Skeleton Notes

Introduction to biological chemistry, Redinbo, M.R.
1
Chapter 1: Life

I . I ntroduction to Biological Chemistry

A. Biochemistry literally means the study of the chemistry of life

B. Life expresses itself through biomolecules

I I . Types of biomoleculesmany are polymers of repeating subunits

Introduction to biological chemistry, Redinbo, M.R. 2
I I I . Questions to ask yourself as a biochemist:

A. What are the chemical and 3-dimensional structures of biomolecules
B. How do biological macromolecules interact with eachother
C. How are biomolecules synthesized and degraded
D. How does the organization of biomolecules influence their activity

VI . Biology obeys the laws of thermodynamics

A. Physical forces influence the behavior of biological systems

Chapter 2: Water

Read prior to lecture
2.1 Weak Interactions in Aqueous Systems
Hydrogen bonding gives water its unusual properties
Water forms Hydrogen bonds with polar solutes
Water interacts electrostatically with charged solutes
Entropy Increases as crystalline substances dissolve
Nonpolar gases are poorly soluble in water
Nonpolar compounds force energetically unfavorable changes in the
structure of water
Van der Waals interactions are weak interatomic attractions
Guided reading questions:
1. what are the structural and physical properties that make water
ideal as a solvent for life? List a few characteristics.
2. How strong are Hydrogen bonds and how do they contribute to
waters physical properties
3. What is Entropy and why, in thermodynamic terms, does it
dictate the solubility/insolubility of molecules and gases? How
does this relate to the structure of water.
4. Why are Van der Waals forces critical for micelle formation and
what is the role of entropy in the process
5. Define in your own words Ka, pKa, and pI

I . Physical properties of water


I I . Structure of water

A. Water has Hydrogen bonding (H-bond.) potential

1. H-bond: Noncovalent interaction in which a H is partially shared
between 2 electronegative atoms

B. Water is a rapidly moving collection of H-bonds

C. H-bonding plays an important role in the interaction of biomolecules

I I I . Role of water as a solvent

A. Interaction with polar solutes: Does overall entropy for this process
increase or decrease? Is S positive or negative?

B. Interaction with nonpolar solutes: Does overall entropy for this process
increase or decrease? Is S positive or negative?

C. Interaction with amphiphillic solutes: Where is entropy increasing and
where is there a decrease in entropy? Why do these spontaneously form in
vivo?


In class review of titrations

CHAPTER 3: AMI NO ACI DS
Read Prior to class:
3.1 Amino Acids
Amino acids share common structural features
The amino acid residues in proteins are L stereoisomers
Amino Acids can be classified by R group
Amino acids have characteristic titration curves
Titration curves predict the electric harge of amino acids
Amino acids differ in their acid-base properties
Omit uncommon amino acids

Guided reading questions Chapter 3 and 4
1. What are the acid/base characteristics of the 20 amino acids used
to determine charge at a given pH
2. How are the amino acids classified? Using table 3-1 in Lehninger,
what do each of the pK values tell you about the acid/base
characteristics of an amino acid? Why do some have 2 and others 3
3. Can one amino acid belong to more than one subcategory? Cite an
example (i.e. both acid and aromatic?)
4. Redefine pKa in light of the terms pK1, pK2 and pKr. How do you
find the pI of an amino acid with a nonpolar R-group versus one
with an acidic R-group?
5. Why is the pK1 value of glycine different from that of acetic acid
(both contain a single COO
-
function attached to a single carbon)
6. What kind of bond is the peptide bond? What is a condensation
reaction?
7. What role does resonance play as it relates to the peptide bond in
polypeptides?

I. Introduction and Background

A. Biochemists have been studying proteins since the 1800s

B. Abundance in cell

Water
Proteins
Nucleic Acids
Carbohydrates
Lipids
Other

C. Proteins play a role in just about every biological process

1.

2.

3.

4.

II. Proteins are built from Amino Acids

A. General structure of Amino Acids and factors influencing charge

Can you identify and mathematically express each of the following important points in
the titration curve:


pK
1
:

pK
2
:

pI :

B. The titration curve of Lysine

Draw the species that predominates below pK
1

Draw the species that predominates above pK
1

Draw the species that predominates below pK
R

Draw the species that predominates above pK
R

What species would predominate at pH 9.5


III. Amino Acids, the Peptide Bond, and Peptides

Draw the condensation reaction resulting in a dipeptide (use R1 and R2 as the side chain
R groups) and the resonance structures of the peptide bond.

Recall the geometry about atoms involved in amide linakages. Place a box around the
atoms that form a rigid plane and are coplanar on your diagram above

A. Peptides and proteins have directionality

NH
3
+
--------------------------------------------------------------COO
-

1.

2.

Calculating the pI of a polypeptideIn class group assignment

(a)
(b) Using the stylized format on the right and table 3-1, draw which species of the
peptide exists at pH 1, pH 3.5, pH 4.2, pH 8.5, pH 10, and pH 12.
(c) Would Lys-Glu-Ser have the sam pI as Ser-Glu-Lys?
(d) What is the pI of Lys-Glu-Ser?

IV. Characteristics of the peptide bond

A. How can an amide plane rotate? Is it possible?
B. Certain combinations of and are forbidden

1. Why? Each and appear to have 360
o
of free rotation.

C. What are the allowed combinations of and ?

CHAPTER 4: AMI NO ACI DS, POLYPEPTI DES, AND HI GHER LEVELS OF
PROTEI N ARCHI TECTURE.

Read prior to class:
4.1 Overview of Protein Structure
A proteins conformation is stabilized largely by weak interactions
The peptide bond is rigid and planar
4.2 Protein Secondary Structure
The alpha Helix is a common Protein Secondary Structure
Amino Acid Sequence Affects stability of the alpha Helix
The Beta Conformation organizes polypeptide chains into sheets
Skip Beta turns
Common secondary structures have characteristic dihedral angles
4.3 Protein Tertiary and Quaternary Structures
Fibrous proteins are adapted for a structureal function (focus on alpha keratin)
Skip ahead to: globular proteins have a variety of tertiary structures (stop after
reading about motifs, folds, supersecondary structures, and domains)
4.4 Protein Denaturation and Folding
Amino acid sequence determines tertiary structure
Polypeptides fold rapidly by a stepwise process. Some proteins undergo
assisted folding (only 1
st
two paragraphs and figure 4-40, Chaperones in
protein folding)
Guided reading
1. List and describe 5 forces that stabilize protein 3-D structure
2. How do amide planes and primary sequence influence the possible
conformations of a fully folded protein?
3. What are the common stabilizing forces of the two main types of secondary
structures?
4. How is the H-bonding pattern in the alpha helix similar to the H-bonding
pattern in the beta-sheet?
5. What is the H-bonding pattern in the alpha helix? How many residues/turn
in an alpha helix?
6. What are the differences and similarities between parallel and antiparallel
beta sheets?
7. Define in your own words tertiary and quaternary structure
8. What are domains and how are they different from motifs?
9. In figure 4-26, why is the thermal denaturation curve of Ribonuclease A left-
shifted compared to Apomyoglobin? Why is the curve sigmoidal?
10. How did Anfinsens experiment solve Levinthals paradox?

I. Levels of structural organization

More details about:

a.) Primary structure:

example: 60 residue protein and we have 20 naturally occurring amino acids. What is
the total possible number of combinations of primary sequence for this protein?

b.) Secondary structure:

1.) Alpha helix: repeating pattern of weak forces = stability

In Class Group Problem:

2.) Beta Sheet

c.) Tertiary structure:

Tertiary structure determined by:

1.

2.

3.

4.

5.


General characteristics of a fully folded protein:

d.) Quaternary structure:

1.

Example: We will examine hemoglobin in much more detail later in this class

II. Protein Folding

example: Cyrus Levinthal 100 residue protein.


2. Proposed sequence of events in protein folding

Compare the four folding funnels above. What information can we glean about the
folding process in terms of stability and intermediates as they all head to the N
state? If proteins fold spontaneously, what does that mean with respect to S and
G?

D. In vivohow do proteins fold properly?

In class group problem:
Draw a reaction coordinate diagram for a protein starting in the unfolded state and ending
in the folded state. Your X-axis should be reaction progress and your Y-axis should be
energy. (Think back to general/organic chemistry and transition state theory)

III. Denatured (unfolded) proteins can be refolded

A. Ways to unfold and/ or precipitate proteins

In class example:
Examine the two graphs below. Both Urea and Ammonium Sulfate are in the family of
Chaeotropic Salts. (a) Explain why the nature of the Urea vs. Native protein plot in
molecular terms, i.e. how is urea affecting the protein to give us these data. (b) Examine
the % proteins in solution vs. Ammonium Sulfate plot. What is the chemical nature (use
words like: pI, amino acid composition, hydrophilic, hydrophobic, polar, etc.) of the
proteins that begin to precipitate early (see arrow #1)? What is it about the proteins at
arrow #2 that prevent them from every coming out of solution?

a.

b.

%proteins in
solution
[Ammonium Sulfate]

%proteins folded
in native state
[Urea]
Famous experiment by Christian Anfinsen RNase A

Protein purification
3.3 Working with proteins
Proteins can be separated and purified
Proteins can be separated and characterized by electrophoresis (omit
purification tables, isoelectric focusing, and 2-D electrophoresis)
1. How does a high vs. low [salt] affect protein solubility? Will all proteins have
the same solubility properties at a given [salt]?
2. How do you know if a protein is positively or negatively charged just by
knowing its pI value?
3. Have a functional and working understanding of the differences between:
Ion-exchange chromatography, size exclusion chromatography, and affinity
chromatography
4. What is the chemical importance of SDS in visualizing proteins by SDS-
PAGE?
5. How do proteins migrate (separate) in an electrophoretic field via SDS-
PAGE

I. Characteristics of Proteins

A. Every Protein has a unique primary sequence. Hence, every protein has several
unique characteristics

III. Methods of cell disruption

A. There are a number of ways to prepare a protein extract from prokaryotic and
eukaryotic cells.


I I . Protein Purification Methods Review

A biochemist is attempting to separate a DNA-binding protein (protein X) from other
proteins in a solution. Only three other proteins (A, B, and C) are present. The proteins
have the following properties:
_____________________pI Size M
r
Bind to DNA?
protein A 7.4 82,000 yes
protein B 3.8 21,500 yes
protein C 7.9 23,000 no
protein X 7.8 22,000 yes
_________________________________________________________
Briefly explain (1 or 2 non-run-on sentences) what type of protein separation techniques
might she use to separate

(a) protein X from protein A?

(b) protein X from protein B?

(c) protein X from protein C?


I I I . Visualizing Proteins: Proteins are charged; therefore, they will migrate in
an electric field.

Problem: Will acidic and basic proteins migrate in the same manner?

A. SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis)

Structure of polyacrylamide at a molecular level:
http://www.ucl.ac.uk/~ucbcdab/enzpur/SDS-PAGE.htm

A protein has been shown to have a molecular weight = 140,000g/mol. You observe the
following when your run your SDS-PAGE both in the absence of BME (lane A) and the
presence of BME (lane B). Lane C is a molecular weight marker. Using simple
geometric symbols, draw the native protein in terms of number of subunits, stoichiometry
of subunits, and the kinds of bonding (covalent: use s-s-, noncovalent: use ) that exist.


PROTEIN FUNCTION

Read prior to class:
5.1 Reversible Binding of a Protien to a Ligand: Oxygen-Binding Proteins
Oxygan can bind to a Heme prosthetic group
globins are a family of oxygen-binding proteins
myglobin has a single binding site for oxygen
protein ligand interactions can be described quantitatively (know what
theta is, but you don't need to derive eqtn 5-4 and 5-5)
proteins structure affects how ligands bind
Hb transports O
2
in blood
Hb and Mb are structurally similar
Hb undergoes a structural change on binding O
2

Hb binds O
2
cooperatively---stop once you read the definition of
allosteric protein (no homotropic/heterotropic control, hill plots, etc.
Hb also transports H
+
and CO
2
(Bohr effect in here, we will not cover
CO
2
binding)
O
2
binding to Hb is regulated by BPG
Sickle-cell anemia is a molecular disease of Hb


1. Compare and contrast the structural and functional similarities between
Myoglobin (Mb) and Hemoglobin (Hb). Make a list.
2. How does the Mb O
2
binding curve differ from that of Hbs O
2
binding
curve? What does the shape of each curve tell us about ligand binding?
What does low affinity versus high affinity mean in terms of p50 values on a
theta plot (theta on Y and pO
2
on X-axis)
3. Define for yourself allosterism, cooperativity, dynamic equilibrium,
and conformational state as they relate to Hb.
4. How do the T and R states of Hb differ both in overall structure and O
2

affinity?
5. What is the role of pO
2
in oxygen transport from lung to tissue?
6. What is the Bohr effect? What are the Bohr protons and how do they
stabilize modulate T and R state interconversion? What role does pH play
in oxygen transport from lung to tissue?
7. What is BPG? Where does it bind to Hb? What forces stabilize its binding?
What role does BPG play in oxygen transport from lung to tissue?
8. Why does a single mutation in Hb lead to such a dramatic pathology? Are
there other Hb mutations that have little to no disease associated?

We will use the examples of Myoglobin and Hemoglobin to discuss how protein tertiary
and quaternary structure influence function.

A. Myoglobin:

B. Hemoglobin:


C. Cooperativity, Conformational changes, and oxygen binding in
Hemoglobin

1. Hemoglobin has two conformational states:

T-state

R-state
Hb has two conformational states The Bohr effect

A. Binding of O
2
to Hb stimulates the release of H
+
from Hb (the Bohr effect)

Graph time: O
2
binding by Hb at different pH values in vitro

Group in class problem:
When you are working late in lab one night, you discover: in oxygenated Hb, the pKr for
the His residues at position 146 of the Beta chain is 6.6. In deoxygenated Hb, the pKr =
8.2 for the same His residue (His 146 of the Beta chain). Describe these findings
focusing on the Bohr effect and the conformational states of Hb.

2,3-bisphosphoglycerate (BPG) modulates the conformational changes within Hb

A. Highly purified (stripped) Hb has a higher affinity for O
2
than Hb in
whole blood (similar to Mb !!!)

B. Blood must contain some other compound that affects O
2
binding to Hb

C. The metabolite BPG is that compound.

Graph time: make a theta plot of stripped Hb and blood Hb. What purpose does BPG
serve?

A new O
2
transport protein has been discovered. The deoxy protein structure reveals a
dimeric (homodimer) structure shown below in figure a. The individual monmeric
subunits interact via salt bridges
between the N- and C-termini.
Helices A and C are stabilized by a
salt bridge between Histidine 13
and Aspartic acid 85. The O
2
site
is between the rigid linked helices
A and C (see figure b). In the
deoxy state, the space between the
Fe
2+
atoms is too small to bind O
2
,
thus the atoms must be forced apart
when O
2
is bound

Answer the following questions,
briefly explaining your answer in
terms of the structure

(a) Will this molecule show cooperative oxygen binding?

(b) Will this molecule exhibit a Bohr effect

(c) Would the addition of BPG have an effect on O2 binding

(d) What would be the effect of a mutation which replaced aspartic acid 85 with
a Lysine residue (pKR = 10.53)


2. A single mutation in hemoglobin can lead to altered structure and function (Sickle
Cell Anemia).

Chapter 6: ENZYME CATALYSIS

6.1 Intro to enzymes
most enzymes are proteins
enzymes are classified by the reactions they catalyze
6.2 how enzymes work,
enzyme affect reaction rates, not equilibria
reaction rates and equilibria have precise thermodynamic definitions
a few principles explain the catalytic power and specificity of enzymes
weak interactions between enzyme and substrate are optimized in the
transition state
binding energy contributes to reaction specificity and catalysis
6.3 enzyme kinetics as an approach to understating mechanism
Substrate concentration affects the rate of enzyme catalyzed reactions
The relationship between substrate concentration and reaction rate can
be expressed quantitatively,
Kinetic parameters are used to compare enzyme activities (1
st

paragraphs and turnover number and M-M equation)
NO: catalytic efficiency or specificity constant
Skip ahead to: enzymes are subject to reversible or irreversible
inhibition.
Focus on: Competitive inhibition AND under mixed inhibitor you will
find noncompetitive inhibition. Competitive and noncompetitive are
the only two we will cover (no noncompetitive or all mixed)
6.4 examples of enzymatic reactions
The chymotrypsin mechanism involves acylation and deacylation of a
Ser residue
NOTE: dont memorize the reaction on p. 217-ish, be able to follow
where you see the different types of catalytic mechanismscovalent
catalysis, acid base, etc
ONLY DO THE END OF CHAPTER QUESTIONS THAT ARE ASSOCIATED
WITH THESE TOPICS.
1. What kinds of molecules are enzymes and why are they necessary for
biochemical reactions in the cell?
2. Thermodynamically, how do enzymes catalyze reactions? What do they
change and what do they NOT change (fig. 6-2)
3. What is the difference between a binding pocket and an active site?
4. Define in your own words: binding energy and induced fit
5. What are the specific types of catalysis that enzymes utilize? List and define
three from your book.
6. (For next lecture, kinetics What were the assumptions Miichaelis and
Menten made when deriving their equation? What is Km mathematically
and conceptually?

I. General properties of enzymes

Enzymes: catalysts in biological systems that increase the rate of reactions, but are not
changed at the end of the process

Enzymes allow for:

1.

2.

3.

4.

II. The six general classes of enzymes

A.
ENZYME CLASS TYPE OF REACTION
1. Oxidoreductases Redox (e- transfer reactions)
2. Transferases Transfer functional group
3. Hydrolases Hydrolysis (break bond with H
2
O)
4. Lyases Add or remove groups to make or break
C=C bonds
5. Isomerasses Rearrange C-skeleton
6. Ligases Form: C-C; C-N; C-O; C-S bonds at the
expense of ATP hydrolysis to ADP

B. Enzymes as catalysts:

1. Enzymes do not change the ______ for a reaction

2. Enzymes alter the _______ of a reaction

Example: Reaction coordinate diagram

III. Catalytic Mechanisms: factors contributing to enzymes catalytic activity. Note:
enzymes may use one or more of these general strategies.

a. Proximity and orientation

b. Preferential transition state binding

c. General Acid-Base chemistry

d. Covalent catalysis

e. Electrostatic catalysis

f. Metal ion catalysis


I V. Catalytic mechanism--chymotrypsin

A. The catalytic mechanism of chymotrypsin. A long winded guide to
understanding catalytic mechanisms. Enjoy the show.

Group exercise:
The figure below shows one of the possible reaction mechanisms for the enzyme,
Lysozyme. Looking at the panels from left to right, List at least 4 types of enzymatic
mechanisms/strategies demonstrated in this figure? Please state your answer in
the form, Glu35 is demonstrating ________ catalysis, and Asp52 is
demonstratingetc. You can also refer to the entire enzyme if it is demonstrating a
catalytic strategy as a whole.

Solving enzyme structure function problems:
The structure of the enzyme Tryptophan Synthetase has been studied extensively by a
variety of methods.
In a series of studies, Yanofsky and co-workers examined the effect on enzyme activity
of various amino acid changes in the protein sequence (Federation Proceedings, 22:75
(1963) and Science 146:1593 (1964)). Altered amino acids are shown in bold. "Wild-
type" is the normal strain isolated from the wild.

Postulate TWO possible explanations for these data
1.
2.

Yanofsky & co. collected more mutants and examined their proteins to determine which
of the above explanations was more likely to be correct:

part 2. Which of your two models is supported by these data? explain?
Alterations of amino acids at another location in the protein were found to interact with
alterations at position A.

part 3. Explain the behavior of mutant 6 in terms of your model of part b.
ENZYME KINETICS, INHIBITION, AND REGULATION

I V. I I . Enzyme catalyzed reactions in biological systems
A. For a simple enzyme catalyzed reaction:

S = substrate and P = products

B. Biological reactions are generally more complex

1. S
1
+ S
2
P
2. S P
1
+ P
2

3. S
1
+ S
2
P
1
+ P
2

In class group excercise
For reaction 1:
What is the equilibrium constant for this reaction (not the number but Keq = ?)
Where do the rate constant(s) for this reaction go?
Can you write a rate equation for this reaction

C. In the simple case of S being converted to P in an enzyme catalyzed reaction, the
enzyme [E] must form a complex with substrate [S] to yield an enzyme-substrate
complex [ES] in order to form product [P].

I. Michaelis and Menten (M & M): Their pioneering work on enzyme
kinetics began around 1913

A. M & M began investigating the effects of [S] on the formation of
the ES complex. They examined the effects by measuring the
initial reaction velocity (
o
).

Question: What if we keep the [E] constant and vary the [S]?

Simple Concept Example:

E + S ES P


I I . Derivation of the M & M equation:
Assumption 1 (assumption of equilibrium): Early in the reaction, little P has
accumulated so k
-2
can be ignored.

P/time = (
o
) = k
2
[ES]

Eqtn. 1 Formation of ES: rate = k
1
[E] [S]

Eqtn. 2 Breakdown of ES: rate = k
-1
[ES]

rate = k
2
[ES]

Steady State Assumption: Once reaction gets started, the [ES] remains constant. As a
result, the formation of ES must equal the Breakdown of ES:

Eqtn. 3

Eqtn. 4

Question: Knowing this, write a mathematical expression showing the Steady State
Assumption and then show it graphically. X-axis is reaction progress, Y-axis is
amount or concentration. Show E, S, and ES

Now rewrite this expression in terms of ES:
Eqtn. 5 Define K
m

lots of algebra (see your text or online resources) for eqtns. 6-9

Ta Da!, the Michaelis-Menten equation is:

Eqtn. 10

SO WHO CARES?

Consider the special case when
o
= Vmax

Eqtn. 11

Use this space to get from eqtn. 11 to 15 (hint: get rid of Vmax first)

Eqtn. 15

When
o
= Vmax, K
m
= [S] that gives Vmax

I I I . Importance of K
m
and Vmax
A. K
m
is a measure of an enzymes affinity for its substrate.

B. K
m
is unique for each enzyme-substrate pair

1. Enzyme with high K
m

2. Enzyme with low K
m


C. Determining K
m
and Vmax experimentally

1. Difficult to determine K
m
and Vmax from:

2. Lineweaver-Burk or Double Reciprocal Plot. (think mx+b = y)

Y-intercept =

X-intercept =

K
M

V
max

1
[S]
+
1
V
max

=
1
v
i

Using Km, Vmax, and k
cat
in practice

a. k cat = # of rxns an enzyme can carry out; most commonly the rate determining
step in an enzyme catalyzed reaction. This is also referred to as Turnover
number = Vmax/[E
total
] (units = mins
-1
or s
-1
)

The enzyme dihydrolipoyl transferase is absolutely required for aerobic respiration. It is
a large enzyme with a M.W. = 120,000g/mol. It enzymatically transfers an acetyl group
from pyruvate to Coenzyme A in the eukaryotic mitochondrion. If 40 g of pure
dihydrolipoyl transferase catalyzes the transfer of 1.2g of substrate to product (M.W. =
87.0 g/mol) in 4 minutes while functioning at Vmax, what is the turnover number (k
cat
) of
this enzyme in units of min
-1
?

IV. Enzyme Inhibition

A. Competitive Inhibition

Effects of a competitive inhibitor on Km and Vmax:

Effects of a noncompetitive inhibitor on Km and Vmax:

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