BIOCHEM 501 1st Edition

Exam # 1 Study Guide

Lecture 1 // SEPT 3
 Cellular basics
 Carbon chemistry and stereochemistry
 Free energy and how cells use it

Carbon
- 4 valence electrons
- forms stable bonds with other H, C, O and N atomts
- 3 different types of geometries: 109.5, 120, 180 degrees
- 3 different depictions of molecules: schematic, ball and stick cartoon, space filling (actual look)

Stereochemistry
- Chiral molecules
- chiral center: 4 different substituents attached to the carbon center
- when rotated, it cannot be superimposed on its mirror image
- achiral center: carbon center can have the same substituents attached
- when rotated, it can be superimposed on its mirror image

Enantiomers: stereoisomers with same chemical formula but different order

-MIRROR images of each other

Free Energy
Second Law of Thermodynamics: the tendency in nature to go towards greater disorder

Entropy: degree of disorder in chemical system

Gibbs Free Energy

G = H - TS

H = enthalpy bonding energy

S = entropy randomness

Energetic Coupling: unfavorable process is driven via coupling to a highly favorable one
Mechanical example: G > 0  work is done by raising the block up the slope
G < 0  work not done, less potential energy of position, block is sliding down
slope

Chemical example: ATP breakdown

- ATP  ADP + P
 - Keq = [ADP] [P]
[ATP]
- large Keq means the reaction tends to proceed until almost all reactant has been
converted to product
- in cells, concentration is far from equilibrium because of the large amount of energy
used
- G measures the distance of system from equilibrium
- G = 

Standard Free Energy:

G = - RTlnKeq

Lecture 2 // SEPT 5
 Properties of water
 Weak interactions between molecules in aqueous solvent
- hydrogen bonds
- ionic interactions
- hydrophobic effect
 Ionization of water, weak acids and bases
- pH, pKa, Henderson-Hasselbach equation

Water
- requires much more energy when converting from liquid to gas
- has a high melting and boiling points relative to many others
- the structure makes it a perfect H bond donor and acceptor
- 104.5 
- two partial negative pair of electrons
- two partial positive H's attached to the Oxygen
- has ability to form a lot of H bonds, building up makes it strong

- in ICE, water molecules forms an H-bonded network that fully satisfies the H-bonding potential
- there are 4 H bonds per water molecule in ice
- versus 3.4 H bonds per water molecule in liquid water
- makes liquid H2O more dense than ice
- when a cup of water has been frozen and the ice level is exactly at the top of the cup.
When the ice melts, the water level is below the rim

- a linear H bond is stronger than an angled H bond

- is an excellent polar solvent

- shields ionic interactions by charge-dipole interactions
- is a poor solvent for NON-POLAR solvents fatty acids, lipids
 - hydrophobic components with water reduces the entropy of water and is
thermodynamically unstable
- hydrophilic head loves water
- hydrophobic tail  hates water, cannot mix
- so then lipids cluster together, release water molecules to the surrounding water which
raises entropy levels to help molecules get together

Dispersion of lipids by HO  clusters of lipid molecules  micelles

Lipids force surrounding HO  only lipids at the edge of  all hydrophobic groups are
molecules to become highly cluster forces ordering of away from HO. ordered shell
ordered water, fewer HO is ordered, of HO molecules is
so entropy rises minimized and entropy is
further increased

- is a reactant in hydrolysis and condensation reactions

Ionization of water: slight tendency of HO molecules to ionize

- HO / H+ + OH-
- Keq = 1.8 x 10^-16 = [H+] [OH-]
[HO]
- Kw = 1.8 x 10^-14 at 25 C  the ion product of water
- for pure water, [H+] = [OH-]
- [H+]^2 = Kw
- [H+] = 1 x 10^-7

Hydrogen ion concentration

- Kw = [H+] [OH-] = 1.8 x 10^-14
- can be used for calculating concentrations of H and OH for strong acids and bases
- EX: 0.1 M HCl is fully ionized so that [OH-] = 10^-13
- biological H ion concentrations vary from 1.5 x 10^-3 to 10^-8

It's more convenient to define [H+] in terms of a log scale

- pH = -log [H+]
- for neutral H2O, pH = -log [1 x 10^-7 ] = 7
- EX: pH of 0.1 M HCl is 1

Dissociation of weak acids and bases

- acids: proton donors or electron pair acceptors
- bases: proton acceptors and electron pair donors
- weak acids and bases are only partially ionized in water while strong acids and bases are
completely ionized
- HA / H+ + A-
- Ka = [H+][A-] / [HA]
- pKa = - log [Ka]

Henderson-Hasselbach Equation
pH = pKa + log [A-]/[HA]
-  pKa = weaker acid stronger base
-  pKa = stronger acid weaker base

- the covalent bond breaks when ionizing water, not the H bond

-EX of pH calculation: what is pH of 0.1 M acetic acid (pKa = 4.76) and 0.1 M of conjugate base
sodium acetate?
- use HH equation:
-pH = pKa + log [A-]/[HA]
= 4.76 + log [1]
= 4.76

- what happens after adding an equal volume of 0.05 M HCl?

- [HA] = 0.15 / 2 [A-] = 0.05 / 2
pH = 4.76 + log [0.025] / [0.075]
= 4.76 - .48 = 4.28

Biological buffers:
- pH is carefully controlled because activity of enzymes is sensitive to pH
- blood plasma ~7.4
- 2 important buffers in blood are phosphate and bicarbonate

Lecture 3 // SEPT 8
 Proteins: the most abundant macromolecules in cells
 Amino acids and their properties
 Amino acid and peptide isoelectric points
 Post-translational modifications

Proteins
- All are polymers of amino acids (AA)
- only 20 AA are defined by the genetic code
- can be small (dozens of AA) or large (thousands)
- built from 20 α-AA
- central alpha carbon, chiral center
- all AA in proteins are L-stereoisomers (except glycine because it doesn’t have a chiral center)
Amino acids
1. Nonpolar, aliphatic R groups
-hydrocarbon groups that are hydrophobic
1. glycine—smallest
2. alanine—1 hydrocarbon
3. proline—only 1 that loops back and connects, 3 hydrocarbons
4. valine—has 3 hydrocarbons
5. leucine—4 hydrocarbons
6. isoleucine—3 hydrocarbons, 1 carbon
7. methionine—methylated sulfur in side chain

2. Aromatic R groups
8. Phenylaline
9. tyrosine—can donate/accept a H bond because of the -OH
10. tryptophan—can donate/accept H bond because of the NH

3. Polar, uncharged R groups

-partial positive and negative charges
11. serine
12. threonine
13. cysteine
14. aspargine—has amino group
15. glutamine—has amino group

4. Positively charged R groups

16. lysine
17. arginine
18. histidine—imidazole group, can turn into an NH+

5. Negatively charged R groups

19. aspartate
20. glutamate—negatively charged carboxyl group

Nonionic form (rare) vs. Zwitterionic form (net charge = 0)

--NH2 --N+H3

Glysine (the exception) has 3 charge states

+NH3-CH2-COOH / +NH3-CH2-COO- / NH2-CH2-COO-
pK1 pK2

Isoelectric point
- pH net charge = 0
- the middle inflection point of the 2 inflection points that show up for glycine
- pKa is the pH where [HA] = [A-]
- can be derived approximately from the pKa’s of the individual side chains
- average the pKa values of all groups that are ionizing around the pH adds up to be neutral

Peptide bond formation is a condensation reaction while peptide bond breakage is a hydrolysis
reaction

Post translational modifications

- after a protein is made (translation), it can be chemically modified by enzymes
- this adds chemical diversity to proteins
- these enzymes recognize specific target peptide sequences on the protein
- forming certain bonds that are critical for different functions, changes their properties

Lecture 4 // SEPT 10
 Protein purification
 Sequencing

Protein purification
Step 1. Choice of protein source: usually out of convenience and quantity
Step 2. Gently disrupt the cell to solubilize the protein: grinding, sonication, etc
Step 3. Purification

Strategies
Charge: ion exchange chromatography, electrophoresis
Hydrophobicity: hydrophobic interaction chromatography
Size: dialysis and ultra filtration, gel electrophoresis
Specificity: affinity chromatography

Detecting proteins: absorption of light by molecules

Spectrophotometer: detect if protein is present
UV: detect protein (tryptophan absorbs UV light more efficiently than other AA)
Chromatography: uses pigment/dyes to mark and collects proteins and separates them
based on physical properties
1. ion exchange: positively charged proteins stick to negatively charged beads
2. size exclusion: a porous column acts as a molecular sieve, in which smaller
molecules get stuck in pores and take longer to travel through the column. The largest proteins
come off first

3. affinity: protein is isolated by binding to a ligand. Elution is achieved with a

high concentration of free ligand
Electrophoresis: separation on the basis of charge by application of an electric field
(denature and unfold protein to interact hydrophobically to become uniform for which you can
decode the protein then)
 Isoelectric focusing: protein sample may be applied to one end of a gel strip with an
immobilized pH gradient. Or a protein in ampholytes solution is used to rehydrate a dehydrated
gel strip
Protein sequencing aka Edman Degradation: use chemistry to remove 1 N-terminal AA at
a time
- time consuming, laborious
- identify mutations (disease), understand shape and function (homology)
- some AA changes are not harmful. Some are natural genetic heterogeneity that
arise from spontaneous point mutations. Most have no effect on function.
Mass spectrometry: separate ions by mass in a vacuum (lighter ones fly farther)
Tandem mass spec: identify fragments of unique mass

Lecture 5 // SEPT 12
 Protein structure
 Features of primary, secondary and tertiary structure
 Forces that stabilize protein structure

Levels of protein structure

1. primary: the sequences of information (AA) that just rotate around the phi and psi bonds
- carbonyl oxygen has a partial negative charge
- amide nitrogen has a partial positive charge, this makes a small electric dipole
- has a big effect on 2°, 3°, 4° (restricts backbone because they are planar)
- Ramachandran plot: displays allowed regions of protein folding space

2. secondary:
- α-helix properties: right handed, H bond between C=O (n)…H-N(n+4)
- 1st AA bond to the 5th AA bond, 3rd to the 7th…etc.
- compact
- side chains protrude from the helix every 100
- β-sheet (R groups project outward in alternating directions)
- two types:
- parallel (point in same direction )
- anti-parallel (point in opposite directions )
- H bonds are formed between strands
- side chains are on alternate sides to form a pleated sheet
- no flat, have a characteristic twist
- contain few AA residues

Protein structure
1. fibrous
- highly extended, exhibit repeating helical or  sheet structure
- abundant, structural
 - keratin (hair, feathers, skin) : extended  helices, cross linked by disulfide bonds,
composed of many hydrophobic residues, high tensile strength
- collagen (bone, cartilage, connective tissue) : triple helix "coiled-coil" of a
polymer with repeating motif (Gly, Pro, Hyp), most abundant protein in humans, high tensile
strength

2. globular
- compact

Protein structure solutions

Xray diffraction pattern  electron density map model building  structure

Forces that stabilize proteins

Stability is the difference in free energy between the folded and unfolded state
- typical protein is ~20 kcal/mol
- H bond is ~2 kcal/mol
Primary source is the hydrophobic effect : the sequestering of hydrophobic
side chains from aqueous environment in the denatured state to the interior of the protein in
the folded state
-folding to release waters
-ion pairs are formed by interactions between oppositely charged side chains

3. tertiary:  loop, a 3D version of secondary

-  barrel, all beta sheets, makes up the loop

4. quaternary structure: multimers (multiple subunits)

- EX: hemoglobin, 22 heterotetramer ("# of subunits" 4 total)
- offers stability and control

Limit to protein size:

1. more efficient to build large structures from small units
2. error rate in protein synthesis is 1 mistake per 10,000 AA

Denaturation/Renaturation: Anfinsen's protein folding experiment

-purify large amounts of proteins, degrade into nucleotides, denatured and reduced 
EITHER oxidize to re-form crosslinks while denaturant is
presentscrambled structure, no activity
OR remove denaturant, oxidize to reform crosslinks, active protein

Lecture 6 // SEPT 15

Protein function
Protein-ligand interaction: all ligands have a specific binding site on the protein they interact
with
- DNA – charge
- Protein + charge

Examples:
Myoglobin serves the role of oxygen storage and transport, abundant in muscle tissues of
mammals
- necessary because O is not very soluble in water, need an O transport system
- O is bound to an inorganic metal (iron) since no AA side chains have this ability
- 153 AA residues, 8  helices

Heme: porphyrin coordinated to an iron atom to stabilize it

- flat ring, provides 4 nitrogen ligands

Stabilization of Fe2+ state is important since this binds O2 while the Fe3+ does not.
- heme stabilizes this 2+ state

Protein fold influences ligand binding

- CO2 binds to free heme 20,000 stronger than O2 (only binds 200 times stronger)
- improves specificity for oxygen
- O2 binds in a bent, angled formation
- CO2 binds in a linear formation, making it stronger

Oxygen binding curve

- hyperbolic
- protein binds very tightly to ligand Mb (myoglobin) + O2 / MbO2

Equilibrium expression for protein (P) binding ligand (L)

- P + L / PL
- Eq. association constant: Ka = [PL]
[P][L]
- Fraction of protein bound/saturation =  = [PL]
[PL] + [P]

 = [L]
[L] + 1/Ka

1/Ka = Kd (the dissociation constant) measures how

tight a protein binds to its
ligand

- when [L] is equal to Kd, half of the binding sites are occupied
Expression for O binding as a function of partial pressure of oxygen (predicts how much O is
bound to protein)
-  = pO2
pO2 + P50 = [L]
[L] + Kd

Myoglobin (mg) would be a poor O transporter (P50 for myoglobin is 0.26 kPa)
- concentration of O2 in lungs is ~13 kPa while in the tissues, it's ~4 kPa
-  = 13/13.26 = 98% in lungs
- 94 % in tissues
- ability to transfer O from lungs to tissues is proportional to the difference in binding
-binds O too tightly, not suitable

Hemoglobin (hg) is the O transporter in blood

-in erythrocytes
-arterial blood, hg is 96% saturated,
-venous blood, ~64%

Hg and mg have similar tertiary structures. Hemoglobin is a tetramer (has a quaternary

structure), formed from 2 different subunits (2 heterotetramer) while myoglobin is a
monomer

Structural changes induced by binding O

- Hg exists in 2 structural states:
1. T-state: has more interactions between the subunits, changes occur at the
interface between  heterodimers, favored when lack of oxygen
2. R-state (relaxed state) : oxygen binds more strongly and stabilizes R, favored
when highly oxidated environment

Oxygen binding alters location of Helix F

- moves the proximal histidine which pulls on helix "F" which changes interface conformation
- iron atom is not centered around heme

Hemoglobin binding curve

- signoidal as a result of cooperativity between the 4 subunits
- binding affinity is much more sensitive than pO2 than myoglobin and can function at O
transporter (bind O2 in lungs and release in tissues)

Cooperativity: communication between the 4 subunits that form the heterotetramer

- alters conformation to favor the R-state, so that it can bind more tightly
- provides a route to regulating affinity of hg for O by interactions with other ligands

Bohr Effect: favors uptake of protons and CO2 in tissue which has lower pH during respiration
and release in the lungs, favors transition from RT
Lecture 7 // SEPT 17
 General properties of enzymes
 Catalytic strategies
 Kinetics: michaelis-menton equation

Enzymes
1. usually proteins (except catalytic RNA/riboyzmes)
2. incredible catalysts, increases rates of reaction under very mild conditions in aq solns
1. general acid-base catalysis: lowers proximity effects (alters pKas) so reaction can
undergo
2. covalent: forming covalent bonds
3. metal ion: to stabilize a negative charge, 1/3 of all enzymes use this, zinc proteases
(breaks peptide bond, coordinates a Zn2+ to activate the H2O molecule and turns it into a
hydroxyl group to cleave for a nucleophilic attack

- lowers activation barriers

- changes the rate of reaction, not equilibrium

3. specific for substrates

4. some are regulated to provide control over many metabolic processes
5. 3D structure is essential for activity

Michaelis-Menton Equation

Vo = Vmax [S]
Km + [S]

Vo = initial velocity
S = substrate
E + S (k1)/(k-1) ES (k2)/(k-2) E + P

Vo is determined by rate of breakdown of ES to form product: Vo = k2 [ES]

- hyperbolic function
(k2 + k1)
k1 = Km, the Michaelis constant

- lowest Km amount needs LEAST amount of [S] to reach Vmax  creates the fast catalytic rate

Lineweaver-Burke Plots: changes the hyperbolic function into linear by taking inverse of
everything
Vo = Vmax [S] 
Km + [S]  1/Vo = Km + [S] or 1/Vo = Km + 1/Vmax
 Vmax[S] Vmax [S]

The ratio of kcat to Km is useful indicator since [S] << Km

Vo = kcat/Km [Et] [S]

Very efficient enzymes are only limited by the rate of diffusion!!

Lecture 8 // SEPT 19
 Types of enzyme inhibition
 Chymotrypsin mechanism
 Regulatory enzymes

Enzyme inhibition:
Reversible inhibitors: generally small molecules that bind in or close to the active site
1. competitive
E + S / ES E + P
+
I
/ (k1)
EI
- inhibited, blocking substrate
-Vmax does NOT change, Km changes
- attained at very high [S], plateaus on curve/slope change

2. uncompetitive
E + S / ES E + P
+
I
/ (k1)
ESI
- inhibitor is no longer competing for same spot on enzyme
-Km and Vmax are affected equally

3. mixed
E + S / ES E + P
+ +
I I
/ (k1) / (k1)
EI + S ESI
- both Km and Vmax affected, UNequally

Irreversible inhibitors: covalently attached to enzyme, aka suicide substrates, binds to

site and when it's done, it degrades and gets recycled so new proteins can be made
 - pharmaceutical agents
- enzyme specificity: small changes in substrate structure have large effects on
enzymatic parameters

Chymotrypsin: protease that digests proteins containing aromatic AA residues

Regulatory enzymes
- must exhibit increased or decreased activity in responses to certain signals
1. allosteric enzymes: bind regulatory compounds (allosteric modulators) non-covalently
(reversibily)
2. covalently modified: regulatory compounds are covalently attached in a reversible manner
3. zymogens: made as inactive precursors that need to be cleaved to become active

Metabolic pathways are often kept in balance through the use of allosteric enzymes and
feedback inhibition (product shuts off its synthesis by negatively regulating an enzyme in
synthesis path)

Allosteric enzymes have sigmoidal kinetic behavior (stimulated by own substrate)

Lecture 9 // SEPT 24
 Lipids

Biological lipids: insoluble in water

- principal form of energy storage
- membranes
- cofactors, electron carriers, pigments, etc

Fatty acid structure:

Polar head, hydrophilic (carboxyl group) ---------------aliphatic tail, hydrophobic (hydrocarbon
chain)

Saturated
- linear
- pack together efficiently
- lower melting points
Unsaturated
- has kinks, creates an angled structure
- inefficient packing
- higher melting points

Energy stores
Triacylglycerols: efficient sources of energy
1. highly reduced
2. provide >2 x the energy as carbohydrates
 3. dehydrated, take up less space and weight than carbs

Structural lipids in membranes

1. gylcerophospholipids
- prostaglandins: derivatives of membrane gylcero. Containing a 20C fatty acid
- influence a range of bio functions (inflammatory response, pain, fever, etc)
2. sphingolipids
- important immunogenic determinatants in blood
- similar structure to glycerol.
3. sterols
- cholesterol: contains fused ring system that is predominantly hydrophobic, alkyl tail
and polar head group
- similar in size and shape as other lipids
- influence bile acids, testosterone, estradiol, etc
- vitamin D: regulates calcium uptake in bone

Lecture 10 // SEPT 26
 Membrane structure and function

Membranes provide compartments and allow redirection of the nergy flow into useful
metabolic processes
Structure: lipid bilayers
- 3 nm thick
- very different composition, each does different biological functions
- integral proteins
- associated with membrane, dissociated by gentle means (high salt)
- vs peripheral proteins
- tightly associated with membrane
- require detergent for removal from the lipid bilayer
- nenver invert (flip flop) spontaneously

Detergent: tricks the integral protein to thinking it still is in the hydrophobic

environment

Uncatalyzed transbilayer (flip flop) diffusion: very slow, takes days

Uncatalyzed lateral diffusion: very fast, micrometer/sec

Fatty acids self-aggregate to sequester their hydrophobic tails away from water
- individual units are wedge-shaped and spontaneously form a micelle
Lipids self-aggregate to spontaneously form bilayers
- individual units are cylindrical and form a bilayer
- bilayers wrap around to form continuous, spherical particles called
liposomes
- no energy is needed, all due to hydrophobic effect
 Compartments: selective, barriers to diffusion, origin of active transport

Membrane dynamics
a. liquid ordered state Lo
b. liquid disordered state Ld

-cells maintain a balance between these two extremes by changing lipid content in membrane
bilayers to maintain it

 barrel membrane proteins

- build almost entirely from beta sheets
- hydrophobic residues face lipid bilayer
- hydrophilic residues line the pore and upper and lower/outer surfaces
- allows selective diffusion of ions and small molecules

Transport across membranes

1. passive transport
- diffusion along gradient
- simple diffusion
- facilitated diffusion: requires a protein carrier

Facilitated diffusion by channels:

- simple pores, "Porins", as sieve based on molecular weight
- passive transporters: glucose transporter in RBC
- using existing gradient of glucose that is outside
- helices form a hydrophilic channel
- gated channels: controlled by external signal
- neurotransmission
- facilitated but coupled to a primary active transporter, utilizing a Na+ gradient

2. active transport
-accumulation of solute on one side of membrane
-thermodynamically unfavorable
- against a gradient
- primary: requires energy, ATP driven
- secondary: using existing gradient made by primary transporter

Na+/K+ pump: outside system is rich in Na+, inside is rich in K+

- 2 K+ for every 3 Na+
- maintains lo Na+ and high K+ concentrations in animal cells
- hydrolysis of ATP into ADP
- establish an electrochemical gradient for secondary
- maintain osmotic pressure in cells