Biochemistry Exam 1

Biochemistry

the chemical analysis of biomolecules and biological systems/pathways

chemical analysis

bonds, stereochemistry, & chemical properties

• biochemistry considers the chemistry within and between molecules and their environment. These factors can affect the structure and function of biomolecules and their systems/pathways

biomolecules

4 different classes

• Proteins

• Nucleic Acids

• Lipids

• Carbohydrates

proteins: definition & function

polymers of amino acids

Functions:

• signaling/receptors

• structural

• motility

• immune system

• enzymes

nucleic acids

store and transfer genetic information

• basic unit is nucleotide

nucleotide

composed of phosphoryl group(s), five-carbon sugar, and base

types of 5-carbon sugars

• RNA (ribose)

• DNA (deoxyribose)

types of bases

• pyrimidines (one ring structure)

• purines (two ring structure)

pyrimidines

one ring structure

• cytosine (nitrate group no carbonyl)

• thymine (methyl group)

• uracil (carbonyl but no methyl)

purines

two ring structures

• adenine

• guanine (carbonyl group)

lipids

• amphipathic nature (hydrophilic head and hydrophobic tail)

• form barriers

• source of energy/storage

• cell signaling

• protein modifications

carbohydrates

• source of energy/storage

• cell recognition

• protein modifications

reverse transciptase inhibitors

a major class of antiviral drugs used mainly to treat HIV by stopping the virus from copying its genetic material

covalent bonds

sharing of electrons

non-covalent bonds

no sharing of electrons

• much weaker, reversible, repeated

ionic bonds

a type of chemical link formed through the electrostatic attraction between oppositely charged ions

• “salt” bridges

hydrogen bonds

• between Hydrogen and electroNEGATIVE atoms (usually oxygen and nitrogen)

• can be disrupted by water

Van der Waals Interactions

electrostatic interactions of partial charges

hydrophobic interactions

the tendency of nonpolar molecules to cluster together in water rather than mix

functional groups

• hydrophobic (non-polar)

• hydroxyl

• aldehyde

• keto

• carboxyl

• amino

• phosphate

• sulfhydryl

hydrophobic (non-polar)

hydrocarbon chains

more hydrophobic means…?

higher melting point

trans vs. cis conformation on melting point

trans- higher melting point

cis- lower melting point

less hydrophobic

lower melting point

carbonyl group functional groups

• Hydroxyl- Alcohols (R-OH

• Aldehyde- Aldehydes (R-C-H

• Keto- Ketones (R - C - R

• Carboxyl- Carboxylic Acids (R- C- OH)

Amino, Phosphate, and Sulfhydryl

• Amino- Amines (R-NH2)

• Phosphate- Organic Phosphates

• Sulfhydryl- Thiols (R-SH)

polar

charge distribution is not equal

non polar

sharing of electrons is equal

water (H2O)

• Polar (Oxygen is electronegative, H is electropositive)

• Involved in Hydrogen bonding

• Solvent for any charged/polar molecule

• Cannot dissolve hydrophobic/ non-polar molecule

• Can dissociate

pH definition

a measure of free hydrogen ion (proton) concentration

• log scale of proton concentration [H+]

importance of pH in biological systems

pH can affect electrostatic interactions (bonding) which can then affect the structure and function of a biomolecule

equilibrium constant, Keq

measures the extent to which reactants are converted to products by the reaction at equilibrium

• rxn has reached a point where the concentrations of the reactant(s) and product are unchanging

Keq for H2O

[H+][OH-]/[H2O]

pH = ?

-log [H+] or log (1/[H+])

lower pH means…?

more acidic

• higher concentration of free H+

acids

proton donors

• forms conjugate base when loses hydrogen ion

bases

proton acceptor

• forms conjugate acid when gains hydrogen ion

Ka = ?

[H+][A-]/ [HA]

pKa

measures the strength of an acid

tells us how easily a molecule gives up a proton

lower pKa…?

the stronger the acid

easier it loses a proton

Henderson-Hasselbach equation

pH = pKa + log ([A-]/[HA])

if [A-] equals [HA]…?

pH = pKa

pKa is the pH at which the acid is…?

• half-associated

• half dissociated

• half-protonated

• half unprotonated

what can we use the Henderson Hasselbach equation for?

to calculate the relative amount of protonation of acids/bases in a give pH when given the pKa of a functional group and the surrounding pH

if pH > pKa

more [A-] than [HA]; basic

• log ([A-] / [HA]) will be a positive value

if pKa is > pH…?

more [HA] than [A-]; acidic

• log ([A-] / [HA]) will be a negative value

why is pKa and pH important in biochem?

enzymes function optimally at a given pH due to the correct protonation states of certain functional groups

• if a functional group is not protonated correctly, the enzyme will not function properly

amino acid functions

• signaling

• “building blocks” for proteins

• precursors for other biomolecules

parts of amino acids

• alpha carbon, chiral (L or D isomers)

• carboxylic acid group (COO-)

• amino group (NH3+)

• R group, “side chain”

L vs. D isomers in amino acids

Orient the carboxylic acid group at the top and the side-chain (R group) at the bottom. If the amino group (–NH₂) on the alpha carbon is on the right, it is D; if it is on the left, it is L

• amino group is #1 priority, carbox group is #2

• D goes clockwise

• L goes counterclockwise

at neutral pH, amino acids exist as…?

dipolar ions (zwitterion)

how many amino acids are commonly found in proteins?

20

how do we group amino acids based on side chain properties?

1. Hydrophobic, non-polar

2. Negatively-charged (at pH=7) (acidic)

3. Positively-charged (at pH=7) (basic)

4. Polar, neutral but charge is not evenly distributed

hydrophobic, non-polar amino acids

have hydrocarbon side chains that repel water and cluster in the interior of proteins to stabilize their structure

• Glycine, Alanine, Valine, Leucine, Isoleucine, Methionine, Proline, Phenylalanine, Tryptophan

negatively-charged amino acids

• Aspartate and Glutamate

• Hydrophilic

positively-charged amino acids

molecules with side chains that accept protons (H⁺), giving them a net positive charge at normal body (physiological) pH

• Lysine, Arginine, Guanidinium, Histidine, Imidazole

polar, neutral amino acids

hydrophilic building blocks of proteins that have uncharged, polar side chains at physiological pH

• Serine, Threonine, Tyrosine, Cysteine, Asparagine, Glutamine

non-essential amino acids (humans can synthesize)

SPY DANGER QC:

• Serine, Proline, Tyrosine,

• Aspartate, Alanine, Asparagine, Glycine, Glutamate, Arginine,

• Glutamine, Cysteine

essential amino acids (humans need these)

F(reedom) V(oice) WITH MLK

• Phenylalanine

• Valine

• Tryptophan, Isoleucine, Tryptophan, Histidine

• Methionine, Leucine, Lysine

ionizable parts of individual amino acids

1. terminal alpha-carboxyl group (all)

2. terminal alpha-amino group (all)

3. side chains

   1. D, Aspartate, (COO-)

   2. R, Arginine (Guanidinium)

   3. C, Cysteine (-S-)

   4. H, Histidine (Imidazole)

   5. E, Glutamate (COO-)

   6. K, Lysine (NH3+)

   7. Y, Tyrosine (-OH)

typical pKa values for ionizable groups in proteins

• Terminal alpha-carboxyl group: 3.1

• Aspartic acid/Glutamic acid: 4.1

• Histidine: 6.0

• Terminal alpha-amino group: 8.0

• Cysteine: 8.3

• Tyrosine: 10.9

• Lysine: 10.8

• Arginine: 12.5

isoelectric pH (pI)

the pH at which the molecule is neutral in charge

• Identify the ionizable groups

• Find the pH “range” where the molecule is isoelectrically neutral (no net charge)

• Average the two pKa of the “range”

protein gel electrophoresis

a lab technique used to separate proteins by moving them through a gel using an electric current (usually polymerized acrylamide)

PAGE stands for?

Polyacrylamide Gel Electrophoresis

what’s the purpose of protein gel electrophoresis?

A gel converts protein sample into a visible “band”

• A band represents many copies of protein molecules that migrated to the same position

  • Allows us to check protein presence, purity, and approx amount

  • Compare samples before and after purification

  • Estimate molecular weight using protein ladder

  • Evaluate complexes

mechanism of separation for gel electrophoresis

proteins migrate in an electric field through a gel matrix

• charged molecules move towards the electrode with the opposite charge

• pores in the gel restrict movement, especially for larger proteins

• faster movement usually means farther travel down the gel

  • Native gel: charge, size, shape

  • Denaturing gel: size

native gel (native PAGE)

run without strong denaturants, so proteins can retain structure and interactions

• protein remains folded (enzymatic activity)

• protein complexes can remain together

• migration depends on native charge, size, and shape

denaturing gel (SDS-PAGE)

• sodium dodecyl sulfate (SDS) is added to the gel and protein samples

  • SDS disrupts non-covalent interactions and coats the proteins with a negative charge

• the samples may also contain Dithiothreitol (DTT) or beta-mercaptoethanol (beta-ME) which reduces disulfide bonds

• separation is primarily by molecular mass

  • smaller proteins travel farther

• Cathode (-) up top with Anode (+) on bottom

isoelectric focusing (IEF)

proteins stop “moving” when they are neutral

• Acidic amino acids are negatively-charged when UNprotonated, neutral when protonated

• Basic amino acids are neutral when UNprotonated, positively-charged when protonated

  • if needed we can reverse the polarity and pH gradient to achieve desired state

primary protein structure

amino acids are linked by peptide bonds (planar)

• Condensation reaction; loss of water (dehydration)

• Carboxyl carbon of AA1 (residue) forms a covalent bond with amino nitrogen group of AA2

• Contains Start N-terminus (alpha-amino group), and End C-terminus (carboxylic acid group

• “Backbone” of repeated atoms

peptide bond

has “double-bond characteristics”

• distance: 1.32 A

• uncharged

• two alpha-carbon configurations: cis & trans

torsion angles

Phi: N-C (alpha)

Psi: C (alpha)- carbonyl carbon

ramachandran diagram

A Ramachandran plot maps the values of \(\psi \) on the y-axis against \(\phi \) on the x-axis

• Shaded zones where specific \((\phi, \psi)\) angle pairs allow the atoms to sit comfortably without bumping into each other. These regions perfectly align with standard secondary structures:

  • Top Left: Extended beta-sheets (\(\beta \)-strands) and collagen helices.

  • Middle/Bottom Left: Right-handed alpha-helices (\(\alpha \)-helices).

  • Top Right: Left-handed alpha-helices (rare in nature).

• Disallowed Regions: The blank, unshaded areas where the combination of angles forces atoms too close together, making the conformation energetically impossible.

disulfide bonds

cysteine residues can covalently link chains in an oxidation reaction

• can be broken using a reducing agent, B-mercaptoethanol

approx. sizes for peptide, oligopeptide, polypeptide, and protein?

peptide: 2 amino acids

oligopeptide: 2-20

polypeptide: 10 to 50

protein: >50 (implies biological function)

size of protein and amino acids (unit of measurement)

protein: Dalton (Da): 1 Da = 1 g/mol

amino acid is about 110 Da

primary sequence determines…?

the 3-D structure of a protein

alpha helix

Coiled backbone, R-groups directed outward

• mostly right-handed

Specifications:

• 3.6 residues per turn

• rise 1.5 A per amino acid or 5.4 A per helical turn

• usually less than 45 A long

what are residues not commonly found in alpha-helices?

• Proline: lacks an amide hydrogen (which is necessary for forming the hydrogen bonds that stabilize the alpha-helix structure

• Glycine: is extremely flexible (has difficulty maintaining the restricted

• Branched chain amino acids (ex. Thr, Val, Ile) (can cause steric crowding near the protein backbone, making the alpha-helical conformation less favorable)

beta (pleated) sheets

consists of two or more B-strands which can be in parallel or anti-parallel orientation

• R-groups are pointing away from the backbone

• stabilized by hydrogen bonds between the carbonyl oxygen and amide hydrogen from different strands

which is more stable parallel or anti-parallel? why?

anti-parallel because of perpendicular nature of H-bonds

alpha-Keratin

two, right-handed alpha-helices intertwined to from a single left-hand “superhelix”

• two helices are held together by van der Waals forces, ionic interactions, and disulfide bonds

  • primary component of wool and hair

  • tertiary structure

collagen

• very abundant in humans (25% of protein)

• fibrous component in skin, bone, tendon, cartilage, and teeth

• Tropocollagen (“collagen” superhelix)

tropocollagen components

consists of 3 helical polypeptide (procollagen) chains

• 3 residues per turn

• Gly every third base

• Abundant in hydroxyproline (Hyp) and proline

• Frequent repeats of - [Gly- Pro - HyP]

• No intrastrand hydrogen bonds within the helix

tropocollagen is stabilized by…?

1. INTERstrand hydrogen bonds between the procollagen chains (the hydrogen of the alpha-amino group of Gly with the carbonyl oxygen on a different procollagen chain)

2. Steric repulsion of the pyrrolidine rings of proline and hydroxyroline

chain for collagens

procollagen chain → procollagen → tropocollagen → collagen fibril → collagen fibre

prolyl hydroxylase

synthesizes hydroxyproline from proline

• requires a Fe2+ (ferrous) ion to activate O2 in the reaction

• in the formation of hydroxyproline, Fe2+ becomes oxidized to Fe3+ (ferric) and prolyl hydroxylase become inactive

importance of vitamin C

1. hydroxyproline is essential for the stability of collagen

2. prolyl hydroxylase synthesizes hydroxyproline from proline

3. vitamin C reduces Fe3+ (ferric) ion back to Fe2+ (ferrous) ion

4. human are unable to synthesize vitamin C and must acquire it from their diet

5. lack of vitamin C can lead to scurvy which results from less-stable collagen

types of tertiary structure

coiled coils, binding pockets, & B-barrel

binding pocket

1. 8 alpha-helices

2. Fe-bound heme prosthetic group held in a “mostly hydrophobic pocket”

3. Polar, charged residues on outer surface make the molecule water soluble

ex. myoglobin

B-barrel

1. class of bacterial membrane channel protein

2. Beta-barrel

   1. B-sheet “tube” consisting of anti-parallel B-strands connected together by hydrogen bonds

   2. embedded in the lipid membrane

   3. hydrophobic residues oriented outward lipid membrane

   4. hydrophilic residues oriented inward toward the center of the barrel

ex. Porins, Canis familiaries allergen 1 (50-75% of dog-allergic are subject to Canis)

quaternary structure

arrangements/interactions of subunits (polypeptides)

• ex. hemoglobin

hemoglobin

• consists of 4 subunits: 2 alpha and 2 B subunits

• involves several ionic interactions between subunits

structure of myoglobin (Mb)

1. 153 amino acids - MW 17 kDa

2. one polypeptide chain: 1, 2, 3 structure

3. 8 alpha-helices (A-H)

4. hydrophobic pocket; contains heme prosthetic group

5. heme

6. oxidation: Fe2+ → Fe3+

heme

• heterocyclic ring structure of 4 pyrrole groups (A-D) connected by methine bridges

• Fe2+ coordinated by 4 porphyrin N atoms and N atom of His F8

• O2 binds to Fe2+ at the 6th ligand position

• His E7 hydrogen bonds to O2

• Additional structural stability provided by 2 hydrophobic side chains Val E11 and Phe CD1

oxidation Fe2+ → Fe3+

1. Fe2+ heme is called myoglobin (can bind to oxygen)

2. Myoglobin structure (hydrophobic pocket) prevents oxidation of Fe2+; allows reversible O2 binding

3. When O2 is bound (oxymyglobin), it appear bright red in color

4. Fe3+ (oxidized) heme is called “metmyoglobin”; Fe3+ prevents O2 binding and appears brown/grey in color

carbon monoxide binds ____ than O2?

tighter

• keeps meat appearing red for longer periods of time

function of myoglobin

• O2 storage

• Facilitate O2 diffusion in muscle

structure of hemoglobin (Hb)

1. Tetramer (4 subunits with structure a2B2)

2. Has 1, 2, 3, and 4 structure

3. alpha and B subunits are structurally similar to Mb

   1. alpha subunit: 141 amino acids, has 7 alpha-helices

   2. B-subunit: 146 amino acids, 8 alpha-helices