Biochemistry Section of Bio 41

1
Biochemistry Section of Bio 41 (Fall 2009, 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. Dont
memorize structures 5. Dont memorize equations
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Proteins
(Polymers of amino acids)
(1-20)
? amino acid
found in proteins
not found in proteins but are found in nature
pp. 27
4
The 20 amino acids present in proteins
Nutritionally required in humans
http//www.jbc.org/cgi/content/full/277/37/e25
pp. 33
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Proteins
(Polymers of amino acids)
(1-20)
? amino acid
found in proteins
not found in proteins but are found in nature
pp. 27
6
Amino Acid R-groups Very Diverse
1. 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
7
R-group Polarity Types
Hydrophobic (non-polar) water hating Hydrophilic
(polar) water loving Charged (polar) water
loving Energetics Important
8
Amino Acids
hydrogen
carbon
nitrogen
oxygen
pp. 28
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Hydrophobic R-groups
sulfur
pp. 29
10
Hydrophobic, aromatic amino acids
pp. 30
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The Basic Amino Acids
pp. 32
12
Acidic Amino Acids
pp. 33
13
Amino acids are linked by the peptide bond
Every amino acid linked in the same way
pp. 34
14
The peptide backbone
pp. 35
15
The direction of the peptide chain
1
2
3
5..
.
C-terminal
N-terminal
pp. 35
16
Ionization of dibasic amino acids
Dipolar or Zwitterion
pH 2
pH 9.5
pH 7
fully protonated half protonated fully
deprotonated
17
Ionization State Varies with pH
(Dibasic amino acids)
pp. 27
18
Consider amino acids as acids or bases
What is pH? pH log10(1/H)
-log10H Consider a weak acid, HA lt-gt H
A- The equilibrium constant, Ka, for this rxn
is Ka HA-/HA What is pK? pKa
-logKa log(1/Ka) pK is the pH at which a
group is 50 ionized
19
Evaluating ionization state with pH and pK
The Henderson-Hasselbalch Equation pH pKa
log(A-/HA)
20
Dibasic
21
Titration of dibasic amino acid
What is a buffer?
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The amino and carboxyl are in peptide bond
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Tribasic amino acids
25
Titration of tribasic amino acids
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The Tribasic Amino Acids
Acidic Basic glutamic lysine aspartic arginine
histidine
COO- Glutamic (glu,E) H3N-C-CH2-C
H2-COO- H
Isoelectric point
pK1 (?-COOH) 2.2 Isoelectric pH pK2 (?-COOH)
4.3 (average 2 closest) pK3 (?-NH3)
9.7 3.25
COO- NH3 COO- NH3 H3N
COO-
27
Another type of covalent bond in proteins
disulfide bond
pp. 36
28
Proline an imino acid
pp. 29
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Protein Features
1. Diversity of function 2. Specificity of
action 3. Complexity of structure
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Specificity
HO
X
galactose
No reaction
32
How to explain?
1. Diversity of function 2. Specificity of
action 3. Complexity of structure
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(Genetically determined)
R1 R2 R3 R4 R5 H3N COO-
N
C
N
C
Oligomeric proteins 4 polypeptides or
subunits or protomers
34
(Stein and Moore- Nobel Prize) www.jbc/org/cgi/con
tent/full/280/9/e6
... .
SO3 3-_
.
Single amino acids
100
Fraction number
1
35
N-ala-gly-asp-phe-arg-gly-C
(ala,arg,asp,gly2,phe)
pp. 78
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(not much)
37
pp. 79
38
Break protein into small peptides to sequence
Specific protein cleavage methods proteolytic
enzymes trypsin cleaves after lysine,
arginine chymotrypsin cleaves after phe,
tyr,trp, leu, met chemical cyanogen
bromide cleaves after methionine
pp. 81
39
Trypsin Cleavage
pp. 80
40
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
41
(Over 100,000 protein sequences are known)
5. Compare to other known sequences learn
function all sequences in databases, easy to
compare 6. Comparisons to similar proteins from
other species provide evolutionary insight
42
Alternative to protein sequencing
Often easier to sequence gene and deduce protein
sequence
protein sequence DNA sequence inform each
other
pp. 83
43
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
44
InsulinComparative 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
45
Amino acid differences in insulin
8
9
Bovine (cow) insulin
pp. 36
46
Insulin Sequence Variation
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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 gt1.5 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
49
Comparison of cytochrome c sequences
50
Cytochrome-c similar sequences- similar
structures-same function
bacteria
bacteria
fish
50 amino acid difference
pp. 520
51
Molecular Clock
amino acid Evolutionary divergence differe
nces (millions of year) Human-monkey 1 50-60 H
orse-cow 3 60-75 Human-horse
12 70-75 Human-dog 10 70-75 Mammals-birds
10-15 280 Mammals-fish 17-21 400 Vertebrates
-yeast 43-48 1,100
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Molecular Evolution
53
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
hemoglobin 2. Function to carry oxygen from lungs
to tissues in red blood cells 3. Oligomeric
protein
4 polypeptides or subunits or protomers Each
?-subunit 141 amino acids Each ?-subunit 146
amino acids 1 heme (O2 carrier/subunit)
?
?
??
?
4. Many human hemoglobin mutations known, many
benign 5. Sickle cell disease, a molecular disease
55
Structure of Hemoglobin
56
Red blood cells sickle in low O2
low O2
Disease cells get trapped in small blood
vessels severe anemia, organ damage, death
57
RBC Flow thru capillary
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HbS forms filaments in absence of O2
59
HbS Filaments
60
Electrophoresis detects difference between HbA
HbS
HbA/HbA HbA/HbS HbS/HbS

migration
-
61
Electrophoresis of HbA and HbS
migration
-

62
Electrophoresis shows HbA(normal) HbS (sickle)
What is/are the difference(s) and how to
determine? 1. ? -subunits HbA HbS 2.
?-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
63
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)
65
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
67
Mercaptoethanol breaks disulfide bonds
pp. 51
68
Unfolding and refolding of ribonuclease primary
structure is sufficient Self assembly
Denature
Renature
Most stable structure
pp. 51-52
69
Insulin violates principle of self-assembly?
urea mercaptoethanol
X
native mature insulin
denatured mature insulin
remove urea mercaptoethanol
70
Insulin is made as precursor and processed
71
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
72
Summary of Primary Structure
1. Every protein of unique function has unique
sequence 2. Homologous proteins from different
species have very similar sequences and
structures insulin cytochrome-c 3. Sequence
differences between homologous proteins are 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
73
Higher Order Structure, 3-D 2o, 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 diffraction Protein
crystals Source of X-rays, 1.5 angstroms
wavelength Detector
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Larger atom, higher electron density
X-rays
pp. 96
75
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
free rotation
O C - C - N - C H
4. Built models, CPK models, predicted ?-helix
76
The peptide bond is planar
pp. 37
77
?-helix
3.6 amino acids/turn
pp. 41
78
Why does ?-helix form? (Energetically favorable)
1. Pauling and Corey showed that hydrogen bonds
stabilize the helix What are hydrogen bonds?
?-
Water O H H
?
pp. 9
79
Hydrogen bonds are weak H--------O 1-3
kcal/mole H O 100 kcal/mole
pp. 8
80
Hydrogen bonds in ?-helix
pp. 41
81
Each amino acid hydrogen bonds to an amino acid 4
down the chain
pp. 41
82
2o brings some amino acids closer together
pp. 41
83
Amount of helix varies 0-100
Ferritin
Coiled coil
Keratin (Hair)
pp. 42
84
2o structure, ?-sheets
1. Pauling and Corey also predicted ?-sheets 2.
Hydrogen bonding between chains
pp. 43
85
pp. 52
86
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
87
2o structure can be predicted?
1. Empirical data, which amino acids appear in
certain structures 2. Theoretical, energy
minimization, not so good
88
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 bound heme 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
89
Crystals of sperm whale myoglobin
90
Larger atom, higher electron density
X-rays
pp. 96
91
X-ray reflection pattern (intensities and
positions)
pp. 97
92
Electron density map (fourier transform)
pp. 97
93
Backbone atoms (John Kendrew, 1957)
1. 8 regions of helix, 70 helix 2. Proline and
other helix destablizing amino acids at
bends 3. Extremely compact, no room for water
inside 4. Hydrophobic R-groups inside 5.
Hydrophilic R-groups outside 6. Myoglobins from
different species have similar sequences
and similar structures 7. Final structure is most
stable
pp. 47
94
Polar amino acids outside hydrophobic inside
intact molecule
slice of molecule
charged
hydrophobic
pp. 47
95
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 surface salt, pH oligomer
protomers 5. Hemoglobin good example
96
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
97
Hemoglobin evolved from myoglobin
heme
Striking structural similarity with only 24/141
identical amino acids between myoglobin, Hb ?,
Hb ?
98
Hemoglobin, ?2 ?2
99
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
100
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 metabolism 4. Hb
picks up CO2 and H , products of metabolism,
and return to lungs 5. Hb releases CO2 and H
and picks up O2 How can Hb both bind and
release O2?
101
Perutz noted structure of oxy and deoxy
hemoglobin differ
?
?
O2
?
?
?
?
?
?
crystals crack
deoxy Hb oxy Hb
Not true for myoglobin!
102
Structural change upon oxygenation
O2
(tense)
(relaxed)
deoxy Hb oxy Hb
pp. 189
103
Structural change on O2 binding subtle
104
O2 binding by Hb is cooperative, allosteric (O2
binding regulated by O2) (homotropic regulation)
hyperbolic curve
sigmoid curve
pp187
105
The Concerted model for allosteric proteins
Monod, Wyman and Changeux, MWC model
T-state, low O2 binding
R-state, high O2 binding
pp189
106
The sequential model for allosteric
proteins Daniel Koshland
R-state
T-state
Increasing O2 binding affinity
pp190
107
Oxygen binding by Hb is a cooperative, allosteric
process (homotropic regulation)
pp. 188
108
Hb O2 binding regulated by H (heterotropic
regulation)
lungs
muscle
pp.192
109
Hb O2 binding regulated by CO2 (heterotropic
regulation)
pp. 193
110
Hb O2 binding regulated by BPG 2,3
bisphosphoglycerate (heterotropic regulation)
lungs
tissues
pp.191
111
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
?
?
?
?
112
Hb F binds O2 more tightly than HbA
pp. 192
HbF binds BPG less well than HbA
113
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
114
Summary of proteins
1. Primary structure, sequence, determines all
higher order structures self
assembly 2.Peptide backbone can form 2o
structures, ? helix, ? 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
115
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
116
Study of enzymes in vitro 1. find a source 2.
prepare cell free extract 3. develop
assay measure the reaction catalyzed 4. purify
and study
117
Study of enzymes in vitro 1. find a source 2.
prepare cell free extract 3. develop
assay measure the reaction catalyzed 4. purify
and study
118
The enzyme assay an example
The enzyme, ?-galactosidase ?-linkage
(substrate) (products)
Nitrophenyl-glucose nitrophenol
glucose (colorless) yellow Measure
rate of appearance of yellow color
119
Enzyme Classification
1. Types of reactions catalyzed
120
2. Cofactor requirement -simple enzymes amino
acid R-groups -complex enzymes protein
cofactor
Vitamins Vit. B1 Riboflavin Niacin Vit.
B6 Pantothenate Biotin Vit B12 folate


pp. 207
121
Examples of coenzymes for oxidation/reduction
coenzyme
coenzyme
reduction
reduction
enzyme
enzyme
enzyme
oxidation
oxidation
(Reactions from the TCA Cycle)
122
Enzymes enhance rates of reactions
pp. 206
123
Enzymes are highly specific
proteolytic enzymes
Trypsin
Thrombin
pp. 207
124
How are enzymes such powerful and specific
catalysts?
Intimate association of the substrate with the
intricate, complex 3-D structure of the enzyme
125
Role of Free Energy, G, in reactions
(Review 1st and 2nd Laws and Entropy. pp 11-13)
Consider the reaction A B C D ?G
?Go RT ln CD/AB In biological
systems use ?Go, ?Go at pH 7 ?G ?Go
RT ln CD/AB standard free energy
gas constant temperature
126
?G ?Go RT ln CD/AB Since at
equilibrium, ?G 0, rearrange 0 ?Go RT
lnCD/AB or ?Go -RT ln CD/AB
since Keq CD/AB ?Go -RT ln
Keq ?G ?Go RT ln
CD/AB Even if ?Go is positive, ?G
can be negative (Problem Berg, pp. 210)
127
pp. 210
128
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
129
How do enzymes accelerate reaction rates?
1. They do not alter equilibrium or change ?G
values 2. They lower activation energy by
formation/binding of transition state
130
Enzymes lower activation energy
pp. 212
131
Consider a reaction ATP very slow ADP Pi
Energy ATP ATP ADP Pi
Energy ATP ATP ADP Pi Energy
very slow
Enzyme
very fast
ATP transition state, transition structure
132
Linus Pauling strikes again!
I think that enzymes are molecules that are
complementary in 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) 707
pp. 212
133
Enzymes bind substrate transition states
Formation of the enzyme-substrate complex,
ES Enzyme kinetics
Saturation curve, Saturation kinetics Active
site
enzyme catalyzed rxn
V
non-catalyzed rxn
pp. 213
S
134
Evidence for active site and ES complex
1. Saturation kinetics implies an active site
and ES complex A discrete place in the enzyme
where substrate binds
135
Active sites are complementary to the substrate
The lock and key
pp. 215
136
Evidence for active site and ES complex
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
pp. 213
Note active site residues
137
Induced Fit, Daniel Koshland 1958 hand in
glove
pp. 215
138
Induced fit in carboxypeptidase
glu 270
tyr 248
arg 145
139
Induced fit in carboxypeptidase
140
Active site of carboxypeptidase
OH
enzyme
141
Why do enzymes have such complex
structures? Active site, catalytic residues come
from entire molecule (carboxypeptidase)
his arg glu arg asn arg his tyr glu
C
N
1 69 71 72 127
144 145 196 248 270
307
but only 9/307 so why are 307 necessary?
142
Michaelis-Menten (1913) Model Accounts for Enzyme
Kinetics
Vo
S
k1
k2
E S ES E P
k-1
k-2
pp. 213
143
k1
k2
E S ES E P
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 dES/dt 0 4. E
ltltltS. Does NOT mean enzyme is saturated with
substrate From these assumptions and simple rate
equations derive M-M equation Berg, pp. 201-203
Vo Vmax S Km S
144
Steady State Kinetics
msec
145
k1
k2
E S ES E P
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 dES/dt 0 4. E
ltltltS. Does NOT mean enzyme is saturated with
substrate From these assumptions and simple rate
equations derive M-M equation Berg, pp. 217-219
v Vo Vmax S Km S
146
k2
k1
E S ES E P
k-1
k-2
v Vo Vmax S Km S
v or Vo dP/dt or -dS/dt initial
velocity Vmax maximum rxn velocity velocity
limit as S infinity Km k-1 k2
Michaelis constant k1 when k2 ltltltltlt k-1
then Km k-1 ES k1
ES Km is measure of affinity of enzyme for
substrate A 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
147
Vo is measure of initial rates
high
low
pp. 217
148
k2
k1
E S ES E P
k-1
k-2
Vo Vmax S Km S
Vo dP/dt or -dS/dt initial
velocity Vmax maximum rxn velocity velocity
limit as S infinity Km k-1 k2
Michaelis constant k1 when k2 ltltltltlt k-1
then Km k-1 ES k1
ES Km is measure of affinity of enzyme for
substrate A 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
149
k1
k2
E S ES E P
k-1
k-2
Vmax Vo at S infinity Km S at 1/2 Vmax
pp. 217
150
Factors that influence enzyme activity
1. Substrate concentration 2. Coenzyme
concentration 3. Temperature 4. pH
a c t i v i t y
urease
trypsin
pepsin
100
enzyme rxn
max activity
chemical rxn
1 2 3 4 5 6 7 8 9 10
25 30 35 40 45 50 55 60 65
pH
Temp
Interesting biology thermophilic
organisms acidophilic organisms
151
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)
152
Lineweaver-Burk Plot for Km and Vmax
1/Vo 1/Vmax Km/Vmax . 1/S
pp. 220
153
The enzyme assay How much enzyme is present?
Use optimal pH, temperature, Saturating
substrate and coenzyme Under saturating S
S gtgtgtgtgt Km
E 2x
P
OR
Vo
E 1x
E
Time
154
Final kinetic parameter turnover
number Molecules substrate converted to
product/per molecule of enzyme per second
pp.221
155
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
156
Not all enzymes obey Michaelis- Menten
kinetics allosteric, regulatory enzymes (more
later)
157
Inhibition of enzyme activity
1. Reversible inhibition -competitive -non-compe
titive 2. Irreversible inhibition
158
Competitive vs non-competitive inhibition
pp. 225
159
Competitive inhibition
1. Inhibitor structurally similar to
substrate 2. Can get formation of ES or EI
but not ESI 3. competition for active site
160
Dihydrofolate reductase purines, pyrimidines
(substrate)
competitive inhibitor
161
Kinetics of Competitive Inhibition
Vo
Overcome inhibition with more substrate
pp. 226
162
Kinetics of competitive inhibition
o
Change in apparent Km
No change in apparent Vmax
pp.228
163
Competitive vs non-competitive inhibition
164
Kinetics of non-competitive inhibition
pp. 227
165
Kinetics of non-competitive inhibition
No change in Km
Change in Vmax
pp. 228
Cannot overcome non-competitive inhibition with
more substrate
166
Inhibition of enzyme activity
1. Reversible inhibition -competitive -non-compe
titive 2. Irreversible inhibition
167
Irreversible inhibition
Potent nerve gas, DIPF, blocks
acetylcholinesterase, necessary for transmission
covalent bond
active site serine
Evidence for active site residues
pp. 229
168
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
169
Regulation of enzyme activity
1. Necessity for regulation, 1000s of
biochemical reactions, all metabolism is
interrelated 2. Control efficiency
170
1000s of reactions, all interlinked
171
Two major regulatory strategies
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
172
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
173
Physiology of allosteric enzymes
Consider 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 F
inhibition
F inhibits C-gtD partially inhibits A -gt B
D E F A B C G H I
174
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
175
Threonine Deaminase Homotropic Activation
heterotropic inactivation
homotropic activation
threonine deaminase threonine B C D isoleu
cine proteins proteins
90
It takes a smaller change in S to go from
10 to 90 activity
Vo
M-M enzyme
dB/dt
threonine deaminase
10
threonine
176
Substrate activation of threonine deaminase
active site
substrate
substrate
structural transition
T-state (less active)
R-state (more active)
177
The Concerted model for allosteric proteins
Monod, Wyman and Changeux, MWC model
T-state
R-state
178
Aspartate transcarbamoylase (ATCase) An
allosteric enzyme (The physiological context)
homotropic activation
heterotropic inhibition
ATCase Aspartate
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
179
ATCase homotropic regulation, substrate
activation
sigmoidal curve
180
The Concerted model for allosteric proteins
Monod, Wyman and Changeux, MWC model
T-state
R-state
181
S-shaped curve is combo of R-state and
T-state A simulation
pp. 281
182
Aspartate transcarbamoylase (ATCase)Heterotropic
regulation feedback inhibition
heterotropic inhibition
ATCase Aspartate
carbamyl-P X UTP CTP protein RNA DNA
183
Oligomeric structure of ATCase
gentle heat
r
r
r
r
r
r
C catalytic R regulatory
R-state
T-state
184
Kinetics of ATCase
activator
C subunits /- CTP
inhibitor
Normal enzyme
Normal CTP
185
X-ray structure of ATCase
pp. 264
186
Structural Transition of ATCase
T tense R relaxed
pp.281
187
X-ray structure of ATCase- side view
188
CTP binding stabilizes the T-state
189
Inhibition of ATCase by CTP
pp. 282
190
ATP, heterotropic activator of ATCase
pp. 282
191
Summarize ATCase
Aspartate, substrate, is homotropic
activator substrate activator CTP, end
product inhibitor, is heterotropic inhibitor
end product inhibitor ATP,
?????? is heterotropic activator
192
Summary enzyme regulation
1. 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
193
Summary of protein structure
1. Complex structure 2. Diverse, complex
functions 3. High specificity 4. Enzymes most
amazing, important
194
What do enzymes do? Metabolism
1. All biochemical reactions are interrelated,
integrated 2. General discussion bacteria
to humans 3. Strategies important reactions in
pathways energetics important regulation
important
pp. 410
195
Energy and material in the biosphere
Light Phototrophs Heterotrophs Chemotrophs
Chemical oxidations
complex carbon glucose, amino acids, O2
Autotrophs
CO2, H2O
196
Heterotrophic requirements
E . coli Leuconostoc Humans (bacteria)
(bacteria) Carbon/Energy glucose glucose
glucose Nitrogen NH3 NH3
NH3 19 amino acids 9 aa 4
nucleotides 8 vitamins 15 vit.
Elements Na, K, Mg, Ca, Zn, Fe, PO4, SO4 etc.
197
Heterotrophic metabolism
Interconversion of material and energy
Heterotrophic metabolism Catabolism Anaboli
sm (breakdown) (synthesis) yields
energy, requires precursors energy,
precursors
coupled
How are catabolism and anabolism coupled?
198
Bioenergetics/Thermodynamics
catabolism/respiration ?Go -686
kcal/mol C6H12O6 6O2 6CO2 6H2O
(sugar) ?Go 686 kcal/mol
anabolism/photosynthesis
Remember For the rxn A B C D
?G ?Go RT ln CD/AB
199
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
200
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)
201
ATP couples energy between catabolism and
anabolism
anabolism
catabolism
pp. 417
202
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
adenine
phosphoanhydride
ribose
203
ATP is intermediate high energy compound
pp. 417
204
ATP is intermediate high energy compound
?Go
205
Coupling Oxidations/Reductions
catabolism Reduced fuel Oxidized
Fuel NAD(ox) NADH(reduced) Reduced
Products Oxidized Precursors anabolism
206
NAD(ox) NADH(reduced) Nicotinamide adenine
dinucleotide
H (hydride ion)
pp. 420
NADP NADPH
(PO4)
207
Two coupling molecules
ATP/ADP couple energy of catabolism/anabolism NAD
/NADH couple ox/red of catabolism/anabolism
208
Catabolism/Energy Metabolism Overview
209
Glucose catabolism
Glucose 6CO2 6H2O (C6H12O6) (requires
O2) ?Go -686 kcal/mol
Occurs in 3 stages 1. Glycolysis 2. TCA
cycle 3. 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)
210
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
diphosphate rxn 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 pathway Glycolysis
yeast
no O2
muscle
no O2
inhibitor
X
211
What to know about glycolysis?
1. Relate structures to each other, dont
memorize 2. Dont memorize enzyme names, except a
few 3. Know general rxn sequences 4. Follow
carbon, phosphates (ATP/ADP), electrons
(NAD/NADH) 5. Understand rxn energetics 6. Where
in cell rxns take place 7. How rxns are
integrated 8. How rxns are regulated
212
Glycolysis Overview
Stage 1 Stage 2 Stage 3
213
Stage 1 glycolysis
pp. 435
214
Stage 1 Energy input, preparation
Glucose
glycogen/starch
cell membrane
2 ATP 2ADP
many other rxns
entry of many other sugars
regulatory enzyme
215
Stage 2 1 6-carbon sugar to 2 3-carbon compounds
pp. 438
216
Stage 2
Fructose 1, 6, bisphosphate
pp. 438
217
Stage 3 energy yield
pp. 441
218
Stage 3 NADH and ATP Produced
4 ADP 4 ATP
2 NAD 2 NADH
many other rxns
?
219
There is no NET oxidation in glycolysis
Regeneration of NAD critical
pp. 446
220
Regeneration of NAD critical
pp. 447
221
What happens to pyruvate? depends on O2 and
which organism
O2 present
O2 NOT present
muscle
yeast
222
Features of glycolysis
1. All enzymes are soluble in cytoplasm of
cells 2. In some organisms, glycolysis is all
there is Anaerobes facultative organisms in
absence of O2 red blood cells tissues like
muscle in absence of O2 3.End product depends on
organism 4. No NET change in oxidation state 5.
Many side rxns, not all carbon goes to
pyruvate 6. Energy yield glucose 2 ADP 2
lactate 2 ATP theoretically 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
whats next?
223
What happens to pyruvate? depends on O2 and
which organism
O2 present
O2 NOT present
muscle
yeast
224
Metabolism of pyruvate in presence of O2 The
Tricarboxylic Acid (TCA)Cycle
-O2
lactate ethanol
glycolysis
O2
pp. 477
225
The TCA Cycle
2 pyruvate 6CO2 6 H2O
Change in cellular location -eukaryotes move
from cytoplasm to mitochondria -prokaryotes in
cytoplasm with glycolysis
226
Anatomy of a mitochondrion
TCA cycle enzymes
pp. 476
227
A mitochondrion
pp. 476
228
TCA Cycle overview (oxidations)
CO2
Pyruvate (3 carbons)
glycolysis
NADH
pp. 476
229
NADH
pyruvate
CO2
TCA Cycle
pp. 489
230
TCA Cycle provides precursors for many things
glycolysis
alanine
Fatty Acids,Sterols
X
X
pp. 493
231
Summary TCA cycle
1. All carbon lost as CO2 2. Gain 5 X 2e- as 4
NADH, 1 FADH2 3. Gain 1 GTP, ATP equivalent 4.
Occurs prokaryotes, cytoplasm eukaryotes,
mitochondria/matrix 5. Many side
reactions acetyl-CoA -gt -gt fats oxaloacetate
-gt -gt aspartic pyruvate -gt -gt alanine
?-ketoglutaric -gt -gt glutamic So whats left? 5
pairs of electrons!
232
Electron Transport/Oxidative phosphorylation (inn
er mitochondrial membrane)
TCA Cycle
NAD
Back to TCA cycle
ADP ATP ADP ATP ADP ATP
2e-
2e-
FADH2
3 ATP/2e- (from NADH) 2 ATP/2e- (from FADH2)
FAD
Back to TCA cycle
H2O
233
Glucose 6CO2 6H2O ATP energy yield
ATP/glucose, with O2 Glycolysis
2 ATP TCA Cycle 2 ATP (GTP) Electron
transport/ox. phosphorylation 26-30 ATP
234
glucose 6O2 6CO2 6H2O a balance sheet
235
(No Transcript)
236
Overall energy yield
glucose 6O2 6CO2 6H2O ?Go
-686 kcal/mol
30 ATP 30 H2O 30 ADP 30Pi ?Go
-219 kcal/mol
219/686 X 100 32
237
Energy yield /- O2
Growth of E. coli, a facultative organism, on
glucose
O2
growth yield (grams of cells)
-O2
glucose
238
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
?
(ox)
Redox potential tendency to donate or accept
electrons 2H 2e- H2 Eo 0.00 at pH
7 Eo -0.42 volts
239
Using Standard Redox Potentials
Consider NADH H 1/2O2 H2O NAD
?
Write half rxns NAD H 2e- NADH Eo
-0.32 volts 1/2 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
pp 508
?Go -2 X 23 X 1.14 -52kcal/mol
240
Electron Transport Prokaryotes Cytoplasmic
membrane EukaryotesInner mitochondrial
membrane
241
A mitochondrion
TCA cycle
Electron transport
242
The electron transport chain (inner
mitochondrial membrane)
Eo ?Eo ?Go ATP
-0.32
2e-
inner membrane
0.27 -12.1 1
-0.05
0.05 -2.5
0.00 0.22
0.22 -10.1 1

0.04 -1.9
0.26
0.02 -0.9
0.28
0.54 -25 1
0.82
243
Respiratory Complexes
34 proteins, FMN Fe-S proteins
Complex I
Complex II
22 proteins, cytochromes
Complex III
13 proteins Cytochromes, Cu
Complex IV
244
Electron Carriers Quinones
245
Electron Carriers Flavoproteins, Flavins
FADH2, FMN,
246
Electron carriers Flavoproteins
247
Electron carriers Cytochromes
protein
248
Structure of cytochrome oxidase complex IV
13 proteins 2 coppers 2 hemes
249
Inhibitors of electron transport
TCA cycle
250
Electron Transport/Oxidative phosphorylation (inn
er mitochondrial membrane)
TCA Cycle
NAD
Back to TCA cycle
ADP ATP ADP ATP ADP ATP
2e-
2e-
FADH2
3 ATP/2e- (from NADH) 2 ATP/2e- (from FADH2)
FAD
Back to TCA cycle
H2O
251
How does electron transport lead to ATP
synthesis? Oxidative phosphorylation
Peter Mitchell Chemiosmotic coupling
pp. 521
252
Energetics of Ion (Proton) Gradients The Proton
Motive Force
???G RT ln(c2/c1) ZF ?V concentration elect
rical c2/c1 concentration difference across
the membrane Z electrical charge of ion
transported, H 1 F Faraday, electrical
constant V electrical potential across membrane
253
The ATP Synthase
Active in OX Phos
Not active in Ox Phos
254
ATP Synthase, F1Fo ATPase, Proton translocating
ATPase
ADP Pi ATP
ATP synthesis
H
electron transport
H
proton pore
255
Electron transport and oxidative phosphorylation
Mitochondrial inner membrane or bacterial
cytoplasmic membrane
NADH
ATP synthase
Electron transport
256
Uncoupling proteins short circuit H
gradient generate heat
Electron Transport goes faster and faster but
cant catch up!
pp. 533
257
Proton gradient is energy source for many
functions (Peter Mitchell got Nobel Prize)
Oxidative phosphorylation
pp. 535
258
The cost of sequestration mitochondrial
transporters
But mitochondria impermeable to NAD/NADH How do
electrons from glycolysis get to mitochondria?
pp. 533
259
glucose 6O2 6CO2 6H2O a balance sheet
260
(No Transcript)
261
Electrons from cytosolic NADH into
mitochondria The glycerol phosphate shuttle
glycolysis
Electron transport
262
Regulation of Energy Metabolism The Energy
Charge
Glycolysis TCA cycle Electron Transport
Oxidative phosphorylation
Overall response to energy charge or energy
status ATP 1/2ADP OR ATP ATP
ADP AMP ADP
263
Regulation of energy metabolism
glycolysis
TCA cycle
Electron transport, ox. phosphorylation
264
Regulation within the mitochondria
respiratory control
ET
265
Regulation of glycolysis phosphofructose kinase
Regulatory molecules ATP - ADP AMP Citrate
-
266
ATP inhibits phosphofructokinase
pp. 453
267
Phosphofructokinase many sites A tetrameric
protein
pp. 453
268
Regulation of TCA Cycle isocitrate dehydrogenase
glycolysis
isocitrate dehydrogenase
pp. 492
269
Photosynthesis
light
ATP, NADPH
6CO2 6H2O C6H12O6 6 O2 ?Go 686
kcal/mol
Photosynthesis involves two parts 1. Light
reactions generate ATP, NADPH 2. Dark
reactions use ATP, NADPH, CO2 -gt sugar Occurs
in prokaryotes bacteria, blue green
algae, in cytoplasmic membrane eukaryotes
chloroplasts
270
The chloroplast
light rxns
dark rxns
grana
pp. 543
271
Chloroplast grana
pp. 542
272
Light rxns Overview light NADPH ATP
pp. 542
273
?
Light Energy Chemical Energy
Pigments absorb light
Chlorophyll a
pp. 544
274
Absorbtion spectrum of chlorophylls a b
pp. 558
275
Other pigments, antenna pigments, accessory
pigments
276
Other pigments
277
Electron transfer from accessory pigment to rxn
center
Antenna pigments
pp. 557
278
Two photosystems
Photosystem II Photosystem I chlorophyll
a 200 chlorophyll b 200 chlorophyll b
50 chlorophyll b 50 carotenoids 100 carotenoid
s 100 Rxn center pig. 1 (P680) rxn center 1
(P700)
279
Photosystem II
pp. 549
280
Absorbtion of light by pigment
Return to ground state heat
pp. 545
281
Electron transfer charge separation
replace e-
transfer e-
pp.545
282
The Z scheme of photosythesis 2H2O
NADP O2 NADPH
light
proton gradient
pp. 553
O2
283
How is ATP made? photophosphorylation
Jagendorf showed H gradient in chloroplasts
makes ATP
pp. 554
284
light
light
PSII
PSI
cyt bf
pp. 555
285
I
II
III
286
The dark rxns of photosynthesis CO2 fixation
ATP, NADPH
6CO2 6H2O C6H12O6 6O2
6CO2 18 ATP 12 NADPH 12 H2O C6H12O6 18
ADP 18 Pi 12 NADP 6H
3 ATP 2 NADPH/CO2
287
Melvin Calvins Nobel Experiment
Chromatography
algae
14CO2 14C-X
light
identify
pp. 567
288
What is CO2 acceptor ?
3-phosphoglycerate
O 14C O CH2-OH O
CH2-O-P-O O
What is 2-carbon CO2 acceptor?
289
Ribulose bisphosphate carboxylase Rubisco- the
most abundant enzyme on earth
Rubisco
5-carbon 6-carbon 2 3-carbon
290
The Calvin Cycle
Rubisco
carbohydrate scramble
starch
pp. 566
291
Starch synthesis
Starch
Not just the reverse of glycolyis
CO2
pp. 570
292
Lipids and Cell Membranes prokaryotic cells
293
Cell Membranes eukaryotic cells
294
Membrane functions
1. Define cell plasma membrane, cell limit 2.
Compartmentalize organelles, mitochondria
etc. 3. Interaction with environment
permeability barrier solute transport 4.
Organize functions electron transport
295
Common features of all cell membranes
1. Structure phospholipids 2. Function proteins
296
Fluid Mosaic Model of Membrane Structure
(Singer and Nicolson, 1972)
protein
phospholipid
pp. 343
297
Lipids very diverse class of biomolecules
(insoluble in water)
Glycerolipids CH2-OH CH2-OH derivatives
of glycerol CH2-OH 1. Triglycerides (storage
lipid) O CH2-O-C-(CH2)nCH3
O CH2-O-C-(CH2)n-CH3
O CH2-O-C-(CH2)n-CH3 2. Phospholipids
O CH2-O-C-(CH2)n-CH3 O CH2-O-C-(CH2)n
-CH3 O- CH2-O-P-O-R O-
298
Other lipids
Sterols
Pigments chlorophyll, carotene, etc. Fat
soluble vitamins
299
Phospholipid
pp. 322
300
Phospholipid
fatty acids
glycerol
polar R-group
phosphate
(lecithin)
pp. 329
301
Phospholipids fatty acids tails
pp. 328
302
The type of fatty acid makes a difference
Unsaturated fatty acid (one cis double bond)
Saturated fatty acids (no double bonds)
Affects physical and functional properties of
membrane
pp. 338
303
Some phospholipid head groups
Phosphatidyl-X
pp. 330
304
Phospholipid R-groups head groups
(lecithin)
pp. 330
305
Phospholipids are amphipathic
Non-polar tails
Polar head group
hydrophobic hydrophilic tails
head groups
pp. 332
306
Amphipathic molecules in H2O (micelles)
hydrophobic
hydrophilic
pp. 333
307
Phospholipids in H2O liposomes
H2O
H2O
pp. 334
308
Phospholipid bilayer
very hydrophobic and fluid olive oil
pp. 333
309
Phospholipid composition of cell membranes very
complex
Phospholipid structural variables 1. 10-15
different fatty acids 2. 2 positions in glycerol
backbone 3. 6-10 head groups Even in simple
membrane, 50-100 individual molecular species
310
Phospholipid bilayer is very fluid
311
Phospholipid bilayer provides
1. structure 2. matrix, support for proteins 3.
permeability barrier to polar molecules
pp. 335
312
Membrane Proteins
1. Proteins provide membrane functions 2. Each
membrane has unique function, different protein
compositions 3. Protein amounts vary
313
Membrane proteins and the bilayer
Integral
f
Peripheral
pp. 336
314
An integral membrane protein Bacteriorhodopsin
pp. 337
315
Another integral membrane protein
prostaglandin synthase
pp. 339
316
Hydrophobic amino acids anchor membrane proteins
Glycophorin a red cell membrane protein
sugars
pp. 341
317
It is possible to predict membrane spanning
proteins
318
Proteins are fluid too
fast
fast
very slow
319
Fluid Mosaic Model of Membrane Structure
(Singer and Nicolson, 1972)
protein
phospholipid
320
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?