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Biochem 460 Exam 1 Spring 04 Zielger U of Arizona


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Quaternary Structure
Sequence of amino acids, linked together by peptide bonds (amide linkages)
Secondary structure (2° structure)
Local, regular/recognizable conformations
Observed for parts of the peptide backbone of a protein
e.g, a-helix, b conformation, collagen helix
Rate enhancement
Factor by which enzyme increases the rate of a reaction is determined by ΔΔG‡, the magnitude of the decrease in ΔG‡ brought about by the enzyme compared with the uncatalyzed reaction's ΔG‡.
Small organic or metalloorganic molecules (coenzymes) or metal ions
metalloorganic molecules that bind tightly or weakly to enzymes
An enzyme without a cofactor
An enzyme with a cofactor
Prosthetic Group
Tightly bound cofactors (either coenzymes or metals)
Equilibrium Constant

The equilibrium constant
Biochemical Standard Conditions
pH 7.0 and 55.5 M H2O
Standard free energy change
ΔG° = the free energy change for going from standard conditions to equilibrium.

* lets us express equilibrium constant in free energy terms
Actual free energy change
ΔG', depends on 3 things:

1) standard free energy change for that reaction (ΔG°', a "reference" telling where equilibrium lies)
2) actual starting concentrations of reactants and products (mass action ratio)
3) temperature.
Enzymes as catalysts do what?
Enzymes increase RATES of (bio)chemical reactions but have NO EFFECT on Keq (and no effect on overall ΔG) of the reaction.
Express the velocity of a simple reaction in terms of the rate constant and the concentration of the reactant?

Expressing velocity in terms of rate constant and the concentration of the reactant.
An enzyme that increases the rate constant for the forward reaction by a factor of 10^8 must also increase the rate of the backward reaction by how much?
Draw the free energy diagram of a hypothetical reaction and show how a catalyst may increase the rate of the reaction, pointing out ΔG for the overall reaction, ΔG‡uncat, and ΔG‡cat. on the diagram.

Free energy diagram for the reaction.
Rate enhancement (factor by which enzyme increases the rate of a reaction) is determined by?
What reaction parameter do enzymes affect in order to increase the rate?
Enzymes decrease activation energy (ΔG‡) for reactions they catalyze.
test for a unique property of protein of interest (the "goody")
specific activity
Ratio of desired protein/total protein
cell lysis
Disruption of cell membrane to release contents
The released contents of a cell, post lysis
activity after each purification step / total starting activity (expressed as a per cent)
fold purification
specific activity after a given step / starting specific activity
molecule that binds specifically (but usually noncovalently) to a macromolecule (usually a protein)
Explain dialysis
for separating proteins from small molecules & ions
Explain gel filtration
Separation by molecular weight and shape.
The larger and heavier the molecule, the quicker it moves through the column, eluting earlier.
What is ion exchange chromatography?
Separation by net charge
How does charge impact how quick a substance elutes in ion exchange chromatography?
Proteins bind to oppositely charged groups on column matrix; the greater the net (opposite) charge on protein, the more tightly it binds, so the later it elutes.
In what order would proteins elute from an anion exchange column if given isoelctric points?
Proteins witha + net charge won't stick, so they will wash on through and elute before anything else.

Molecules with a - net charge will elute in the order of the pI values, because of the differences in net charge. The one with the pI value furthest from the working pH will elute last.
In what order would proteins elute from a cation exchange column if given the isoelectric points?
A molecule with a - net charge won't stick, so it will wash on through and elute first.

Molecules with a + net charge will elute in the order of the pI values. The most + charged one elutes last.
What is affinity chromatography?
Very specific and selective kind of column chromatography
What is SDS polyacrylamide gel electrophoresis?
The molecular mass appearing on the gel is that of the individual polypeptide chain subunits of the protein.
How would you predict the relative mobilities of polypeptide chains on an SDS gel, given subunit molecular
A protein with 2 non-identical subunits, 1 with mass 20 kd and the other with mass 50 kd (so native Mr is 70 kd) would show 2 separate bands on an SDS gel, with mobilities corresponding to 20 kd and 50 kD.
What is the use of a purification table?
Maximize specific activity while maximizing yield.
What is isoelectric focusing?
separation based on differences in ISOELECTRIC POINT (pI) (so based on CHARGE DIFFERENCES)
What is the isoelectric point?
Isoelectric point (pI) of a protein (or any molecule) = the pH at which its net charge is zero
What 2 separation methods are combined to run a 2-dimensional gel?
First dimension - separate proteins by pI

Second dimension - separating the proteins further, by their polypeptide chain molecular mass
What method is useful for determination of protein molecular weight, specifically, subunit molecular weight?
Gel filtration approximates native molecular weight if the column is run under nondenaturing conditions, or individual subunit molecular weights if a denaturing agent and a reducing agent are present.
What is the relationship between the free energy change for a process (ΔG) and the enthalpy change (ΔH) and the change in entropy (ΔS)?
A negative ΔG represents a decrease in free energy for a process, which is favorable.

Making bonds/interactions gives negative ΔH, which is favorable.

Increase in disorder gives positive ΔS, which is favorable
Draw the structure of a typical amino acid, indicating the following features: α-carbon, α-carboxyl group, α-amino group, side chain (“R group”)

Typical amino acid structure
Draw a hydroxyl
Draw a methyl
Draw a ketone
Draw a aldehyde
Draw a thiol
Draw an amide
Draw a carboxylic acid
Draw an ether
Draw an anhydride
Draw an ester
Draw an amine
Draw a phosphoro
Draw a phenyl
Draw a guanadino
Draw an imidazole
Draw a disulfide
acid + alcohol -->
ester + H20
acid + amine -->
amide + H20
acid + acid -->
anhydride + H20
3 Types of noncovalent bonds are:
1. ionic interactions
2. hydrogen bonds
3. van der Waals interactions
What is an ionic interaction?
Electrostatic attraction between oppositely charged groups, or repulsion between like charges.
What are ionic interactions also known as?
Salt links, salt bridges, and ion pairs.
What are the relative values of the dielectric constants for a nonpolar solvent and a polar solvent?
More polar solvents have a higher dielectric constant.
What is an example of a nonpolar solvent?
What is an example of a polar solvent?
How does solvent polarity affect strength of ionic interactions?
If you are dealing with a polar solvent, it will either increase or decrease the attraction depending upon whether or not the solute is polar.
What is a hydrogen bond?
They are electrostatic in nature.
It is a hydrogen atom covalently bonded to an electronegative atom, the donor, and the lone pair of a non-bonded electrons on another electronegative atom, the acceptor.
what is a hydrogen bond donor?
A hydrogen bond donor is the electronegative atom attacehd to a hydrogen.
what is a hydrogen bond acceptor?
It is the electronegative atom with a lone-pair of electrons.
How does the strength of a hydrogen bond relate to its directionality?
Linear hydrogen bonds are stronger than bent hydrogen bonds.
What are two common hydrogen bond donor groups?
-O-H or -N-H
What are some common hydrogen bond acceptor groups?
:O=C, :O-H, :N-H, =N-, :O=P
What are van der Waals interactions?
They are also electrostatic in nature.
Weak, nonspecific attractive force between ANY two atoms that approach within about 4-5 Ã… of each other.
How (qualitatively, not an equation) does their strength relate to the distance between atoms?
There is a certain distance where they are most effective, but when their electron clouds begin to overlap, they become strongly repulsed.
are such weak, nonspecific interactions important in biochemistry?
Individually they are weak, but sum of many close approaches is very important in steric (molecular shape) complementarity, which can be very specific.
Explain hydrophobic interactions and explain the roles they play in biological systems.
Tendency of nonpolar groups or molecules to associate (cluster together) to minimize exposure to H2O, the "oil drop effect"
Explain the polar properties of water
Asymmetric charge distribution on H2O makes molecule polar.
Explain the hydrogen bonding ability of water
Each H2O molecule has an O atom with 2 unshared pairs of electrons so it can be the acceptor of 2 hydrogen bonds, and it also has 2 O–H bonds and can donate 2 hydrogen bonds as well.
Explain the solvent properties of water.
Because of its polarity and hydrogen bonding properties, H2O is an excellent solvent for many biomolecules:

* ions
* other polar molecules
* molecules with groups that can hydrogen bond to H2O
* H2O (70% of the cell by weight) thus an excellent medium for most of the intracellular environment.
What are some negative effects of water's great solvent ability?
* Weakens interactions between oppositely charged ions (remember it has a high dielectric constant D), and
* Competes with other solute molecules for hydrogen bonding groups in solutes.
Write a general chemical reaction.

General Chemical Reaction
Write the mathematical expression for the general chemical equations equilibrium constant.

Equilibrium Constant Equation
Write the “mass action ratio” for any chemical reaction.

Mass Action Ratio
Briefly explain in conceptual terms what is represented in a change in free energy (ΔG)
Quantitates the energy available to do useful work.

It is related to enthalpy, entropy, and temperature.
Briefly explain in conceptual terms what is represented in a change in
enthalpy (ΔH)
A measure of the energy (heat content) of the system at constant pressure
Briefly explain in conceptual terms what is represented in a change in entropy (ΔS)
a measure of the randomness (disorder) of the system
Write the equation relating ΔG, ΔH, and ΔS.

(Good Hunters Take Shotguns)
How does temperature affect ΔG, and what are the units of T?
T is the absolute temperature in units of K (T = oC + 273).

* If ΔG is negative (ΔG < 0): process goes in direction written (left to right)
* If ΔG = 0: process is at equilibrium
* If ΔG is positive (ΔG > 0): process goes in reverse (right to left)
What are the 2 simplifying assumptions made in biochemistry that are consistent with physiological conditions, and make "biochemical standard conditions" different from standard conditions normally referred to in chemistry?
The concentration of water [H2O] does not change during the reaction, i.e. [H2O] = 55.5 M.

The pH = 7.0 and does not change during the reaction, i.e. [H+] = 10–7 M.
Explain the difference between the equilibrium mass action ratio (Keq') and the actual mass action ratio.
The equilibrium mass action ratio is represented by : ΔG° = - RTlnKeq

The actual mass action ratio, reflecting actual starting conditions, the actual concentrations of reactants and products is represented by : ΔG(actual) = ΔG° + RTln{actual mass action ratio}
Explain what ΔG° is.
Standard conditions ("standard state"): 1 M in each reactant and product (or 1 atm for gaseous reactants or products), with temperature = 25 °C = 298 K.
Explain the difference between ΔG° and ΔG°'
Under these conditions the (constants) [H2O] and [H+] are incorporated into ΔGo to give a new "biochemical" standard free energy change, ΔGo'. The "biochemical" equilibrium constant, related to ΔGo', is designated Keq'.
Interconvert ΔG°' and Keq', given the absolute temperature and the value of R, the gas constant.
lnKeq = – ΔGo/RT, so Keq = e– ΔGo/RT
Explain the relationship of ΔG' to the direction in which a reaction will go spontaneously
If ΔG' is < 0, it will move toward the reactants.
If ΔG' is > 0, it will move toward the products, spontaneously.
If ΔG' is = 0, it will be in a state of equillibrium.
Define endergonic
It is a process that requires the input of free energy.

Unfavorable (positive) free energy change for going in direction of equilibrium.
Define exergonic.
It is a process that has a negative, favorable, free energy change.
Explain free energy coupling
Free energy coupling is the process of attaching an endergonic reaction to an exergonic reaction, allowing the endergonic reaction to proceed.
Why is free energy coupling important in biochemistry?
Free energy coupling, with enzymes as catalysts, is the strategy used in metabolic pathways.
Calculate the overall ΔG'for a coupled reaction given the ΔG' values for the component reactions.
Reaction 1:
Glucose + Pi <=> glucose-6-phosphate + H2O
(ΔGo' = + 13.8 kJ/mol, endergonic)
Reaction 2:
ATP + H2O <=> ADP + Pi
(ΔGo' = - 30.5 kJ/mol, exergonic)
Coupled Reaction 3:
Glucose + ATP <=> glucose-6-phosphate + ADP
(ΔGo' = - 16.7 kJ/mol)
Explain the relation between ΔG' and the rate of a reaction.
There's NO information about rates in the value of a ΔG -- we can't answer this question from bioenergetics.
Explain primary structure
⬢sequence of amino acids, linked together by peptide bonds (amide linkages)
Explain tertiary structure
⬢3-dimensional conformation of whole polypeptide chain in its folded state
What are the ionic forms that predominate at acidic (say, pH 1), neutral (pH 7), and basic (pH 13) pH values.

Ionic Forms Image
and ionic forms that predominate at
acidic (say, pH 1), neutral (pH 7), and basic (pH 13) pH values.

Ionization state
Write the aliphatic side chains (nonpolar)

The longer chains are more hydrophobic
Write the aliphatic side chains (nonpolar), and state if the longer hydrocarbon chains are more or less hydrophobic.
Glycine (Gly)
Alanine (Ala)
Valine (Val)
Leucine (Leu)
Isoleucine (Ile)
Proline (Pro)
Write the aromatic side chained amino acids.
Phenylalanine (Phe)
Tyrosine (Tyr)
Tryptophan (Trp)
Write the hydroxyl-containing amino acids
Serine (Ser)
Threonine (Thr)
Sulfur-containing amino acids
Cysteine (Cys)
Methionine (Met)
Acidic amino acids
Aspartate (Asp)
Glutamate (Glu)
Amide Amino Acids
Asparagine (Asn)
Glutamine (Gln)
Basic side chain amino acids
Lysine (Lys)
Arginine (Arg)
Histidine (His)
Typical pKa value of a terminal alpha-carboxyl group
Typical pKa value of aspartic acid and glutamic acid
Typical pKa value of Histidine
Typical pKa value of a terminal alpha-amino group
Typical pKa value of cysteine
Typical pKa value of tyrosine
Typical pKa value of Lysine
Typical pKa value of arginine
Ionization reaction for a terminal alpha-carboxyl group
Ionization reaction for aspartic acid and glutamic acid
Ionization reaction for histidine
Ionization reaction for a terminal alpha-amino group
Ionization reaction for cysteine
Ionization reaction for tyrosine
Ionization reaction for lysine
Ionization reaction for arginine
Which amino acids are non-polar
Ile, Leu, Val, Ala, Pro, Gly
Which amino acids are more polar?
Asn, Gln, Ser
Which are the most H20-soluble?
Asp, Glu, Lys, Arg, His
Is Phe hydrophobic or not?
It is very hydrophibic
Are Tyr and Trp aromatic or not?
Tyr and Trp have polar portions
Write the chemical equation for formation of a peptide bond.
Explain the relation between the N- and C-terminal residues of a peptide or protein and the numbering of the amino acid residues in the chain,
Starting at the amino end of the chain, ending at the carboxyl terminus convention sequence written left to right from "N-terminus" to "C-terminus"
Write the convention for writing sequences left to right from amino to carboxy terminus.
...-N-Cα1-C(O)-N-Cα2-C(O)-N-Cα3-C(O)-... etc.
Explain how the partial double bond character of the peptide bond and steric effects relate to the
conformation of a polypeptide chain
There are two possible configurations, with the alpha carbons being on the same or opposite sides of the double bond. Molecules much prefer to be in trans conformations as opposed to cis due to the steric hindrance.
Explain which bond rotation angle is defined/described as Φ
Φ is the alpha-carbon to the nitrogen
Explain which bond rotation angle is defined/described as Ψ.
Ψ is alpha-carbon to carbon.
Explain what a Ramachandran plot is, and how it relates to "allowed" combinations of (Φ,Ψ) coordinates for proteins.
What's allowed and what's not sterically possible obviously depends partially on the nature of the R groups on adjacent residues, so depends on local sequence.

The most local sequences shown are in dark green on the plot, while borderline combinations are shown in light green.
Define amino acid residue
They are numbered from amino end of chain toward carboxyl terminus.

Starting at the amino end of the chain, ending at the carboxyl terminus

Convention sequence written left to right from "N-terminus" to "C-terminus"
Define main chain
It is the backbone of the chain. It repeats down the chain:

...-N-Cα1-C(O)-N-Cα2-C(O)-N-Cα3-C(O)-... etc.
Define side chains
They are the R groups on amino acids.
Define conformation
Spatial arrangement of atoms/groups that changes by bond rotation without breaking covalent bonds
Define configuration
Spatial arrangement of atoms/groups that cannot be changed without breaking covalent bonds.
What types of bonds/interactions are involved in holding together/stabilizing each level of protein structure?
Secondary structures are stabilized by all kinds of noncovalent bonds, but especially by hydrogen bonds.
Define the secondary structure of a protein.
major types of secondary structure found in many proteins:

* α helix
* β conformation
* β turns
Describe the α-helix, including what groups serve as hydrogen bond donors and acceptors
Each N-H group donates a hydrogen bond to the oxygen of a C=O group 4 residues behind it in sequence.
How does chirality in proteins usually occur?
Proteins are usually right-handed, meaning the backbone turns clockwise if viewed down helix axis in N-to-C direction
How many residues per turn are there in the alpha helix?
The C=O is at "end" of residue n, and the N-H is at "beginning" of residue n+4, so 3.6 residues per 360° turn of a helix
What is the orientation of the R groups relative to the axis of the helix?
R groups are on the outside of the helix, radiating outward.
How does the helix dipole work?
N-terminal end δ +
C-terminal end δ -
How does the packing density of atoms work?
The interior core is tightly packed, not hollow.
Describe the β-conformation, including which groups serve as hydrogen bond donors and acceptors
Backbone amide N-H and C=O groups again almost fully hydrogen-bonded, but hydrogen bonds can be

between different sections of the backbone, or between sections of backbone on different polypeptide chains. No predictable relationship in the amino acid sequence for what sections are hydrogen bonded to each other
How does the distance between adjacent residues of Beta-conformation sheets compare with the rise of the alpha-helix?
Distance between adjacent AA residues ~3.5 Å (further apart, more stretched out, than in α helix)
How does the orientation of R groups in a Beta pleated sheet work?
Side chains (R groups) point in alternate/opposite directions for adjacent residues in chain
Explain parallel and antiparallel Beta-conformation
In antiparallel B-sheets, Adjacent strands run in opposite directions; note hydrogen bonds between N-H group on one strand/section and C=O group on other strand/section.

In parallel B-sheets,
Adjacent strands run in same direction; note hydrogen bonds between N-H group on one strand/section and C=O group on other strand/section.
Identify the most important noncovalent interactions stabilizing the α-helix and β-conformations.
Hydrogen bonds are most stabilizing
Explain what a β-turn is
abrupt change in direction of polypeptide backbone, at surface of protein
and what types of amino acid residues are often found in β turns.
often involve Gly, Ser, and/or Asn residues (small, hydrophilic R groups) or Pro (cyclic structure has a "built-in" elbow/bend in backbone to help start turn)
Outline 3 principles guiding folding of water-soluble globular proteins
*Minimization of solvent-accessible surface area
*Maximization of intra-protein hydrogen bonds
*Chiral effect
What are the generalizations about protein structure resulting from the 3 principles guiding folding.
* Maximizing "hydrophobic interactions" really means getting hydrophobic groups OUT OF WATER.
* Tendency of extended backbone structural arrangements to be right-handed as a result of having all L-amino acids
Explain supersecondary structure
Groupings of secondary structural elements
Explain domain
structurally independent folding units looking like separate globular proteins but all part of same polypeptide chain
(connected in same primary structure)

* Larger proteins often have 2 or more domains.
Explain subunit
A subunit is a subset polypeptide chain that is part of a greater whole chain.
Describe myoglobin
binds O2 in muscle cells for storage and for intracellular transport, using a heme group (black in structure below,, with purple Fe 2+)

mostly α-helical.
Describe the general structure of an αβ barrel
parallel 8-stranded β barrel on interior, surrounded by α helices, a structural motif found in many different enzymes
Describe how the primary and secondary,
structures of a porin relate to the tertiary structure.
In the amino acid sequence, there are more or less alternating hydrophobic and hydrophilic residues in the Beta-strands.
(adjacent R groups project out from sheet on opposite sides). As a tertiary structure, this produces a protein capable of allowing ions to pass through the lipid bilayer.
Explain quaternary structure
3-dimensional relationship of the different polypeptide chains (subunits) in a multimeric protein, the way the subunits fit together and their symmetry relationships (only in proteins with more than one polypeptide chain; proteins with only one chain have no quaternary structure.)
Explain homotetramer
A homotetramer is a collection of four identical subunits
Explain heterodimer
It is a collection of two different subunits (chains with different amino acid sequences)
Explain rotational symmetry
Individual subunits can be superimposed on other identical subunits (brought into coincidence) by rotation about one or more rotational axes.

Example - If the required rotation is 180 degrees, the protein has a 2-fold axis of symmetry.
Is the folded form of a protein in a higher or lower free energy state than the unfolded state, under
"native" (e.g., physiological) conditions?
The folded is in a lower state of free energy, because it took advantage of the free energy available to do work as an unfolded protein.
Discuss the differences between native and denatured forms of proteins
A native form of protein is the form that the protein would naturally take under regular conditions. The denature form, is the one that takes place after the native form is chemically denatured, using urea for example.
Using Anfinsen’s work on ribonuclease as the example, describe the experimental evidence supporting the conclusion that the information needed to specify the 3-dimensional structure of a protein resides in its amino acid
In order to denature ribonuclease, Anfinsen used both B-mercaptoethanol, and urea. Amino acid sequence dictates where disulfide bonds occur as well as how folding of proteins occur.
What are the effects of a) urea and b) β-mercaptoethanol on protein structure? (They're different!)
Urea somehow disrupts the noncovalent bonds within the protein that stabilize its native tertiary & quaternary structure, while β-mercaptoethanol reduces disulfide bonds in proteins.
Briefly explain how a small amount of abnormally folded protein might cause formation of amyloid plaques (fibrous aggregates) and tangles in neurodegenerative diseases such as the prion diseases (spongiform encephalopathies), Parkinson disease, Alzheimer
Formation of larger aggregates seems to involve "seeding" with small aggregates (dimers, trimers, etc.), which may be more toxic to the cells than the large fibrils.

One theory: Cellular "garbage disposal" apparatus for disposing of abnormally folded proteins, with assistance of chaperones, is overwhelmed in these diseases.
Explain amphipathic
Amphipathic is a compound that has both hydrophobic and hydrophilic sidechains

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