Exam 2 Biochem Flashcards
Nucleic acids are polymers of nucleotides used for:
storage of genetic info (DNA)
transmission of genetic info (mRNA)
processing of genetic information (ribozymes)
protein synthesis (tRNA and rRNA)
Nucleotides are also used in the monomer form for cellular functions:
energy for metabolism (ATP)
enzyme cofactors (NAD+)
signal transduction (cAMP)
Nucleotide =
nitrogeneous base
pentose
phosphate
Nucleoside =
nitrogeneous base
pentose
Phosphate Group
Negatively charged at neutral pH
Typically attached to 5’ position of sugar
Nucleic acids are built using the 5’-triphosphates version of the nucleotide.
ATP, GTP, TTP, CTP
May be attached to other positions for specialized function
Nitrogenous Bases
Derivatives of pyrimidine or purine
Nitrogen-containing heteroaromatic molecules
Planar or almost planar structures
Absorb UV light around 250–270 nm
Pyrimidine Bases
Cytosine, thymine, uracil
All are good H-bond donors and acceptors.
Neutral molecules at pH 7
Purine bases
Adenine and guanin
All are good H-bond donors and acceptors.
Neutral molecules at pH 7
b-N-Glycosidic Bond
the pentose ring is attached to the nitrogenous base via a N-glycosidic bond
The bond is formed:
–to position N1 in pyrimidines
–to position N9 in purines
This bond is quite stable toward hydrolysis, especially in pyrimidines.
Bond cleavage is catalyzed by acid.
Why is neucleoside modification made after DNA synthesis?
Epigenetic marker:
–way to mark own DNA so that cells can degrade foreign DNA (prokaryotes)
–way to mark which genes should be active (eukaryotes)
–could the environment turn genes on and off in an inheritable manner?
Inosine
sometimes found in the “wobble position” of the anticodon in tRNA.
made by de-aminating adenosine
provides richer genetic code
Pseudouridine
found widely in tRNA and rRNA.
more common in eukaryotes but found also in eubacteria
made from uridine by enzymatic isomerization after RNA synthesis
may stabilize the structure of tRNA
may help in folding of rRNA
Polynucleotides in both DNA and RNA/mRNA
Covalent bonds are formed via phosphodiester linkages.
negatively charged backbone
Linear polymers
no branching or cross-links
Directionality
The 5’ end is different from the 3’ end.
We read the sequence from 5’ to 3’.
Polynucleotides in DNA
DNA backbone is fairly stable.
Hydrolysis accelerated by enzymes (DNAse)
Polynucleotides in RNA
RNA backbone is unstable.
In water, RNA lasts for a few years.
In cells, mRNA is degraded in a few hours.
Hydrolysis of RNA
RNA is unstable under alkaline conditions.
Hydrolysis is also catalyzed by enzymes (RNase).
Three types of RNA enzymes
S-RNase in plants prevents inbreeding.
RNase P is a ribozyme (enzyme made of RNA) that processes tRNA precursors.
Dicer is an enzyme that cleaves double-stranded RNA into oligonucleotides.
protection from viral genomes
RNA interference technology
Hydrogen-Bonding Interactions
Two bases can hydrogen bond to form a base pair.
For monomers, a large number of base pairs is possible.
In polynucleotide, only a few possibilities exist.
Purine to pyrimidine
Replication of Genetic Code
Strand separation occurs first.
Each strand serves as a template for the synthesis of a new strand.
Synthesis is catalyzed by enzymes known as DNA polymerases.
A newly made DNA molecule has one daughter strand and one parent strand.
Messenger RNA:
Is synthesized using DNA template and generally occurs as a single strand
Contains ribose instead of deoxyribose
Contains uracil instead of thymine
One mRNA may code for more than one protein
Together with transfer RNA (tRNA), transfers genetic information from DNA to proteins
DNA Denaturation
Covalent bonds remain intact.
Genetic code remains intact.
Hydrogen bonds are broken.
Two strands separate.
Base stacking is lost
UV absorbance increases.
Thermal DNA Denaturation (Melting)
DNA strands dissociate at elevated temperatures.
strands re-anneal when the temperature is lowered.
reversible thermal denaturation and annealing form the basis for the polymerase chain reaction.
commonly monitored by UV spectrophotometry at 260 nm.
Factors affecting DNA denaturation
Melting point depends on base composition, dna length, pH and ionic strength
AT-rich regions melt at lower temperatures
Spontaneous Mutagenesis: Deamination
Amine group is transformed into double bonded oxygen
very slow reactions
large number of residues
The net effect is significant: 100 C = U events/day in a mammalian cell.
Spontaneous Mutagenesis: Depurination
N-glycosidic bond is hydrolyzed
Significant for purines: 10,000 purines lost/day in a mammalian cell
Lose purine base
Spontaneous Mutagenesis: other types
Oxidative damage
—hydroxylation of guanine
—mitochondrial DNA is most susceptible
Chemical alkylation
—methylation of guanine
Spontaneous Mutagenesis: Radiation
dimerization of pyrimidines; this may be the main mechanism for skin cancers.
Ring opening and strand breaking (heard to repair all)
Nucleotides: energy source
With 1+ phosphate group, may provide energy
Other Functions of Nucleotides: Coenzymes
an organic non-protein compound that binds with an enzyme to catalyze a reaction
Proteins: Main Agents of Biological Function
What are those functions?
Catalysis
Transport
Structure
Motion
Amino Acids
Proteins are linear heteropolymers of a-amino acids.
carry out a variety of biological functions:
1. capacity to polymerize
2. useful acid-base properties
3. varied physical properties
4. varied chemical functionality
Amino Acids Have Three Common Functional Groups Attached to the α Carbon
carboxyl group (COOH)
amino group (NH2)
a-Hydrogen
Amino Acids: Classification
nonpolar, aliphatic (7)
aromatic (3)
polar, uncharged (5)
positively charged (3)
negatively charged (2)
nonpolar, aliphatic (7)
glycine
alanine
proline
valine
leucine
isoleucine
methionine
aromatic (3)
phenylalanine
tyrosine
tryptophan
polar, uncharged (5)
serine
threonine
cysteine
asparagine
glutamine
positively charged (3)
lysine
arginine
histidine
negatively charged (2)
aspartate
glutamate
Ionization of Amino Acids
contain at least two ionizable protons, each with its own pKa.
The carboxylic acid has an acidic pKa and will be protonated at an acidic (low) pH:
amino group has a basic pKa and will be protonated until basic pH (high) is achieved
carboxylic acid has an acidic pKa
−COOH = COO− + H+
slightly more acidic than an carboxylic acids
The amino group has a basic pKa
−NH4+ = NH3 + H+
Slightly less basic than in amines
Ionization of Amino Acids
low pH
the amino acid exists in a positively charged form (cation).
Ionization of Amino Acids
high pH
the amino acid exists in a negatively charged form (anion).
zwitterion
Between the pKa for each group, the amino acid exists in a zwitterion form, in which a single molecule has both a positive and negative charge.
Isoelectric Point (equivalence point, pI)
PI = (pK1 + pK2) / 2
In Zwitterions
the net charge is zero.
AA is least soluble in water.
AA does not migrate in electric field.
Amino Acids Can Act as Buffers
Amino acids with uncharged side chains, such as glycine, have two pKa values
As buffers prevent change in pH close to the pKa, glycine can act as a buffer in two pH ranges.
Ionizable Side Chains Also Have pKa and Act as Buffers
Ionizable side chains influence the pI of the amino acid.
Ionizable side chains can be also titrated.
Titration curves are now more complex, as each pKa has a buffering zone of 2 pH units.
How to Calculate the pI When the Side Chain Is Ionizable
Identify species that carries a net zero charge.
Identify the pKa value that defines the acid strength of this zwitterion: (pKR).
Identify the pKa value that defines the base strength of this zwitterion: (pK2).
Take the average of these two pKa values.
Amino Acids Polymerize to Form Peptides
Peptides are small condensation products of amino acids.
They are “small” compared with proteins (Mw < 10 kDa).
Naming Peptides: Start at the N-terminal
Numbering (and naming) starts from the amino terminus (N-terminal).
Using full amino acid names
Using the three-letter code abbreviation
For longer peptides (like proteins) the one- letter code
Peptides: A Variety of Functions
Hormones and pheromones
Neuropeptides
Antibiotics
Protection, e.g., toxins
Proteins are comprised of:
Polypeptides (covalently linked a-amino acids) + possibly:
- cofactors
- coenzymes
- prosthetic groups
- other posttranslational mods
cofactors
functional non-amino acid component
metal ions or organic molecules
coenzymes
organic cofactors
prosthetic groups
covalently attached cofactors
A Mixture of Proteins Can Be Separated
charge
size
affinity for a ligand
solubility
hydrophobicity
thermal stability
Chromatography is commonly used for preparative separation in which the protein is often able to remain fully folded.
Column Chromatography
allows separation of a mixture of proteins over a solid phase (porous matrix) using a liquid phase to mobilize the proteins.
Proteins with a lower affinity for the solid phase will wash off first; proteins with higher affinity will retain on the column longer and wash off later.
Separation by Charge: Ion Exchange
proteins move through columns at rates determined by their net charge at the pH being used.
With cation exchangers, proteins with more negative net charge move faster
Separation by Size: Size Exclusion
Larger molecules pass more freely and appearing in the earlier fractions first using crosslinked polymer
Separation by Binding: Affinity
Protein mixture added to column contained polymer bound ligand for specific protein of interest.
Unwanted proteins are washed through the column and then protein of interested eluted by ligand solution
Electrophoresis for Protein Analysis
The electric field pulls proteins according to their charge.
The gel matrix hinders mobility of proteins according to their size and shape.
The gel is commonly polyacrylamide, so separation of proteins via electrophoresis is often called polyacrylamide gel electrophoresis, or PAGE
SDS PAGE Separates Proteins by Molecular Weight
SDS – sodium dodecyl sulfate – a detergent
SDS micelles bind to proteins and facilitate unfolding.
SDS gives all proteins a uniformly negative charge.
The native shape of proteins does not matter.
The rate of movement will only depend on size: small proteins will move faster.
Isoelectric Focusing Can Be Used to Determine the pI of a Protein
Protein sample may be applied to one end of a gel strip with an immobilized pH gradient.
After staining, proteins are shown to be distributed along pH gradient according to their PI values
Aromatic Amino Acids Absorb Light in a Concentration-Dependent Manner
Proteins typically have UV absorbance maxima around 275–280 nm.
Tryptophan and tyrosine are the strongest chromophores.
For proteins and peptides with known extinction coefficients (or sequences), concentration can be determined by UV-visible spectrophotometry using the Lambert-Beer law: A = ECL
Protein Sequencing
Edman degradation (classical method):
–successive rounds of N-terminal modification, cleavage, and identification
–can be used to identify protein with known sequence
Mass spectrometry (modern method):
–MALDI MS and ESI MS can precisely identify the mass of a peptide, and thus the amino acid sequence
–can be used to determine posttranslational modifications
Protein Sequences as Clues to Evolutionary Relationships
Differences indicate evolutionary divergences.
Analysis of multiple protein families can indicate evolutionary relationships between organisms, ultimately the history of life on Earth.
