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![Page 1: Biochemistry Section of Bio 41 (Fall 2007, Bob Simoni) 1. Lecture material most important 2. Reading in Berg, supplemental assignments for the 6th edition.](https://reader035.fdocuments.us/reader035/viewer/2022070410/56649ea45503460f94ba8f7d/html5/thumbnails/1.jpg)
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
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"Macromolecules" 1-1
• Proteins: most diverse, complex, responsible forall cell functions
• Lipids: Structural, cell membranes, energymetabolism
• Carbohydrates: Structure, energy metabolism
• Nucleic Acids: Genetic material
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(Polymers of amino acids)
amino acid
(1-20)
found in proteins not found in proteinsbut are found in nature
Proteins
pp. 27
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Nutritionally required in humans
The 20 amino acids present in proteins
pp. 33http://www.jbc.org/cgi/content/full/277/37/e25
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(Polymers of amino acids)
amino acid
(1-20)
found in proteins not found in proteinsbut are found in nature
Proteins
pp. 27
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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
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R-group Polarity Types
Hydrophobic (non-polar)water hating
Hydrophilic (polar)water loving
Charged (polar)water lovingEnergetics Important
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nitrogen oxygen
hydrogencarbon
Amino Acids
pp. 28
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Hydrophobic R-groups
sulfur
pp. 29
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Hydrophobic, aromatic amino acids
pp. 30
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The Basic Amino Acids
pp. 32
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Acidic Amino Acids
pp. 33
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Amino acids are linked by the peptide bond
pp. 34
Every amino acid linked in the same way
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The peptide backbone
pp. 35
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The direction of the peptide chain
1 2 3 5……..…….
pp. 35N-terminal C-terminal
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pH ~ 2 pH ~ 9.5
Dipolar or Zwitterion
pH ~ 7
Ionization of dibasic amino acids
fully protonated half protonated fully deprotonated
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Ionization State Varies with pH(Dibasic amino acids)
pp. 27
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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
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Evaluating ionization state with pH and pK
The Henderson-Hasselbalch Equation
pH = pKa + log([A-]/[HA])
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Dibasic
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Titration of dibasic amino acid
What is a buffer?
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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
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The amino and carboxyl are in peptide bond
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Tribasicamino acids
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Titration oftribasic amino acids
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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
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Another type of covalent bond in proteins
disulfide bond
pp. 36
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Proline: an imino acid
pp. 29
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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
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Protein Features
1. Diversity of function
2. Specificity of action
3. Complexity of structure
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Specificity
galactose No reactionX
HO
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How to explain?
1. Diversity of function
2. Specificity of action
3. Complexity of structure
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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
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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
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N-ala-gly-asp-phe-arg-gly-C
(ala,arg,asp,gly2,phe)pp. 78
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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)
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Primary Structure 3-3Amino Acid Sequence
The Edman Degradation
pp. 79
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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
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Trypsin Cleavage
pp. 80
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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
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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)
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Often easier to sequence gene and deduce protein sequence
Alternative to protein sequencing
pp. 83
protein sequence DNA sequence
inform each other
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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
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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
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Amino acid differences in insulin
8 9
Bovine (cow) insulin
pp. 36
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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
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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?
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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
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Comparison of cytochrome c sequences
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Cytochrome-c:
similar sequences- similar structures-same function
50 amino acid difference
pp. 520
bacteriabacteriafish
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“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
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Molecular Evolution
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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
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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
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Structure of Hemoglobin
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Red blood cells sickle in low O2
low O2
Disease: cells get trapped in small blood vesselssevere anemia, organ damage, death
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RBC Flow thru capillary
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HbS forms filaments in absence of O2
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HbS Filaments
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Electrophoresis detects difference between HbA & HbS
migration
-
+
HbA/HbA HbA/HbS HbS/HbS
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migration
+-
Electrophoresis of HbA and HbS
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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
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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
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Sickle cell disease frequency in Africa(correlates with high malaria frequency)
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Summary of hemoglobin mutations
1. 1/146 amino acid changes can cause functional defect
2. Genetic disease depend on genes and environment
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Primary structure determines three-dimensional structure
Ribonuclease: (enzyme digests RNA)
pp. 50
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Mercaptoethanol breaks disulfide bonds
pp. 51
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Unfolding and refolding of ribonuclease: primary structure is sufficient“Self assembly”
Most stable structure
pp. 51-52
“Renature”
“Denature”
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Insulin violates principle of self-assembly?
ureamercaptoethanol
remove ureamercaptoethanol
Xnativematureinsulin
denaturedmature insulin
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Insulin is made as precursor and processed
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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
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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
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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
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Larger atom, higher electron density
pp. 96
X-rays
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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
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The peptide bond is planar
pp. 37
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-helix
3.6 amino acids/turnpp. 41
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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
-
+
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Hydrogen bonds are weak
H--------O ~1-3 kcal/mole
H O ~100 kcal/mole
pp. 8
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Hydrogen bonds in -helix
pp. 41
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Each amino acid hydrogen bonds to an amino acid 4 down the chain
pp. 41
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2o brings some amino acids closer together
pp. 41
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Amount of helix varies 0-100%
Ferritin
Keratin (Hair)Coiled coil
pp. 42
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2o structure, β-sheets
1. Pauling and Corey also predicted β-sheets2. Hydrogen bonding between chains
pp. 43
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pp.52
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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
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2o structure can be predicted?
1. Empirical data, which amino acids appear in certain structures
2. Theoretical, energy minimization, not so good
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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
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Crystals of sperm whale myoglobin
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Larger atom, higher electron density
pp. 96
X-rays
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pp. 97
X-ray reflection pattern(intensities and positions)
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pp. 97
Electron density map (fourier transform)
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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
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= charged
= hydrophobic
Polar amino acids outside; hydrophobic inside
intact molecule slice of molecule
pp. 47
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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
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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
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Hemoglobin evolved from myoglobin
Striking structural similarity with only 24/141 identical amino acids between myoglobin, Hb , Hb β
heme
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Hemoglobin, 2 β2
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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
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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?
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Perutz noted structure of oxy and deoxy hemoglobin differ
β
β
+ O2
β
β
deoxy Hb oxy Hb
crystals crack
Not true for myoglobin!
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O2
deoxy Hb oxy Hb
Structural change upon oxygenation
pp. 189
(tense) (relaxed)
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Structural change on O2 bindingsubtle
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sigmoid curve
hyperbolic curve
O2 binding by Hb is cooperative, allosteric(O2 binding regulated by O2)
(homotropic regulation)
pp187
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The Concerted model for allosteric proteins Monod, Wyman and Changeux, MWC model
T-state, low O2 binding
R-state, high O2 binding
pp189
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The sequential model for allosteric proteinsDaniel Koshland
Increasing O2 binding affinity
T-state R-state
pp190
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Oxygen binding by Hb is a cooperative, allosteric process(homotropic regulation)
pp. 188
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Hb O2 binding regulated by H+
(heterotropic regulation)lungs
muscle
pp.192
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Hb O2 binding regulated by CO2
(heterotropic regulation)
pp. 193
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Hb O2 binding regulated by BPG; 2,3 bisphosphoglycerate(heterotropic regulation)
tissues lungs
pp.191
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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
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Hb F binds O2 more tightly than HbA
HbF binds BPG less well than HbA
pp. 192
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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
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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
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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
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Study of enzymes in vitro
1. find a source
2. prepare cell free extract
3. develop assaymeasure the reaction catalyzed
4. purify and study
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Study of enzymes in vitro
1. find a source
2. prepare cell free extract
3. develop assaymeasure the reaction catalyzed
4. purify and study
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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)
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Enzyme Classification1. Types of reactions catalyzed
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2. Cofactor requirement-simple enzymes; amino acid R-groups -complex enzymes; protein + cofactor
VitaminsVit. B1RiboflavinNiacinVit. B6PantothenateBiotinVit B12folate
pp. 207
**
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Examples of coenzymes for oxidation/reduction
(Reactions from the TCA Cycle)
enzyme
coenzyme
enzyme enzyme
coenzyme
oxidation oxidation
reduction reduction
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Enzymes enhance rates of reactions
pp. 206
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Enzymes are highly specific
Trypsin
Thrombin
proteolytic enzymes
pp. 207
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How are enzymes such powerful and specific catalysts?
Intimate association of the substrate with the intricate,complex 3-D structure of the enzyme
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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)
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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)
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pp. 210
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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
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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”
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Enzymes lower activation energy
pp. 212
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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
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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!
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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”
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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
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Active sites are complementary to the substrate
The lock and key
pp. 215
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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
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Induced Fit, Daniel Koshland ~1958“hand in glove”
pp. 215
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Induced fit in carboxypeptidase
arg 145
tyr 248
glu 270
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Induced fit in carboxypeptidase
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Active site of carboxypeptidaseOH
enzyme
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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?
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Michaelis-Menten (1913) Model Accounts for Enzyme Kinetics
Vo
[S]
E + S ES E + Pk1 k2
k-1 k-2 pp. 213
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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]
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Steady State Kinetics
msec
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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]
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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
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Vo is measure of initial rates
low
high
pp. 217
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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
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E + S ES E + Pk1 k2
k-1 k-2
Vmax = Vo at [S] = infinity
Km = [S] at 1/2 Vmax
pp. 217
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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
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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)
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Lineweaver-Burk Plot for Km and Vmax
pp. 220
1/Vo = 1/Vmax + Km/Vmax . 1/[S]
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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
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Final kinetic parameter: turnover numberMolecules substrate converted to product/per molecule of enzyme per second
pp.221
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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
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Not all enzymes obey Michaelis- Menten kinetics:allosteric, regulatory enzymes (more later)
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Inhibition of enzyme activity
1. Reversible inhibition-competitive-non-competitive
2. Irreversible inhibition
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Competitive vs non-competitive inhibition
pp. 225
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Competitive inhibition
1. Inhibitor structurally similar to substrate
2. Can get formation of [ES] or [EI] but not [ESI]
3. “competition” for active site
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Dihydrofolate reductase: purines, pyrimidines
(substrate)
competitive inhibitor
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Kinetics of Competitive Inhibition
pp. 226Overcome inhibition with more substrate
Vo
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Kinetics of competitive inhibition
Change inapparent Km
No change in apparent Vmax
o
pp.228
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Competitive vs non-competitive inhibition
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Kinetics of non-competitive inhibition
pp. 227
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Kinetics of non-competitive inhibition
No change inKm Change in Vmax
pp. 228
Cannot overcome non-competitive inhibition with more substrate
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Inhibition of enzyme activity
1. Reversible inhibition-competitive-non-competitive
2. Irreversible inhibition
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Irreversible inhibitionPotent nerve gas, DIPF, blocks acetylcholinesterase, necessary for
transmission
active site serine covalentbond
Evidence for active site residues pp. 229
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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
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Regulation of enzyme activity:
1. Necessity for regulation, 1000s of biochemical reactions, all metabolism is interrelated
2. Control = efficiency
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1000s of reactions, all interlinked
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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
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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
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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
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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
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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
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Substrate activation of threonine deaminase
active site
substrate substrate
T-state(less active)
R-state (more active)
structural transition
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The Concerted model for allosteric proteins Monod, Wyman and Changeux, MWC model
T-state
R-state
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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
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ATCase: homotropic regulation, substrate activation
sigmoidal curve
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The Concerted model for allosteric proteins Monod, Wyman and Changeux, MWC model
T-state
R-state
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S-shaped curve is combo of R-state and T-stateA simulation
pp. 281
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Aspartate transcarbamoylase (ATCase):Heterotropic regulation: feedback inhibition
ATCaseAspartate + carbamyl-P X UTP CTP
protein RNA DNA
heterotropic inhibition
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Oligomeric structure of ATCase
R-state T-state
r r r r r r
C = catalyticR = regulatory
gentle heat
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Kinetics of ATCase
inhibitor
activator
“C” subunits+/- CTP
Normal enzyme Normal + CTP
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pp. 264
X-ray structure of ATCase
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Structural Transition of ATCase
T = tenseR = relaxed pp.281
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X-ray structure of ATCase- “side” view
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CTP binding stabilizes the T-state
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Inhibition of ATCase by CTP
pp. 282
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ATP, heterotropic activator of ATCase
pp. 282
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Summarize ATCase
Aspartate, substrate, is: homotropic activator substrate activator
CTP, end product inhibitor, is heterotropic inhibitor end product inhibitor
ATP, ?????? is heterotropic activator
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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
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Summary of protein structure
1. Complex structure
2. Diverse, complex functions
3. High specificity
4. Enzymes most amazing, important
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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
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Light
Phototrophs HeterotrophsChemotrophs
Chemical oxidations
complex carbonglucose, amino acids, O2
CO2, H2O
Energy and material in the biosphere
Autotrophs
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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.
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Heterotrophic metabolismInterconversion of material and energy
Heterotrophic metabolism
Catabolism Anabolism(breakdown) (synthesis)yields energy, requiresprecursors energy,
precursors
How are catabolism and anabolism coupled?
coupled
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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
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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
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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)
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ATP couples energy between catabolism and anabolism
pp. 417
catabolism
anabolism
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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
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ATP is intermediate “high energy” compound
pp. 417
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ATP is intermediate “high energy” compound
Go’
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Coupling Oxidations/Reductions
catabolismReduced fuel Oxidized Fuel
NAD(ox) NADH(reduced)
Reduced Products Oxidized Precursors anabolism
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NAD+(ox) NADH(reduced)Nicotinamide adenine dinucleotide
NADP NADPH(PO4)pp. 420
H: (hydride ion)
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ATP/ADP couple energy of catabolism/anabolismNAD/NADH couple ox/red of catabolism/anabolism
Two coupling molecules
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Catabolism/Energy Metabolism Overview
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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)
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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
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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
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Stage 1 Stage 2 Stage 3
Glycolysis Overview
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Stage 1 glycolysis
pp. 435
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glycogen/starch
cell membrane
Glucose
many otherrxns
2 ATP 2ADP
regulatoryenzyme
Stage 1: Energy input, preparation
entry of many other sugars
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Stage 2: 1 6-carbon sugar to 2 3-carbon compounds
pp. 438
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Stage 2
pp. 438
Fructose 1, 6, bisphosphate
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Stage 3: energy yield
pp. 441
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Stage 3: NADH and ATP Produced
4 ADP 4 ATP
2 NAD 2 NADH
?
many other rxns
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There is no NET oxidation in glycolysis
pp. 446
Regeneration of NAD+ critical
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Regeneration of NAD+ critical
pp. 447
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What happens to pyruvate?depends on O2 and which organism
muscle
yeast
O2 presentO2 NOT present
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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
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What happens to pyruvate?depends on O2 and which organism
muscle
yeast
O2 presentO2 NOT present
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Metabolism of pyruvate in presence of O2
The Tricarboxylic Acid (TCA)Cycle
glycolysis -O2 lactateethanol
+ O2
pp. 477
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The TCA Cycle2 pyruvate 6CO2 + 6 H2O
Change in cellular location-eukaryotes; move from cytoplasm to mitochondria-prokaryotes; in cytoplasm with glycolysis
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Anatomy of a mitochondrion
TCA cycle enzymes
pp. 476
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A mitochondrion
pp. 476
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TCA Cycle overview (oxidations)
Pyruvate(3 carbons)
CO2
pp. 476
glycolysisNADH
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pp. 489
TCA Cycle
pyruvate
CO2
NADH
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TCA Cycle provides precursors for many things
Fatty Acids,Sterols
X
alanine
pp. 493
glycolysis
X
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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!
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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)
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Glucose 6CO2 + 6H2O + ATPenergy yield
ATP/glucose, with O2
Glycolysis 2 ATP
TCA Cycle 2 ATP (GTP)
Electron transport/ox. phosphorylation 26-30 ATP
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glucose + 6O2 6CO2 + 6H2Oa balance sheet
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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%
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Energy yield +/- O2
growth yield(grams of cells)
[glucose]
-O2
+O2
Growth of E. coli, a facultative organism, on glucose
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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)
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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
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Electron Transport: Prokaryotes: Cytoplasmic membrane Eukaryotes:Inner mitochondrial membrane
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TCA cycleElectron transport
A mitochondrion
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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-
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Complex I
Complex III
Complex IV
Respiratory Complexes
34 proteins, FMNFe-S proteins
Complex II
22 proteins, cytochromes
13 proteinsCytochromes,Cu
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Electron Carriers: Quinones
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Electron Carriers: Flavoproteins, Flavins: FADH2, FMN,
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Electron carriers: Flavoproteins
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Electron carriers: Cytochromes
protein
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Structure of cytochrome oxidasecomplex IV
13 proteins2 coppers2 hemes
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Inhibitors of electron transport
TCA cycle
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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)
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How does electron transport lead to ATP synthesis?Oxidative phosphorylation
Peter Mitchell: Chemiosmotic coupling
pp. 521
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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
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The ATP Synthase
Active in OX Phos
Not active in Ox Phos
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ATP Synthase, F1Fo ATPase,Proton translocating ATPase
electrontransport
[H+]
[H+] proton pore
ATP synthesis
ADP + Pi ATP
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NADH
Electron transport ATP synthase
Electron transport and oxidative phosphorylation
Mitochondrial inner membrane or bacterial cytoplasmic membrane
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Uncoupling proteins short circuit H+ gradientgenerate heat
pp. 533
Electron Transport goes faster and faster but can’t catch up!
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Proton gradient is energy source for many functions(Peter Mitchell got Nobel Prize)
Oxidative phosphorylation
pp. 535
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The cost of sequestration: mitochondrial transporters
But mitochondria impermeable to NAD/NADHHow do electrons from glycolysis get to mitochondria?
pp. 533
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glucose + 6O2 6CO2 + 6H2Oa balance sheet
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Electrons from cytosolic NADH into mitochondria: The glycerol phosphate shuttle
Electron transport
glycolysis
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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]
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Regulation of energy metabolism
glycolysis
TCA cycle
Electron transport,ox. phosphorylation
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Regulation within the mitochondria respiratory control
ET
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Regulation of glycolysis: phosphofructose kinase
Regulatory molecules
ATP -ADP +AMP +
Citrate -
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ATP inhibits phosphofructokinase
pp. 453
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Phosphofructokinase: many sitesA tetrameric protein
pp. 453
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Regulation of TCA Cycle: isocitrate dehydrogenase
isocitrate dehydrogenase
pp. 492
glycolysis
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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
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light rxns
The chloroplast
grana
dark rxns
pp. 543
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Chloroplast “grana”
pp. 542
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Light rxns: Overviewlight NADPH + ATP
pp. 542
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Light Energy Chemical Energy?
Pigments absorb light
Chlorophyll a
pp. 544
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Absorbtion spectrum of chlorophylls a & b
pp. 558
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Other pigments, antenna pigments, accessory pigments
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Other pigments
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Electron transfer from accessory pigment to rxn center
Antenna pigments
pp. 557
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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)
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Photosystem II
pp. 549
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Absorbtion of light by pigment
Return to ground stateheat
pp. 545
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Electron transfer: charge separation
replace e-
transfer e-
pp.545
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The “Z” scheme of photosythesis
2H2O + NADP+ O2 + NADPH
O2
light
proton gradient
pp. 553
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How is ATP made? photophosphorylation
Jagendorf showed H+ gradientin chloroplasts makes ATP
pp. 554
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PSII PSIcyt bf
light
light
pp. 555
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I II III
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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
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Melvin Calvin’s Nobel Experiment
14CO214C-X
Chromatography
algae
identify
light
pp. 567
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What is CO2 acceptor ?
O14C O
CH2-OH O CH2-O-P-O O
3-phosphoglycerate
What is 2-carbon CO2 acceptor?
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Ribulose bisphosphate carboxylase“Rubisco”- the most abundant enzyme on earth
Rubisco
5-carbon 6-carbon 2 3-carbon
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The Calvin Cycle
Rubisco
carbohydrate scramble
starch
pp. 566
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Starch
Starch synthesis
pp. 570
CO2
Not just the reverse of glycolyis
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Lipids and Cell Membranes prokaryotic cells
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Cell Membranes eukaryotic cells
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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
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Common features of all cell membranes
1. Structure: phospholipids
2. Function: proteins
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Fluid Mosaic Model of Membrane Structure (Singer and Nicolson, 1972)
phospholipidprotein
pp. 343
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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-
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Other lipids
Sterols
Pigments: chlorophyll, carotene, etc.
Fat soluble vitamins
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Phospholipid
pp. 322
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Phospholipid
pp. 329
(lecithin)
polar R-groupphosphate
glycerolfatty acids
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pp. 328
Phospholipids: fatty acids “tails”
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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
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Some phospholipid “head groups”
Phosphatidyl-X
pp. 330
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Phospholipid: R-groups“head groups”
pp. 330
(lecithin)
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Phospholipids are amphipathic
Polar “head group”Non-polar “tails”
hydrophobic hydrophilic “tails” “head groups”
pp. 332
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Amphipathic molecules in H2O (micelles)
hydrophobic
hydrophilic
pp. 333
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Phospholipids in H2O“liposomes”
H2O
H2O
pp. 334
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Phospholipid bilayer
pp. 333
very hydrophobic and fluid olive oil
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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
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Phospholipid bilayer is very fluid:
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Phospholipid bilayer provides:
1. structure
2. matrix, support for proteins
3. permeability barrier to polar molecules
pp. 335
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Membrane Proteins
1. Proteins provide membrane functions
2. Each membrane has unique function,different protein compositions
3. Protein amounts vary
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Membrane proteins and the bilayer
Integral
Peripheralf
pp. 336
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An integral membrane protein: Bacteriorhodopsin
pp. 337
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Another “integral” membrane protein: prostaglandin synthase
pp. 339
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Hydrophobic amino acids anchor membrane proteins
Glycophorin: a red cell membrane protein
sugars pp. 341
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It is possible to predict membrane spanning proteins
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Proteins are “fluid” too
fast
fast
very slow
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Fluid Mosaic Model of Membrane Structure (Singer and Nicolson, 1972)
phospholipidprotein
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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?