Exam 2 Biochem

Question 1

Nucleic acids are polymers of nucleotides used for:

Answer 1

storage of genetic info (DNA)

transmission of genetic info (mRNA)

processing of genetic information (ribozymes)

protein synthesis (tRNA and rRNA)

Question 2

Nucleotides are also used in the monomer form for cellular functions:

Answer 2

energy for metabolism (ATP)

enzyme cofactors (NAD+)

signal transduction (cAMP)

Question 3

Nucleotide =

Answer 3

nitrogeneous base
pentose
phosphate

Question 4

Nucleoside =

Answer 4

nitrogeneous base
pentose

Question 5

Phosphate Group

Answer 5

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

Question 6

Nitrogenous Bases

Answer 6

Derivatives of pyrimidine or purine

Nitrogen-containing heteroaromatic molecules

Planar or almost planar structures

Absorb UV light around 250–270 nm

Question 7

Pyrimidine Bases

Answer 7

Cytosine, thymine, uracil

All are good H-bond donors and acceptors.

Neutral molecules at pH 7

Question 8

Purine bases

Answer 8

Adenine and guanin

All are good H-bond donors and acceptors.

Neutral molecules at pH 7

Question 9

b-N-Glycosidic Bond

Answer 9

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.

Question 10

Why is neucleoside modification made after DNA synthesis?

Answer 10

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?

Question 11

Inosine

Answer 11

sometimes found in the “wobble position” of the anticodon in tRNA.
made by de-aminating adenosine
provides richer genetic code

Question 12

Pseudouridine

Answer 12

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

Question 13

Polynucleotides in both DNA and RNA/mRNA

Answer 13

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’.

Question 14

Polynucleotides in DNA

Answer 14

DNA backbone is fairly stable.
Hydrolysis accelerated by enzymes (DNAse)

Question 15

Polynucleotides in RNA

Answer 15

RNA backbone is unstable.
In water, RNA lasts for a few years.
In cells, mRNA is degraded in a few hours.

Question 16

Hydrolysis of RNA

Answer 16

RNA is unstable under alkaline conditions.
Hydrolysis is also catalyzed by enzymes (RNase).

Question 17

Three types of RNA enzymes

Answer 17

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

Question 18

Hydrogen-Bonding Interactions

Answer 18

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

Question 19

Replication of Genetic Code

Answer 19

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.

Question 20

Messenger RNA:

Answer 20

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

Question 21

DNA Denaturation

Answer 21

Covalent bonds remain intact.
Genetic code remains intact.
Hydrogen bonds are broken.
Two strands separate.
Base stacking is lost
UV absorbance increases.

Question 22

Thermal DNA Denaturation (Melting)

Answer 22

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.

Question 23

Factors affecting DNA denaturation

Answer 23

Melting point depends on base composition, dna length, pH and ionic strength

AT-rich regions melt at lower temperatures

Question 24

Spontaneous Mutagenesis: Deamination

Answer 24

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.

Question 25

Spontaneous Mutagenesis: Depurination

Answer 25

N-glycosidic bond is hydrolyzed Significant for purines: 10,000 purines lost/day in a mammalian cell Lose purine base

Question 26

Spontaneous Mutagenesis: other types

Answer 26

Oxidative damage ---hydroxylation of guanine ---mitochondrial DNA is most susceptible Chemical alkylation ---methylation of guanine

Question 27

Spontaneous Mutagenesis: Radiation

Answer 27

dimerization of pyrimidines; this may be the main mechanism for skin cancers. Ring opening and strand breaking (heard to repair all)

Question 28

Nucleotides: energy source

Answer 28

With 1+ phosphate group, may provide energy

Question 29

Other Functions of Nucleotides: Coenzymes

Answer 29

an organic non-protein compound that binds with an enzyme to catalyze a reaction

Question 30

Proteins: Main Agents of Biological Function What are those functions?

Answer 30

Catalysis Transport Structure Motion

Question 31

Amino Acids

Answer 31

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

Question 32

Amino Acids Have Three Common Functional Groups Attached to the α Carbon

Answer 32

carboxyl group (COOH) amino group (NH2) a-Hydrogen

Question 33

Amino Acids: Classification

Answer 33

nonpolar, aliphatic (7) aromatic (3) polar, uncharged (5) positively charged (3) negatively charged (2)

Question 34

nonpolar, aliphatic (7)

Answer 34

glycine alanine proline valine leucine isoleucine methionine

Question 35

aromatic (3)

Answer 35

phenylalanine tyrosine tryptophan

Question 36

polar, uncharged (5)

Answer 36

serine threonine cysteine asparagine glutamine

Question 37

positively charged (3)

Answer 37

lysine arginine histidine

Question 38

negatively charged (2)

Answer 38

aspartate glutamate

Question 39

Ionization of Amino Acids

Answer 39

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

Question 40

carboxylic acid has an acidic pKa

Answer 40

−COOH = COO− + H+ slightly more acidic than an carboxylic acids

Question 41

The amino group has a basic pKa

Answer 41

−NH4+ = NH3 + H+ Slightly less basic than in amines

Question 42

Ionization of Amino Acids low pH

Answer 42

the amino acid exists in a positively charged form (cation).

Question 43

Ionization of Amino Acids high pH

Answer 43

the amino acid exists in a negatively charged form (anion).

Question 44

zwitterion

Answer 44

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.

Question 45

Isoelectric Point (equivalence point, pI)

Answer 45

PI = (pK1 + pK2) / 2 In Zwitterions the net charge is zero. AA is least soluble in water. AA does not migrate in electric field.

Question 46

Amino Acids Can Act as Buffers

Answer 46

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.

Question 47

Ionizable Side Chains Also Have pKa and Act as Buffers

Answer 47

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.

Question 48

How to Calculate the pI When the Side Chain Is Ionizable

Answer 48

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.

Question 49

Amino Acids Polymerize to Form Peptides

Answer 49

Peptides are small condensation products of amino acids. They are “small” compared with proteins (Mw < 10 kDa).

Question 50

Naming Peptides: Start at the N-terminal

Answer 50

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

Question 51

Peptides: A Variety of Functions

Answer 51

Hormones and pheromones Neuropeptides Antibiotics Protection, e.g., toxins

Question 52

Proteins are comprised of:

Answer 52

Polypeptides (covalently linked a-amino acids) + possibly: 1. cofactors 2. coenzymes 3. prosthetic groups 4. other posttranslational mods

Question 53

cofactors

Answer 53

functional non-amino acid component metal ions or organic molecules

Question 54

coenzymes

Answer 54

organic cofactors

Question 55

prosthetic groups

Answer 55

covalently attached cofactors

Question 56

A Mixture of Proteins Can Be Separated

Answer 56

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.

Question 57

Column Chromatography

Answer 57

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.

Question 58

Separation by Charge: Ion Exchange

Answer 58

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

Question 59

Separation by Size: Size Exclusion

Answer 59

Larger molecules pass more freely and appearing in the earlier fractions first using crosslinked polymer

Question 60

Separation by Binding: Affinity

Answer 60

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

Question 61

Electrophoresis for Protein Analysis

Answer 61

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

Question 62

SDS PAGE Separates Proteins by Molecular Weight

Answer 62

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.

Question 63

Isoelectric Focusing Can Be Used to Determine the pI of a Protein

Answer 63

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

Question 64

Aromatic Amino Acids Absorb Light in a Concentration-Dependent Manner

Answer 64

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

Question 65

Protein Sequencing

Answer 65

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

Question 66

Protein Sequences as Clues to Evolutionary Relationships

Answer 66

Differences indicate evolutionary divergences. Analysis of multiple protein families can indicate evolutionary relationships between organisms, ultimately the history of life on Earth.