regeneration of NAD+ critical

Regeneration of NAD+ critical

pp. 447

What happens to pyruvate?depends on O2 and which organism

muscle

yeast

O2 presentO2 NOT present

1. All enzymes are soluble: in cytoplasm of cells2. In some organisms, glycolysis is all there is

Anaerobesfacultative organisms in absence of O2

red blood cellstissues like muscle in absence of O2

3.End product depends on organism4. No NET change in oxidation state5. Many side rxns, not all carbon goes to pyruvate6. Energy yield

glucose + 2 ADP 2 lactate + 2 ATPtheoretically: glucose 2 lactate Go’ = - 47 kcal/mol

2ADP 2ATP Go’ = 14.6 kcal/mol 14.6/47 X 100 = 30%

But 47/686 is pretty low! So what’s next?

Features of glycolysis

What happens to pyruvate?depends on O2 and which organism

muscle

yeast

O2 presentO2 NOT present

Metabolism of pyruvate in presence of O2

The Tricarboxylic Acid (TCA)Cycle

glycolysis -O2 lactateethanol

+ O2

pp. 477

The TCA Cycle2 pyruvate 6CO2 + 6 H2O

Change in cellular location-eukaryotes; move from cytoplasm to mitochondria-prokaryotes; in cytoplasm with glycolysis

Anatomy of a mitochondrion

TCA cycle enzymes

pp. 476

A mitochondrion

pp. 476

TCA Cycle overview (oxidations)

Pyruvate(3 carbons)

CO2

pp. 476

glycolysisNADH

pp. 489

TCA Cycle

pyruvate

CO2

NADH

TCA Cycle provides precursors for many things

Fatty Acids,Sterols

X

alanine

pp. 493

glycolysis

X

Summary TCA cycle

1. All carbon lost as CO2

2. Gain: 5 X 2e- as: 4 NADH, 1 FADH2

3. Gain 1 GTP, ATP equivalent4. Occurs: prokaryotes, cytoplasm

eukaryotes, mitochondria/matrix5. Many side reactions:

acetyl-CoA -> -> fatsoxaloacetate -> -> asparticpyruvate -> -> alanine -ketoglutaric -> -> glutamic

So what’s left? 5 pairs of electrons!

Electron Transport/Oxidative phosphorylation(inner mitochondrial membrane)

TCA Cycle

FADH2

2e-

2e-

NAD+

FAD

Back to TCA cycle

Back to TCA cycle

ADP

ATP

ADP

ATP

ADP

ATP H2O

3 ATP/2e-(from NADH)

2 ATP/2e-

(from FADH2)

Glucose 6CO2 + 6H2O + ATPenergy yield

ATP/glucose, with O2

Glycolysis 2 ATP

TCA Cycle 2 ATP (GTP)

Electron transport/ox. phosphorylation 26-30 ATP

glucose + 6O2 6CO2 + 6H2Oa balance sheet

glucose + 6O2 6CO2 + 6H2O Go’ = -686 kcal/mol

Overall energy yield

30 ATP + 30 H2O 30 ADP + 30Pi Go’ = -219 kcal/mol

219/686 X 100 = ~32%

Energy yield +/- O2

growth yield(grams of cells)

[glucose]

-O2

+O2

Growth of E. coli, a facultative organism, on glucose

Oxidation/Reduction (Redox) Rxns and Free Energy

electrical energy (NADH) chemical energy (ATP)

Redox rxns written as reduction reactions

X(oxidized) + ne- X(reduced)

Redox rxns occur in pairs:

pyruvate(ox) + NADH(red) lactate(red) + NAD+?

Redox potential: tendency to donate or accept electrons

2H+ + 2e- H2 Eo = 0.00

at pH 7 Eo’ = -0.42 volts

(ox)

Using Standard Redox Potentials

Consider: NADH + H+ + 1/2O2 H2O + NAD+?Write half rxns:

NAD+ + H+ + 2e- NADH Eo’ = -0.32 volts1/2 O2 + 2H+ + 2e- H2O Eo’ = 0.82 volts

Rewrite in the correct direction:NADH NAD+ + H+ + 2e-

1/2 O2 + H+ + 2e- H2O

NADH + H+ + 1/2O2 H2O + NAD+ Eo’ = + 1.14 volts

Go’ = -nF Eo’number of electrons, Faraday

Go’ = -2 X 23 X 1.14 = -52kcal/molpp 508

Electron Transport: Prokaryotes: Cytoplasmic membrane Eukaryotes:Inner mitochondrial membrane

TCA cycleElectron transport

A mitochondrion

The electron transport chain (inner mitochondrial membrane)

innermembrane

Eo’ Eo’ Go’ ATP-0.32

-0.05

0.27 -12.1 1

+0.26

+0.28

+0.82

0.00

+0.22 0.22 -10.1 1

0.54 -25 1

0.05 -2.5

0.04 -1.9

0.02 -0.9

2e-

Complex I

Complex III

Complex IV

Respiratory Complexes

34 proteins, FMNFe-S proteins

Complex II

22 proteins, cytochromes

13 proteinsCytochromes,Cu

Electron Carriers: Quinones

Electron Carriers: Flavoproteins, Flavins: FADH2, FMN,

Electron carriers: Flavoproteins

Electron carriers: Cytochromes

protein

Structure of cytochrome oxidasecomplex IV

13 proteins2 coppers2 hemes

Inhibitors of electron transport

TCA cycle

Electron Transport/Oxidative phosphorylation(inner mitochondrial membrane)

TCA Cycle

FADH2

2e-

2e-

NAD+

FAD

Back to TCA cycle

Back to TCA cycle

ADP

ATP

ADP

ATP

ADP

ATP H2O

3 ATP/2e-(from NADH)

2 ATP/2e-

(from FADH2)

How does electron transport lead to ATP synthesis?Oxidative phosphorylation

Peter Mitchell: Chemiosmotic coupling

pp. 521

Energetics of Ion (Proton) GradientsThe “Proton Motive Force”

G = RT ln(c2/c1) + ZF V

concentration electrical

c2/c1 = concentration difference across the membraneZ = electrical charge of ion transported, H+ = +1F = Faraday, electrical constantV = electrical potential across membrane

The ATP Synthase

Active in OX Phos

Not active in Ox Phos

ATP Synthase, F1Fo ATPase,Proton translocating ATPase

electrontransport

[H+]

[H+] proton pore

ATP synthesis

ADP + Pi ATP

NADH

Electron transport ATP synthase

Electron transport and oxidative phosphorylation

Mitochondrial inner membrane or bacterial cytoplasmic membrane

Uncoupling proteins short circuit H+ gradientgenerate heat

pp. 533

Electron Transport goes faster and faster but can’t catch up!

Proton gradient is energy source for many functions(Peter Mitchell got Nobel Prize)

Oxidative phosphorylation

pp. 535

The cost of sequestration: mitochondrial transporters

But mitochondria impermeable to NAD/NADHHow do electrons from glycolysis get to mitochondria?

pp. 533

glucose + 6O2 6CO2 + 6H2Oa balance sheet

Electrons from cytosolic NADH into mitochondria: The glycerol phosphate shuttle

Electron transport

glycolysis

Regulation of Energy Metabolism“The Energy Charge”

Glycolysis TCA cycle Electron Transport Oxidative phosphorylation

Overall response to “energy charge” or energy status

[ATP] + 1/2[ADP] OR [ATP][ATP] + [ADP] + [AMP] [ADP]

Regulation of energy metabolism

glycolysis

TCA cycle

Electron transport,ox. phosphorylation

Regulation within the mitochondria respiratory control

ET

Regulation of glycolysis: phosphofructose kinase

Regulatory molecules

ATP -ADP +AMP +

Citrate -

ATP inhibits phosphofructokinase

pp. 453

Phosphofructokinase: many sitesA tetrameric protein

pp. 453

Regulation of TCA Cycle: isocitrate dehydrogenase

isocitrate dehydrogenase

pp. 492

glycolysis

Photosynthesis

6CO2 + 6H2O C6H12O6 + 6 O2 Go’ = 686 kcal/molATP, NADPH

Photosynthesis involves two parts:1. Light reactions

generate ATP, NADPH

2. Dark reactionsuse ATP, NADPH, CO2 -> sugar

Occurs in: prokaryotes; bacteria, blue green algae, in cytoplasmic membrane eukaryotes; chloroplasts

light

light rxns

The chloroplast

grana

dark rxns

pp. 543

Chloroplast “grana”

pp. 542

Light rxns: Overviewlight NADPH + ATP

pp. 542

Light Energy Chemical Energy?

Pigments absorb light

Chlorophyll a

pp. 544

Absorbtion spectrum of chlorophylls a & b

pp. 558

Other pigments, antenna pigments, accessory pigments

Other pigments

Electron transfer from accessory pigment to rxn center

Antenna pigments

pp. 557

Two photosystems

Photosystem II Photosystem I

chlorophyll a 200 chlorophyll b 200chlorophyll b 50 chlorophyll b 50carotenoids 100 carotenoids 100

Rxn center pig. 1 (P680) rxn center 1 (P700)

Photosystem II

pp. 549

Absorbtion of light by pigment

Return to ground stateheat

pp. 545

Electron transfer: charge separation

replace e-

transfer e-

pp.545

The “Z” scheme of photosythesis

2H2O + NADP+ O2 + NADPH

O2

light

proton gradient

pp. 553

How is ATP made? photophosphorylation

Jagendorf showed H+ gradientin chloroplasts makes ATP

pp. 554

PSII PSIcyt bf

light

light

pp. 555

I II III

The “dark” rxns of photosynthesisCO2 fixation

6CO2 + 6H2O C6H12O6 + 6O2

ATP, NADPH

6CO2 + 18 ATP + 12 NADPH + 12 H2OC6H12O6 + 18 ADP + 18 Pi + 12 NADP+ + 6H+

3 ATP + 2 NADPH/CO2

Melvin Calvin’s Nobel Experiment

14CO214C-X

Chromatography

algae

identify

light

pp. 567

What is CO2 acceptor ?

O14C O

CH2-OH O CH2-O-P-O O

3-phosphoglycerate

What is 2-carbon CO2 acceptor?

Ribulose bisphosphate carboxylase“Rubisco”- the most abundant enzyme on earth

Rubisco

5-carbon 6-carbon 2 3-carbon

The Calvin Cycle

Rubisco

carbohydrate scramble

starch

pp. 566

Starch

Starch synthesis

pp. 570

CO2

Not just the reverse of glycolyis

Lipids and Cell Membranes prokaryotic cells

Cell Membranes eukaryotic cells

Membrane functions

1. Define cell: plasma membrane, cell limit

2. Compartmentalize: organelles, mitochondria etc.

3. Interaction with environment: permeability barriersolute transport

4. Organize functions: electron transport

Common features of all cell membranes

1. Structure: phospholipids

2. Function: proteins

Fluid Mosaic Model of Membrane Structure (Singer and Nicolson, 1972)

phospholipidprotein

pp. 343

Lipids: very diverse class of biomolecules (insoluble in water)

Glycerolipids CH2-OHCH2-OH derivatives of glycerolCH2-OH

1. Triglycerides (storage lipid) OCH2-O-C-(CH2)nCH3

OCH2-O-C-(CH2)n-CH3

OCH2-O-C-(CH2)n-CH3

2. Phospholipids OCH2-O-C-(CH2)n-CH3

OCH2-O-C-(CH2)n-CH3

O-

CH2-O-P-O-RO-

Other lipids

Sterols

Pigments: chlorophyll, carotene, etc.

Fat soluble vitamins

Phospholipid

pp. 322

Phospholipid

pp. 329

(lecithin)

polar R-groupphosphate

glycerolfatty acids

pp. 328

Phospholipids: fatty acids “tails”

The type of fatty acid makes a difference

Saturated fatty acids (no double bonds)

Unsaturated fatty acid (one cis double bond)

Affects physical and functional properties of membrane

pp. 338

Some phospholipid “head groups”

Phosphatidyl-X

pp. 330

Phospholipid: R-groups“head groups”

pp. 330

(lecithin)

Phospholipids are amphipathic

Polar “head group”Non-polar “tails”

hydrophobic hydrophilic “tails” “head groups”

pp. 332

Amphipathic molecules in H2O (micelles)

hydrophobic

hydrophilic

pp. 333

Phospholipids in H2O“liposomes”

H2O

H2O

pp. 334

Phospholipid bilayer

pp. 333

very hydrophobic and fluid olive oil

Phospholipid composition of cell membranes very complex

Phospholipid structural variables:

1. 10-15 different fatty acids2. 2 positions in glycerol backbone3. 6-10 head groups

Even in simple membrane, 50-100 individual molecular species

Phospholipid bilayer is very fluid:

Phospholipid bilayer provides:

1. structure

2. matrix, support for proteins

3. permeability barrier to polar molecules

pp. 335

Membrane Proteins

1. Proteins provide membrane functions

2. Each membrane has unique function,different protein compositions

3. Protein amounts vary

Membrane proteins and the bilayer

Integral

Peripheralf

pp. 336

An integral membrane protein: Bacteriorhodopsin

pp. 337

Another “integral” membrane protein: prostaglandin synthase

pp. 339

Hydrophobic amino acids anchor membrane proteins

Glycophorin: a red cell membrane protein

sugars pp. 341

It is possible to predict membrane spanning proteins

Proteins are “fluid” too

fast

fast

very slow

Fluid Mosaic Model of Membrane Structure (Singer and Nicolson, 1972)

phospholipidprotein

Membrane Summary

1. Phospholipid bilayer universal membrane structure

2. Phospholipid composition varies, physical state

3. Protein provides function, type and amount vary

4. Each membrane has unique function, protein composition

5. Fluid Mosaic model, good general description

6. Really interesting question: how are membranes made?