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Course Syllabus
Biochemistry 411
Meeting Time: M-F 11AM or 1PM
Instructor: Dr. Rick Thomas
Phone: 881-7461
Text: Biochemistry 3rd Ed by Voet and Voet
Fall 2005
Place: EPS 317
Office: 301D EPS
Office Hours: 11-12 M-F
Prerequisites: Chem121-2, 341-2, Biol 110-20
Course Content: The course topics are protein structure, enzyme activity and regulation, and metabolism of
carbohydrates, fats, and proteins. The metabolism involves the transfer of the energy content from these
major food components to a usable form (ATP) and the storage of these fuels. The course is intended to
allow the student to develop an understanding of the interconnections among the metabolic pathways and
how they are regulated to serve the needs specific to various conditions (produce ATP for sprint or for rest,
store fuel after meal, and mobilize stored fuel in hunger).
Unit Topics
I: Protein Structure and Activity............……...................................
II: Enzymes and Energy Transfer................………………….…......
III: Overview metabolism, Glycolysis, Gluconeogenesis..,…….......
IV: TCA Cycle, Electron Transport, Oxidative Phosphorylation…...
V: Glycogen, Fatty Acids, Amino Acids,..........................................
VI: Integration of Metabolism...........................................................
* - these chapters will be used only for selected topics.
Chapters
2, 4, 8, 9*,10
3*, 13, 14,15*
16, 17, 23*
21, 22
18, 25, 26
27
Grading: There will be five exams and a comprehensive final exam. The final score will be calculated as
follows:
400 points - four best exams
400 " - final exam
800 "
-total / 0.8= 1000 (maximum possible)
A grade of A will be assigned if the final score is 890 - 1000, B if 790 - 889, C if 690 - 789, D if 590 - 689, and
F if less than 590.
If possible, exams will be scheduled for the second class meeting after completion of each unit.
Attendance at all scheduled meetings is strongly encouraged. This, along with frequent study of your notes
will make it much easier to get the most out of the course. Readings from the text by Voet and Voet should
be used to supplement topics covered in class and done as early in each unit as possible.
A student who attends all lectures will have a bonus of 10 points added to the total (out of 1000)
THERE WILL BE NO MAKEUP EXAMS
Absence from an exam will result in a score of zero. The lowest test score or one zero will be dropped. A
student that misses exactly two exams will have one of them replaced with a score equivalent to that on the
final exam. A student that misses more than two exams will have one or more zeroes included in the final
average.
Biochemistry 419 Laboratory Spring 2006
The lab is NOT a REQUIRED part of the course. The lab is strongly recommended by UT Memphis for
pre-pharmacy students. All students who are interested in the content are encouraged to enroll. The
sequence of experiments will include most of the following, based on student preferences:
1. Fractionation of a Red Blood Cell lysate, Partial purification of hemoglobin by ion-exchange
chromatography
2. Estimation of hemoglobin molecular weight by gel permeation chromatography
3. Estimation of hemoglobin molecular weight by SDS polyacrylamide gel electrophoresis; subunit
composition of Hb
4. Enzyme kinetics and Inhibition and the affinity constant for acid phosphatase
5. Evolutionary relationships among serum proteins by immunodiffusion and electrophoresis and detection of
sickle cell anemia by electrophoresis
6. PCR, the polymerase chain reaction to amplify specific DNA
7. Genetic engineering including:
a. restriction endonuclease digestion of target and plasmid DNA
b. production of recombinant DNA by ligation
c. transformation of bacteria with recombinant DNA
d. isolation of recombinant DNA and analysis by agarose gel electrophoresis
8. Isolation and properties of eukaryotic DNA.
9. Production, isolation and purification of a “valuable”, fluorescent recombinant protein
Grading will be based on the same scale as lecture (89-100:A, etc), and:
30 percent on performing experiments and quality of results
30 percent on lab reports
40 percent on quizzes
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BIOCHEMISTRY 411
UNIT I Protein Strucure and Function
Variety of protein functions: enzymes, transport and storage, muscle contraction, immunity, hormone and
neurotransmitter receptors, membrane transport, control of gene expression, control of growth and
differentiation. Proteins are the active and controlling agents in nearly all biological processes.
Chapter 2.Water Properties and Solutions
I.A.1. polar: the water molecule is bent, not linear. The "O end" is relatively negative and the "H ends" are
relatively positive. (Fig 2-1)
2. cohesive: each molecule in liquid water is Hydrogen bonded to 3 or 4 others (3.4=average over time)
and in solid water (ice) to 4 others. (Fig 2-2, 3)
3. dynamic: H bonds give liquid water an ice-like structure over short times and short distances. But the
water molecules move and reorient about a trillion (1012) times per second.
B.1. Water's polarity enables it to dissolve polar molecules and ions by hydrating them (Fig 2-5).
Interaction with water competes with ion pairing and H bonding between other molecules or groups. (Fig 2-6)
2. Nonpolar molecules (hydrocarbons) do not dissolve in water ("like dissolves like"). However, when a
mostly nonpolar molecule has a polar or ionic group it may. Fatty acids have long Carbon chains with a
carboxylate group on the end. (Fig 2-7) These aggregate to form globules (micelles) or bilayers (as in
membranes) in which the negatively charged carboxylates interact with water on the surface and the Carbon
chains interact with each other on the interior away from water (fig 2-8). A micelle is “a model” for globular
protein structure.
(Biologically important properties of water arising from polarity and cohesiveness: 1.high heat capacity allows
body to absorb much heat, as when jogging, with very small temperature change. 2.high heat of vaporization
allows body to release a lot of heat by evaporation of sweat or panting. 3.expansion upon freezing allows it to
float, insulating lakes, preventing freeze through. Otherwise all water on earth would freeze, no life!)
Chapter 4.Amino Acids (AAs) (alpha amino carboxylic acids) (Fig 4-1)
I.A.ion formation, zwitterions (Fig 4-2)
B.peptide bonds; polypeptide chains of proteins: linear (unbranched) polymers. (Fig 4-3) (residue: water is
lost when peptide bond forms, the rest of the AA is the "residue" remaining in the di-, tri-, ...oligo(3 to 10)- or
poly- peptide)
C.Groupings by character of side chain (Table 4-1, p66,67)
1.nonpolar: gly, ala, val, leu, ile, pro, phe, trp, met
2.polar: a.hydroxyl: ser, thr, tyr b.amide: asn, gln c.thiol: cys (note disulfide bonds)
3.ionic: a.positive (basic): lys, arg, his b.negative (acidic): asp, glu
D.Acidic/basic side chains - titration curves: charge state vs. pH
II. stereochemistry: only L-AAs occur in proteins
Chapter 8. Protein Structure: A protein’s native structure is the one it has under the conditions in the cells
in which it naturally occurs. Some proteins have more than one native structure, but they are inactive or
nonfunctional in any other conformation. (fig17-5, 21-18) (conformation = "shape" dependent on bond
rotations, Fig 8-5)
I.Secondary Structure: local conformational structure, which may be repeated over a long distance.
(Primary structure is sequence of AA residues and location of disulfides)
A.peptide bond does not rotate (resonance). The chain behaves as one made up of 6-atom planes in
which the peptide group is trans, not cis. (Fig 8-1, 2). This leaves the 2 bonds to the alpha C as the only
ones which can rotate in each residue (these rotational angles are called "phi" and "psi": fig 8-4). Steric
factors limit the possible values for pairs of (phi, psi) (figs 8-6,7,8 and 9).
B.Alpha Helix: In a helix, for each 360 degree cycle, the chain extends along the axis around which the
helix winds (like a spiral staircase, the threads of a screw, the spring in a ballpoint pen, a phone cord or a
"slinky") (fig 8-10). In the alpha helix there are 3.6 residues per right handed turn (5.4A/turn, so 1.5A helix
length per residue). (Fig 8-11, 12 and 14b). The side chains project slightly down the chain and outward
from the “helical rod” (fig 8-12 in stereo).
C.Beta pleated sheet: the rotations are such that the peptide chain extends (3.5A/residue) in a zig-zag
pattern in a given direction (fig 8-16, 17 and 18).
D.Beta bends: turns from one segment of alpha helix or beta sheet to another (fig 8-22)
E.Omega loops (fig 8-23) occur on the surfaces of most proteins (recognition?)
F.Factors (interactions) that stabilize the alpha helix and beta sheet
1.alpha helix: a. each O of C=O forms a H bond with the H of the N-H of the fourth residue up the same
chain. b)each C=O and H-N of the backbone is a dipole and all of the C=O and H-N bonds point the same
way along the helix. These dipoles interact favorably, and the interactions add up. This increases stability
relative to all other conformations
2.beta sheet: each O of C=O forms a H bond with an H of an N-H on an adjacent chain or "looped back"
portion of the same chain.
3. each C=O and H-N of the backbone is a dipole and all of the C=O….H-N pairings are aligned dipoles
that point the same way along the helix. These aligned dipoles interact favorably, and the interactions add
up. This increases stability relative to all other conformations
4. alpha helix and beta sheet: a)in each case, the pair of phi, psi rotational angles that is repeated is one
which minimizes steric overlap (at least for small side chains).
5.favorable interactions of neighboring side groups: if a conformation places side groups in position to
have favorable interactions, these interactions will help to hold the molecule in this conformation.
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IMPORTANT POINT: The alpha helix has the same number of H bonds (its C=O......H-N) as these same
atoms would make with water if the chain were not in alpha helix (and all of these atoms were exposed to
water). This is what is meant by saying the alpha helix is “not stabilized by H bonds”. The alpha helix is not
stabilized more by these H bonds than the other conformation is by its H bonds to water.
Stability is relative: A is more or less stable than B (or equally stable). The H bonds do have a stabilizing
effect on the alpha helix, but the stabilizing effect is equal to that for the conformation with all of these atoms
exposed to water. And there is a sense in which the alpha helix and beta sheet are stabilized by H bonds:
relative to all conformations with fewer H bonds. These points apply in exactly the same way for β sheet.
E. Factors destabilizing:
1. steric hinderance: bulky side chains which would overlap if chain coiled into alpha helix or approached
other chain to form beta sheet. (Polyisoleucine can NOT be alpha helical)
2. electrostatic repulsion: like charged side groups too close (polyglutamic acid at pH = 7 is polyglutamate
- cannot be in alpha helix, but at pH = 2 it is poly glutamic acid, can be)
3. some AAs can not take on corresponding phi, psi (proline's alpha C is in a ring - no free rotation)
F.Nonrepetitive structures: on average, about half of a protein will not be in alpha helix or beta sheet. The
conformation at these locations is referred to as "loop" or "coil", but is specific and relatively constant for that
protein as opposed to the rapidly changing "random coil". Many proteins also have segments that are free
to change conformation.
II.Fibrous Proteins (which form fibers)
A.alpha Keratin of hair, wool (feathers, etc)
1.Consists mainly of alpha helical strands distorted slightly by twisting in pairs, the "coiled coil" (fig 8-27).
The two helices interact by contact of nonpolar side chains, which are spaced along each chain so as to
place them along the interface between the two chains. Neighboring coiled coils are linked by disulfide
bonds. The more S-S bonds, the harder the keratin (horns, nails).
2.Perms: the shape of a hair can be changed by chemically reducing (breaking) disulfide bonds between
chains, putting the hair in the desired shape, and reoxidizing (reforming) different disulfide bonds.
3.After reducing S-S bonds, hair and wool can be stretched to more than twice their length by breaking H
bonds (more easily with heat and water), illustrating the coiled nature of its alpha helices.
B.silk fibroin has a beta sheet structure (fig 8-17). It is almost fully extended, so it cannot be stretched. Its
length changes little as the stretching force increases until it breaks as covalent bonds of chains break.
III.Folded Structure of Globular Protein: each polypeptide chain of most proteins folds into a compact,
"globular" shape, its tertiary structure. Each molecule of a particular protein folds in exactly the same way.
Different proteins contain different proportions of alpha helix, beta sheet, and "loop" or “coil”, but most
contain some of each (fig 8-39c, 40, 41 and 45
A.General features: chains fold so that: (see part IV below for interactions)
1.a.nonpolar side chains point to the interior where they interact with each other (London) and minimize
contact with water (hydrophobic).
b.charged side chains are usually on the surface where they interact with water. (Or they may interact
with other side chains by ionic or H bonds.)
c.polar side chains are usually on the surface where they H bond with water. (Or they may H bond with
other polar side chains) (fig 7-45 for parts a,b, and c)
2.the atoms in the interior are very tightly packed, as in a crystal, rather than being separated by empty
spaces. This maximizes the London interactions among them.
3.domains, each consisting of a large chunk of the single chain, fold up independently of each other
without intersecting or overlapping. The domains interact with and fit closely to each other. Often each
domain will have a specific role in the overall activity of the protein (Fig 8-45).
B.Each protein folds into a specific pattern unique to that protein and identical for each molecule of
that protein. WHY? HOW? What causes this?
1.This occurs so as to:(see IV below for interactions)
a.maximize the favorable interactions among the side chains (H bonds, ionic, London)
b.minimize unfavorable interactions among side chains (ion repulsion, steric overlap)
c.maximize favorable interactions of side chains with water (polar and charged groups on
exterior surface H bond with water)
d.minimize unfavorable “interactions” of side chains with water (hydrophobic HCs to interior,
water excluded from interior)
2.The sum of the energies for these hundreds/thousands of interactions in a large protein is many
thousands of kJ. But a slightly different structure may involve breaking a few interactions and forming a few
others, so that the difference between two conformations may be only a few dozen kJ or less.
C. A protein's native tertiary structure may be altered by a variety of denaturing agents, such as high
temperature, acid/base, alcohols, detergents, and salts. These effects may result in a precipitation as well,
and always result in loss of activity. (Precipitation, like some other denaturing effects, may be reversible:
redissolve.)
IV. Energy Analysis for a Protein Folding Into Its Native Structure: a protein folds the way it does because
that is its most stable structure, the one that forms with the greatest energy release.
1. It is extremely unfavorable for a protein to have a specific folded structure as to the protein chain’s
entropy (ΔS < 0 for the chain.). (Entropy is the amount of disorder, which means change in position of
particles as time passes: Everything “wants to” move freely! The ions of a salt like NaCl or AgCl will move
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freely in an aqueous solution unless the ionic bonds that would hold them together in a solid crystal are
strong enough to overcome this tendency and the ion-dipole interactions with water.) In proteins, all (or
almost all) of the atoms have a fixed position in relation to each other, so even though the molecule has
random movements in the aqueous medium, this has a much lower entropy than random coil.
2. The tendency to fold: random coil ↔ native, depends on ΔG, and [ΔG = ΔH - T(ΔS)]. Both the ΔH and
the ΔS involve the changes for the polypeptide chain and for the water. [ΔG = -RT ln K]
3. The enthalpy change (ΔH) results from bonds (and interactions) forming and breaking. The ΔH for a
reaction can be calculated from the change in the bond energy.
For the reaction in which ethanol is “burned” in metabolism:
CH3CH2OH + 3 O2  2 CO2 + 3 H2O
bonds broken (+: energy must be put in)
bonds formed (-: energy released)
1 C-C..................1(347) = 347
4 O=C..........4(799) = 3196
5 C-H..................5(413) = 2065
6 O-H...........6(467) = 2802
1 C-O..................1(358) = 358
- 5998kJ
1 O-H..................1(467) = 467
Energy must be put in (+) to break a bond and so
3 O=O.................3(495) = 1485
Energy is released (-) when a bond is formed. So:
+4722
deltaH = +4722 -5998 = -1276kJ. This is very close
to the value that would be obtained by measuring heat
released in the burning of ethanol!
4. The ΔG of the protein and the ΔG of water add together to give the overall deltaG for the folding
reaction. SUMMARY: the protein folds the way it does because this conformation has the best overall result
for: [Interactions: (within the protein) + (between the protein and water) + (within water)] + [Freedom of
movement: (of water) + (of protein)]. This is the same as the concept in III.B.1. above.
Noncovalent (“Weak” or “Reversible”) Interactions
A.Contrast with covalent bonds: <30 kJ/mol vs. ~120-1000 kJ/mol. Also recall that heat energy (enthalpy) is
released when a bond (interaction) forms (energy must be "put in" to break a bond). A process is
"spontaneous" (can occur) if energy is released (ΔG < 0) (and is not spontaneous (will not occur without
proportional energy input) as written if energy is absorbed (ΔG > 0). If a process, say A ---> B, absorbs
energy and is not spontaneous, then the reverse, B ---> A releases energy and is spontaneous). So a
protein’s conformational structure will be maintained (stable) only to the extent that energy is released in
folding in that particular way.
B.Types (those with * are the most important of the side-chain interactions in stabilizing protein structure)
*1.ionic bond: (also known as ion pair, or salt bridge): oppositely charged ions attract (~20-40 kJ/mol in aq.
environment), (like charges repel). Vary with 1/r2, r = distance between Charges (atom nuclei).
*2.Hydrogen bonds: N-H or O-H ----:O or :N (Figs 2-2, 3 and 5)(~10-40 kJ/mol). Vary with 1/r3. H bonds are
stronger when one atom in the bond is in a charged group like -CO2- or -NH3+.
3.Dipole - dipole: polar molecules attract/(repel) other polar molecules as positions of unlike/(like) polarity
approach. (~10 kJ/mol) Vary with 1/r3. (Fig 8-57)
*4.London dispersion forces (instantaneous dipole-induced dipole): atoms of nonpolar groups
(hydrocarbons) attract (~4 kJ/mol) (at room temperature, thermal motion easily breaks bonds of ~2.5 kJ/mol)
Vary with 1/r6.
5.ion - dipole: water dipole interacts with charged group (but water forms strong H bonds with the side
chains of charged AAs.
(All five types of interactions are as strong as indicated only if the distance is optimal, much weaker as
distance increases or, for H bond, if D-H bond is not aligned with orbital of unshared pair on :A).
**6.hydrophobic effect: HCs associate with each other and "stay away" from the water as much as possible
(oil droplets float rather than dissolve).
a. Nonpolar hydrocarbons (HCs) inserted into water interfere with water-water interactions. To attain the
maximum water-water interaction at water-HC contacts, the water molecules form ordered arrangements
around the HC molecules. (figs 8-58, 59)
b. These ordered structures are unchanging, as if the water was frozen in an ice crystal, so they are
lower in entropy than the usual water structure. Processes will tend to occur spontaneously in the direction
in which entropy increases (other factors being equal). (The enthalpy change for water-HC contact, which
involves the changes in bonding (interactions) is negative, which favors oil dissolving in water.)
These ordered structures are lower in entropy than the usual water structure. Processes will tend to occur
spontaneously in the direction in which entropy increases (other factors being equal). (The enthalpy change
for water-HC contact, which involves the changes in bonding (interactions) is negative, which favors oil
dissolving in water. Table 8-5)
c. The hydrophobic effect, which is based on the tendency of water to attain the state of maximal entropy
by not being in contact with HCs, is the greatest net contributor to tertiary structure.
d. The hydrophobic effect is NOT an interaction or bond. But we will always include it along with those
above, as applicable, when considering the interactions involved in any situation.
Entropy is disorder. But what is disorder? It is the level of unpredictability of the future locations of particles.
Each ion in an NaCl crystal is in an almost fixed location over time: highly predictable, very low entropy. Each
ion in aqueous NaCl changes position often, as water molecules do (1012 times/second): highly
unpredictable, high entropy.
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V. The quaternary structure of a protein is the number and arrangement of individual polypeptide chains
(subunits). The various subunits are synthesized separately and fold into their tertiary structures
independently before associating with each other. (More later about specific examples).
Chapter 9 Protein Folding
I. What determines the tertiary structure of a protein? This is determined by its primary structure. That is, a
protein will fold on its own (“self-assembly”) into its most stable structure based on the interactions of its
side chains with water and each other. Exactly what tertiary structure will result from this for a given
polypeptide depends on the sequence of AA residues along the chain. (As explained in III A and B in Ch. 8
above).
A. The experimental evidence for this is summarized as follows: If reagents are added so as to cause the
protein to unfold it will become inactive. If conditions are then changed back to allow refolding, the protein
will revert to its native conformation, and regain its activity. fig 9-2
Experimental details:
1.treat protein (the enzyme RNase) with beta-mercaptoethanol (breaks -S-S- ---> -SH + HS-) and 8M
urea (interferes with weak interactions, especially the hydrophobic effect because the solvent, water is
“changed”)
2.result: "denatured", "random coil" conformation of protein molecule (constantly changing
conformation), no enzymatic activity in this medium
3.remove beta-mercaptoethanol, then later remove urea - result: 1% of enzymatic activity regained
4.remove urea before, or at same time as beta-mercaptoethanol - result: nearly all of enzymatic activity
regained.
5.Interpretation: in 3., the -S-S- bonds reform at random when the molecule is in the random coil form.
There are 105 different sets of 4 -S-S- bonds that can form from 8 cysteines, and only 1 of the 105 is the
same as in the native, functional arrangement. So only 1 of 105 (=1%) molecules can refold later, when the
urea is removed to native, active form. The 104 “incorrect” disulfide pairings prevent native folding.
On the other hand, in 4., the chain can refold through weak interactions of side chains ("fast reaction") before
the disulfides can reform ("slow reaction" with oxygen). The weak interactions of the side chains fold the
chain into the native form, "steering" the "correct" pairings of the disulfides. The native state results
from side chain interactions.
B. Interaction of residues on the protein’s interior is the major factor in the self-assembly of a folded
protein. Experiment: Link 8-residue chains of ala (these chains are water soluble) to each of the 11 free
amino groups on the surface of RNase. Then perform the experiment above. The same results are obtained:
major alterations in a significant fraction of surface residues and the interactions they have did not alter the
activity or conformation. Apparently, the hydrophobic effects involved in the interaction of the nonpolar
residues on the interior are the major factor in folding.
II. What are the steps in the folding process?
A. Protein Structure is Hierarchical
1. Proteins consist of small folded chunks that associate with each other. Each chunk consists of
consecutive residues along the length of the chain and usually associates with the chunks in the adjacent
segments of the chain. This would occur as consecutive segments are synthesized on a ribosome. Then
these larger chunks associate with similar larger chunks.
2. A protein usually consists of several domains, each of which consists of subdomains, which consist
of sub-subdomains...fig 9-5
B. The Folding Process
1. Folding of a denatured protein begins with a rapid “burst phase” in the first ~5 ms, in which much of
the secondary structure forms and most of the nonpolar side chains associate as a “molten globule” and
exclude most of the water in the interior of the folding protein. 8-Anilino-1-naphthalene sulfonate (ANS, pg
283) has three aromatic rings and is highly hydrophobic; its fluorescence is enhanced when in a nonpolar
environment. Much of this enhancement occurs during the burst phase as a result of binding to the
hydrophobic core when it is in a solution in which a denatured protein is folding.
2. Then, in the remainder of the first second, small but frequent movements allow the nonpolar side
chains on the interior to find their most stable packing and release any remaining water while the small
chunks of local structure associate into larger, more stable ones. Additional ANS binding occurs in this
phase.
3. Finally, the last movements bring all the groups into their most stable conformations during the next
few seconds.
(Note that the folding of some proteins in vivo (in living cells) involves interactions with folding accessory
proteins (skip details)).
Prelude to Protein Function.
1. A protein's ACTIVITY is what that protein does. For example, the reaction catalysed (including the
substrate(s) (reactants) and products it acts on) describes an enzyme's activity.
2. All protein activities involve specific binding, which is the interaction of the protein molecule with one or a
few other molecular structures. The specificity of these interactions, often allowing a protein to "recognize"
one specific compound among the many thousands it encounters in a living system, is based on exactly the
same principles as the formation of the unique tertiary structure of the protein: complimentary reversible
interactions and complimentary shape (see Ch. 8, IV for interactions).
3. Many proteins have variable activity. The variations occur as a result of subtle changes in conformation in
response to the binding of "signal" molecules. This is the basis of metabolic regulation. Specific details of
this process for hemoglobin will be covered.
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Chapter 10: Myoglobin (Mb) and Hemoglobin (Hb)
I.Reasons for study
A.Illustrate globular protein structure and structure -function relationship.
B.Illustrate the modulation of protein activity by "signal" molecules, one of the most important concepts in
biochemistry.(3. In prelude)
C.Importance of oxygen in energy yielding metabolism.
II.Myoglobin: the oxygen binding/storing protein of muscle
A.Oxygen binds to iron (II) ion of heme group (fig 10-1). Heme structure is mostly hydrophobic, consisting
of 4 pyrrole rings linked through methenes (-CH=) to form a "superring", with the iron ion coordinated to a N
of each pyrrole ring. Heme, a prosthetic group, is bound within a hydrophobic cleft of the Mb polypeptide (fig
10-11, 8-63). (Many proteins have tightly bound prosthetic groups. Heme is a typical one: 1. in being the
site where the chemical “reaction” occurs and 2. in having it's activity modified by being bound to the
polypeptide (see C. below).)
B.Mb has a polypeptide chain of about 150 AA residues is about 75 % alpha helical, has 8 helical segments
of 7 to 24 residues each (see Fig. 10-11 for backbone structure").
C.Since it is the heme that binds O2, why expend a large amount of energy to synthesize a polypeptide for
the heme to bind to?
1.Iron ion of free heme is oxidized (to 3+) by oxygen, destroying the ability to bind O2 reversibly. (For this
oxidation to occur, a heme-O2-heme "sandwich" must form. This was shown by preventing sandwich with
blocking groups, fig 10-5, which allows heme to bind oxygen reversibly. Similarly, by having the heme
bound in a crevice of Mb, this oxidation is prevented.)
2.(Not in Voet and Voet) Free heme binds CO (carbon monoxide) 25000 times as strongly as it binds
oxygen. In Mb (and Hb) the fifth position around the iron is coordinated to a his N and there is another his
side chain near the oxygen binding site at the sixth position (the distal his, E7, fig 10-12). This distal his
stericly decreases CO binding strength by 100x but does not weaken O2 binding. (Heme DOES NOT bind
carbon dioxide)
III.Hemoglobin: the O2 carrier protein of blood (is red, like Mb, and makes up the vast majority of the protein
of the red blood cell (RBC))
A.*Hb consists of 4 "subunits", each of which has a tertiary structure very similar to that of Mb. (Fig 8-63, 1013) (The subunits are each synthesized separately and then fold into their tertiary structures before
associating with each other non- covalently. Each subunit binds one heme, one O2.) Everything about Mb in
A.-C. above also applies to Hb.
B.*The 4 subunits are 2 identical subunits of one type and 2 of another. (Identical in having the same AA
sequence.) At various times during embryonic and fetal development, new RBC populations replace the
existing ones. The new RBCs contain Hb in which one or both types of subunit are different than in the
replaced RBCs. *After birth, human Hb has 2 alpha subunits and 2 beta. (alpha and beta as used here have
no connection with their use in alpha helix and beta sheet) (We will discuss the reason for this Hb/RBC
"switch" below.) *Each alpha chain has many points of contact with each beta chain, but there is little
alpha-alpha or beta-beta contact (see Fig 10-13). *One alpha-beta pair shifts in position relative to the other
upon O2 binding.
*: These statements describe the quaternary structure of Hb.
C. The tertiary structure of Mb is very similar to that of any Hb alpha or beta chain. The quaternary
structures of Hbs from different species are also very similar. However the AA sequences are quite different
for Mb vs. Hb or for Hbs from different species. These differences result from evolutionary changes in the
corresponding genes. There are certain residues which are invariant however, and these are vital to the
function of the molecule. For example, both his residues near the heme iron occur in approximately the
same location in all Hbs and Mbs.
Before we get into the O2 binding properties of Hb, let’s consider the idea of binding affinity for a protein, P
that has a site on it that binds either one of 2 substances, A or B:
PA ↔ P + A
Kd = [P][A]
[PA]
PB ↔ P + B
Kd = [P][B]
[PB]
If the dissociation constant, Kd has a value of 1.0 x 10-5 for PA and a value of 1.0 x 10-2 for PB, then we can
calculate the value of ΔG for each from ΔG = -RT ln K = - (8.314J/molK)(298K)(ln 1.0 x 10-5) = 28.5 kJ for PA
(and 11.4 kJ for PB). The entropy change, ΔS for each of these reactions is about the same, and [ΔG = ΔH T(ΔS)], so the difference is mainly in the ΔH. Therefore, breaking the bonds between P and A requires about
17 kJ more than breaking the bonds between P and B. So there are either stronger bonds between P and A
or there are more bonds than P to B. This is the idea of affinity. Note that that the higher affinity of P for A
(than for B) is associated with a lower value for the dissociation constant. Note also that when [A] = 1.0 x 10-5
M: 1.0 x 10-5 = [P](1.0 x 10-5)/[PA] or 1 = [P]/[PA] or [P] = [PA] = ½ [P]total. That is, the dissociation constant
is equal to the concentration of the substance (that binds to the protein) that causes half of the protein to be
bound to it. This concentration is lower for A than for B because P has a higher affinity for A.
D.1. The oxygen binding properties of Hb, without which large animals could not exist, result from
interactions among the 4 subunits. The binding of oxygen to Hb is "cooperative." This means that the Hb02
molecule binds the next O2 more tightly (with "greater affinity") than Hb does, and so on, with Hb(O2)3 binding
the next oxygen most tightly. The result is that Hb is able to release most of its oxygen in the capillaries near
active muscle (and other tissues), which it could not do if its O2 binding was like Mbs. So the lower affinity of
7
Hb for oxygen and the variation of affinity are essential to its function, which is to bind oxygen in the lungs
and release it at other places in the body. (see Fig 10-3)
Skip the Hill equation page 218,219 but note the results, in fig 10-4 (Y is the fraction of Hb’s sites that have
oxygen bound; slope indicates extent of subunit cooperativity, and Y=0.5 intercept indicates affinity:
a.At very low [O2] the slope is one, which indicates there is no effect of the subunits on each other before
first O2 binds. This extrapolates to a high P50, which indicates a low affinity.
b. At intermediate [O2], slope is about 3.0, so subunits are "working together". (slope of 4 is max possible
for protein with 4 subunits, all binding/releasing at same time)
c. At high [O2], slope goes back to 1.0. Once 3 oxygens are bound to "all" molecules of Hb they each
have one site left which is same for each Hb. Extrapolates to very low P50, high affinity.
2. In addition, the affinity of Hb for oxygen decreases as the concentrations of CO 2 and H+ increase. This
further enhances the release of O2 in muscle, where these levels are high and the binding of O2 in lungs
where these levels are low. It is not by chance that these 2 species regulate Hb affinity for O 2. Rather, the
CO2 and H+ specifically and unambiguously indicate the need for O2 (and the need for Hb to release it)
because they are directly related to O2 utilization:
C6H12O6 + 6 O2 ----> 6 CO2 + 6 H2O: CO2 is produced in proportion to O2 consumption; and
CO2 + H2O <----> H2CO3 <----> H+ + HCO3- : [H+] increases when and where [CO2] increases.
(other “fuel burning” also produces CO2 in proportion to O2 consumption; and other acids are also
produced in tissues where metabolism is active and O2 utilization is high.) (We will refer to this relationship
between regulatory substances ("effectors") and the function of the protein they regulate as "metabolic
logic". The effector indicates a condition to which the protein can and must respond in order to meet the
needs of the organism. In this case, a high [CO2] (and a high [H+]) indicates (only occurs where) there is a
high rate of O2 utilization and so a need for O2. Hb can respond by increasing the release of O2 in such
locations.)(fig 10-6: pH effect, fig 10-8: CO2)
3. The low affinity of Hb for O2 results partly from the binding of a molecule of 2,3 bisphosphoglycerate
(BPG) within the central cavity of the Hb molecule (Fig 10-22). When O2 binds, Hb's quaternary structure
changes, BPG is released (fig 10-13) and the O2 affinity increases.
E. Molecular basis of Hb function: The differences between Mb and Hb in binding O2 all result from
interactions among the Hb subunits. These are allosteric effects; O2 binding at one heme group affects O2
binding properties of the other hemes. Allosteric (allo-other, steric-place) effects are those in which events at
one location on a protein molecule change the properties at a different location on the same protein
molecule. These effects have been observed only in multi-subunit proteins.
1. There is a shift of one alpha-beta pair of subunits relative to the other pair upon the first (or second (or
third)) oxygenation. This is a change in quaternary structure from the T (tensed) to the R (relaxed) form.
2. This shift results from the following "sequence" of events (which occur in a concerted manner) : i) when
the O2 binds it pulls the iron down into the plane of the heme ring (Fig 10-16) ii) this pulls the his side chain
(to which the iron is bound) along with it. iii) this pulls the F helix along, causing other segments of the chain
to also move. This is a slight shift in the tertiary structure from the t (tensed) to the r (relaxed) form. (In the t
form heme ring stericly blocks O2 from close approach to Fe ion, bond is long and weak when Hb stays in t.)
iiii) this results in a shift of this subunit relative to the others, breaking "salt bridges" among the subunits
(fig 9-18 and 9-19) and between the subunits and BPG, pushing the BPG out of the central cavity (Fig 9-13)
The evidence for this mechanism (the “Perutz mechanism”) is as follows: i) when two compounds which
“pull” the molecule in opposite directions were added, the bond between the Fe and the proximal his broke:
NO which binds more strongly than O2 and pulls to R, and IHP, which binds more strongly than 2,3 BPG and
pulls to T)
ii) removing the arg #141 at the C terminal of the alpha chains greatly reduces cooperativity, and further
removing the his #146 from the C terminal of the beta chains eliminates it. These residues are involved in
many of the intersubunit interactions that exist in T and are broken in shift to R.
3. The affinity depends on the net change in energy upon binding. This results mainly from the change in
interactions (gained: -, lost: +. Review IV in Ch.8 and AFFINITY, above)
a. If Hb remains in the T form when the first O2 binds, the Fe(II)-O2 bond is long and weak because
steric interference from the heme ring blocks the O2 from close approach to the Fe ion. This weak bond has
a small negative energy so the affinity is low.
b. If Hb changes from T to R when the first (or second) oxygen binds, a strong bond at the O2 binding
site (iron-O2) and several intersubunit interactions are formed (large negative energy sum), but these are
mostly offset by the breaking of the salt links between subunits and from the subunits to BPG (large +
sum). The small net gain in interactions for the first/second O2 bound corresponds to a low energy change
and a low affinity.
c. Once the first (or second) O2 is bound (and T--->R occurs), there are no interactions broken to offset
those formed for each additional O2 that makes a strong bond to Fe(II). So, the energy change is a large
negative and the affinity is high for these later (2nd, 3rd, 4th) O2.
4. a. The CO2 effect is also allosteric. (CO2 lowers the affinity of the O2 binding sites by binding at a
place other than the O2 binding site) The CO2 binds to the amino terminals of the chains (as carbamate) and
is involved in salt links which must be broken during T--->R. This makes the energy more +/less- in 3b
above and so lowers the affinity. When oxygenated Hb in the R form binds CO2, this increases the tendency
for an R ---> T shift and for O2 release from the T form that binds it weakly. The carbamate occupies the
same place as the Cl- in fig 10-18, 19a.
b. The "regulatory" effect of Cl- reflects the [CO2], and occurs at the same site on the Hb molecule.
When HCO3- leaves the RBC it is replaced by a Cl- that enters; this is more frequent when [CO2] is high, so
the [Cl-] is high in the RBC when the [CO2] is high.
c. The CO2 also has an indirect effect in that it produces H+. (D.2. above)
8
5. The H+ effect is also allosteric. It has greater affinity for Hb in the T form than in the R, and its binding
helps keep Hb in the deoxy (T) form.
a. Its greater affinity for the T form results from interaction of the protonated amino terminal (fig 10-18)
with Cl- or carbamate in T Hb, from interaction of protonated side chains of his 146 of the beta chains with
asp 94 side chains of the same subunit in T Hb, and from similar effects on other groups. When these
groups are NOT protonated, these interactions do not occur.
b. In other words, when these groups are protonated, these interactions stabilize the tensed, deoxy
form of Hb since they must be broken in the T--->R transition. As for CO2, this makes the overall energy
change for O2 binding and T  R more+/less- and so lowers the affinity.
Note that these groups have a lower affinity for protons in the R form because in this form the protons “bind
to only one group”, but in the T form they “bind to two groups”.
6. The fetal Hb (HbF) has a higher affinity for O2 than adult Hb (HbA). This is because HbF has a lower
affinity for BPG than HbA has. The higher affinity for O2 allows the transfer of O2 from the mother's Hb to
that of the fetus in such a way that the HbF is more saturated than the HbA. The lower affinity of HbF for
BPG results from the fact that HbF has a ser projecting toward BPG in the central cavity where HbA has his
143of its beta chains (fig 9-22), his+...-BPG, but ser side chain doesn’t contact BPG.
IV. Abnormal Hbs-only HbS covered.
1. HbS is a "mutant" form of HbA in which glu 6 of HbA's beta chain is changed to val. Fig. 10-26 b and c
show that this val is located so as to fit into a hydrophobic pocket on another HbS molecule. This allows HbS
to aggregate into huge fibers ( Figs 10-24, -25 and -28 ), causing the RBCs to sickle. In this form the RBCs
carry O=O poorly and are inflexible, causing them to clog/block flow through the capillaries.
Skip Part 4. Allosteric Regulation Models
Chapter 6 Protein properties (briefly)
I.Precipitation:Proteins are least soluble at their isoelectric point (pI), the pH at which the the total charge
equals zero (# of + charges = # of - charges). Certain salts cause the protein's solubility to decrease as the
salt concentration increases. At a certain pH and [salt], some proteins in a mixture will salt out (precipitate),
while others will not.
II.Chromatography
A.Ion-exchange: separates based on charge or charge density. Charged protein molecules stick to
oppositely charged stationary phase particles. The proteins are then removed by eluting ("washing through"
the column) with salt solution, which provides ions to compete for binding to the stationary phase. The
greater the net charge on a protein, the higher the salt concentration required to wash it off the solid. By
manipulating the pH, the charge on the protein can be controlled (+ at pH < pI, - at pH > pI). Then, changing
the pH of the eluting buffer toward the pI can decrease the charge until the protein no longer sticks.
B.Gel permeation (aka gel "filtration"): the stationary phase is tiny porous spheres, allowing molecules to
permeate the gel beads. Molecules larger than the pores will not permeate and will remain in the mobile
phase that flows between the beads, coming through before molecules that do permeate. Smaller molecules
will permeate the beads to an extent that depends on thier size, with the smallest molecules and ions coming
through last because they spend about twice as long inside the stationary phase as they do in the mobile
phase.
C.Dialysis: "filtration" through pores of a specific sizeseparates proteins based on size (into 2 groups)
D.Affinity: attached to the stationary phase is a substance to which only the protein of interest will stick
(such as the substrate of an enzyme). All other proteins wash through. Then the conditions are changed to
interrupt the interaction and the desired protein washes through. This gives greatest purification in a single
step.
III.Electrophoresis: the movement of charged particles in an electric field
A.Native: proteins are charged as a result of their content of asp, glu, his, lys, and arg side chains. The
greater the net charge on the protein molecule, the faster it will move. The larger the protein molecule, the
slower it will move. The actual rate of movement is determined by both of these effects (charge/mass ratio).
B.SDS (sodium dodecyl sulfate): SDS binds to proteins, causing them to unfold and take on a linear shape
(12 carbon dodecyl groups overcome hydrophobic effect). The negative charges on the many sulfates
cause the overall charge to be negative and about the same per unit length for all proteins. This eliminates
the charge difference effect and the rates of movement depend only on the size factor (as above).
Unknowns are run at the same time as known standards, MW can be estimated.
C.Isoelectric focusing:a substance in the gel sets up a continuous pH gradient. A protein migrates toward
the point at which the pH is equal to its pI, and stays there, uncharged.
IV.Ultracentrifugation: separates based on density and molecular weight
A.Protein can be "harvested" as a "pellet" at the bottom of tube, since protein is denser than water.
B.Proteins in a mixture can be separated, with each protein moving as a "band". Band for largest and/or
densest moving fastest.
C.Equilibrium density gradient: the density of the liquid varies slightly and continuously over the length of the
tube. A protein will move to a location where the density is equal to its density and then will remain there.
How to Access Reserve Readings Online
You may wish to access Library Reserve items online, either to read them or print them out. Here are the
steps to follow:
1. Open the UTM homepage: www.utm.edu
2. From the items on the left of the homepage, click Library.
3. From the items on the left of the Library page, click Catalog.
4. From the buttons on the Catalog page, click Course Reserves.
9
5. On the Course Reserves page, you may click on Course and type in the course name or number; or you
may click on Professor and type in the professor’s name (my name is spelled “Thomas”). If you use the
Professor method, you will see all of his/her courses listed, and you must then click on your particular
course.
6. Each copy of each item is listed, so you'll need to look for the copy that says it is at the circulation desk
AND on the web. The ones on the web are indicated by the words, “Circ desk & web.”
7. Click on the linked title.
8. You will now be looking at a screen that has all copies of that title
listed. At the top, under the heading “Click on the following to:”, is an URL that is very complicated and ends
in a two or three digit number. Click on that URL.
9. A screen with language about copyrights comes on. Just
click Accept (in the lower left corner of the page).
10. Wait for Acrobat Reader to load and you're off and running. You can only print out the article or read it.
Because it's in PDF format you cannot edit it.
Course Syllabus Biochemistry 412 Spring
Course Content: The course revolves around DNA and the molecular machinery involved in processes
related to it. The structure of DNA and biologically and technologically important alterations in the structure
are considered. DNA as the genetic material, involving the processes by which DNA directs its replication
and the biosynthesis of proteins, is the major focus. Two areas in which these concepts are reviewed,
viruses and molecular immunology, will complete the content of the course.
Unit Topics
Chapters
I: DNA structure, transcription I........................... 5, 29, 31
II: Transcription II, Translation............................. 31, 32
III: DNA Replication, Repair, Recombination........ 30
IV: Recombinant DNA, Eukaryotic Genes............ 5, 34
V: Viruses, molecular immunology, AIDS/HIV....... 33,35