Biochemistry Chapter 3

Proteins are constructed from a common set of what?

20 amino acids

Amino acids are joined in linear sequences through what type of bond linkage

An amide linkage, the peptide bond.

Proteins can be separated based on what?

Differences in their chemical and functional properties.

Amino acids sequences are a key resource to understanding what theory?

Evolutionary relationships

Proteins are what? Be specific.

Linear heteropolymers of alpha-amino acids

Amino acids have properties that are well suited to carry out a variety of biological functions such as what?

• Capacity to polmerize

• Useful acid-base properties

• Varied physical properties

• Varied chemical functionality

What is a protein complex?

More than one protein

What is the primary function of proteins?

Means by which the genetic information is expressed and propagated

What are the main biological functions of proteins? And what are some examples of proteins that serve these functions?

• Catalysis

  • Enolase (Glycolytic pathway)

  • DNA polymerase (DNA replication)

• Transport

  • Hemoglobin (O2 through blood)

  • Lactose permease (lactose across cell membrane)

• Structure

  • Collagen (connective tissue)

  • Keratin (hair, nails, feathers, horns)

• Motion

  • Myosin (muscle tissue)

  • Actin (muscle tissue, cell motility)

What dictates protein sequence?

DNA sequence

The alpha carbon of amino acids always has how many substituents? And is what shape?

Four and tetrahedral

What groups do all amino acids (except proline)?

1. Acidic carboxyl group on alpha carbon

2. Basic amino group on alpha carbon

3. Alpha hydrogen connect to the alpha carbon

4. R unique to each acid

What is the simplest amino acid?

Glycine, Gly, G

Amino acid residues in proteins are what type of stereoisomer?

L stereoisomer

D and L system specifies what?

Absolute configuration

What is another term for two possible stereoisomers?

Enantiomers

Are amino acids optically active?

Yes

How do you name an amino acid?

Start from the alpha carbon and go down the R-group.

Common amino acids can be placed in five basic groups based on their R substituents. What are those 5 groups and how many amino acids are in each group?

1. Nonpolar, aliphatic (7)

2. Aromatic (3)

3. Polar, uncharged (5)

4. Positively charged (3)

5. Negatively charged (2)

What amino acids make up the non-polar, aliphatic group? And what is characteristic properties of this group?

• Glycine

• Alanine

• Proline

• Valine

• Leucine

• Isoleucine

• Methionine

• They are unable to form hydrogen bonding

• Hydrophobic effect stabilizes them

• Most likely to be found in the center of a protein

What amino acids make up the positively charged group? What are some important notes about this group?

• Lysine

• Arginine

• Histidine

• Structural formula shows predominate form at pH 7.0

• Exception is His, shown uncharged however significant fraction is positively charged

• Basic

What amino acids make up the negatively charged group? And what’s important to note?

• Aspartic acid

• Glutamic acid

• Acidic (COOH) side chains

• Net negative charge at pH 7.0

What amino acids make up the aromatic group? And what’s important to note?

• Phenylalanine

• Tyrosine

• Tryptophan

• R groups absorb UV light at 270-280nm

• Can contribute to hydrophobic effect

What amino acids make up the polar uncharged group? And what’s important to note?

• Serine

• Threonine

• Cysteine

• Asparagine

• Glutamine

• R groups can form hydrogen bonds

• Cysteine can form disulfide bonds

What 3 amino acids can be phosphorylated?

1. Tyrosine

2. Serine

3. Threonine

Which 3 amino acids are heterocyclic?

1. Tryptophan

2. Histidine

3. Proline

How do uncommon amino acids arise In proteins?

By post-translational modifications of proteins

Are uncommon amino acids found in ribosomes?

No, except for Selenocysteine

Are modifications of uncommon amino acids reversible and if so, why?

Yes, especially phosphorylation, they are important in regulation and signaling.

What are 3 times and examples of when modifications of common amino acids occur?

1. After protein synthesis (ex. 4-hydroxyproline in collagen and plant cell wall)

2. During protein synthesis (ex. pyrrolysine contributes to methane biosynthesis)

3. Modified transiently to change protein’s function (ex. phosphorylation)

What 3 properties make up amino acids?

1. Chirality (except glycine)

2. Light absorbance

3. Acidic/Basic

Uncommon amino acids are found often as what?

Free metabolites (ex. ornithine, intermediate in arginine biosynthesis)

Amino acids absorb what kind of light?

All amino acids absorb light in the infrared (IR) region. Only Phe, Tyr, and Trp absorb UV light.

What wavelength is used to quantify proteins?

280nm

What is a zwitterion and when does it occur?

Amino acid form as dipolar ion at a neutral pH with a net charge of zero.

Without an ionizable side chains what point in a titration is when a zwitterion forms? And what can be said about the amino acid property wise?

At the isoelectric point (pI) which is when the amino acid is least soluble in water and does not migrate in an electric field.

Amino acids contain how many ionizable protons each with their own pKa?

At least two

Amino acids without ionizable R chains can act as buffers in how many pH regimes?

2

What is the isoelectric point and how is it calculated?

The pI is the characteristic pH at which the net electric charge is ZERO. pI=1/2(pK1+pK2)

Amino acids with a single alpha NH2 group and a single alpha COOH group with an R groups that does NOT ionize have similar, if not identical pKa in what range for each group?

COOH = 1.8-2.4

NH3+ = 8.8-11.0

For triprotic acids (Asp, Glu, Arg, Lys, His) the pI is the average of which two pKa’s?

The two that dissociate the neutral species

Amino acids are amphoteric which means what?

They have both basic and acidic groups. Allows them to act as base or acid.

What type of product are peptides?

Small condensation products of amino acids.

How are peptides named?

From the amino terminus to the carboxyl terminus

What are some biological functions and examples of peptides?

• Hormones and pheromones

  • Insulin (sugar)

  • Oxytocin (childbirth)

• Neuropeptides

  • Substance P (pain mediator)

• Antibiotics

  • Polymyxin B (Gram - bacteria)

  • Bacitracin (Gram + bacteria)

• Protection (Toxins)

  • Amanitin (mushrooms)

  • Conotoxin (cone snails)

  • Chlorotoxin (scorpions)

What proteins comprised of?

Polypeptides (covalently linked amino acids) and possibly any of the following…

• Cofactors

  • Functional non-amino acid component

  • Metal ions or organic molecules

• Coenzymes

  • Organic cofactors

  • NAD+ or FAD

• Prosthetic groups

  • Covalently attached cofactors

  • Heme in myoglobin

• Other modifications

How can you estimate the number of amino acid residues in a molecule?

# of residues = molecular weight/110

What is the average molecular weight of a free amino acid? And the average of a bound amino acid?

~128 Free

~110 Bound (-18 for water removed in peptides bond)

What are the two primary methods for determining amino acid sequence of a protein? And how do they differ?

• “Genomic” method

  • Sequence the gene

  • Sequence the mRNA

• Biochemical method

  • Isolate protein to homogeneity

  • Then determine amino acid sequence using a direct chemical method

What was the first protein to be fully sequenced?

Insulin

What is the first set of steps in sequencing a protein?

1. Isolate and purify protein of choice

2. Reduce (break) disulfide bonds w/ reducing agent (ex. DTT)

3. Label amino-terminal residue with FDNB or other labeling agent

4. Determine number of peptides in the protein

What are the three common agents used for labeling amino acids?

Dabsyl chloride

Dansyl chloride

FDNB (1-fluoro-2,4-dinitrobenzene)

How does concentrated HCl affect peptides?

Hydrolyzes all the peptide bonds. Useless for further determination of protein sequence.

Sequencing beyond the first residue is done by a method called what? Briefly explain how and up to what length?

The method is called “Edman Degradation”.

It requires an isolated single polypeptide. Which is immobilized by its carboxyl-terminus to a solid support. A PITC reacts with the amino terminus to form a PTC adduct this is repeated for each residue in the chain.

This method can practically sequence ~50 residues.

Oxidation of disulfide bonds by which agents prevent the reformation of these bonds?

• Performic acid

• Carboxymethylation after reduction by DTT

Name 4 ways how permanently reducing disulfide bonds can help us resolve a protein?

1. Cleaving polypeptide chain

2. Sequencing the peptide

3. Determination of order of peptides

4. Locating disulfide bonds

Proteases are used to what?

Cleave proteins to smaller fragments.