MCAT Biochemistry - Amino Acids, Peptides and Proteins

Proteinogenic Amino Acids

- 20 alpha amino acids encoded by the human genetic code.

Denaturation

- A protein loses its 3D structure. - Although it is sometimes reversible, denaturation is often irreversible. - Whether its denaturation is reversible or not, unfolded proteins cannot catalyze reactions. - Two main causes are heat and solutes.

Alpha Helices

- A rodlike structure in which the peptide chain coils clockwise around a central axis. - Stabilized by intramolecular hydrogen bonds between a carbonyl oxygen atom and an amide hydrogen atom four residues down the chain. - The side chains of the amino acids in the alpha-helical conformation point away from the helix core. - An important component in the structure of keratin.

Peptide Bond Hydrolysis

- Amides can be hydrolyzed using acid or base catalysis. - In living organisms, hydrolysis is catalyzed by hydrolytic enzymes such as trypsin and chymotrypsin. Both are specific, in that they only cleave at specific points in the peptide chain. - Trypsin cleaves at the carboxyl end of arginine and lysine, while chymotrypsin cleaves at the carboxyl end of phenylalanine, tryptophan and tyrosine. - They break apart the amide bond by adding a hydrogen atom to the amide nitrogen and an OH group to the carbonyl carbon.

Titration Curve for Glycine - pKa2

- As we add more base, the carboxylate group goes from half-deprotonated to fully deprotonated. The amino acid stops acting like a buffer, and pH starts to increase rapidly during this phase. - When we've added 1.0 equivalent of base, glycine exists exclusively as the zwitterion form. This means that every molecule is now electrically neutral, and thus the pH equals the isoelectric point (pI) of glycine. - As we continue adding base, glycine passes through a second buffering phase as the amino group deprotonates; again, the pH remains relatively constant. - When 1.5 equivalents of base have been added, the concentration of the zwitterion form equals the concentration of the fully deprotonated form, and the pH equals pKa2. - Finally, when we've added 2.0 equivalents of base, the amino acid has become fully deprotonated, all that remains in NH2CH2COO-; additional base will only increase the pH further.

Negatively Charged under Basic Conditions

- At a basic pH, the carboxylate group is already deprotonated and thus remains -COO-. - We are now well above the pKa for the amino group, so it deprotonates too, becoming -NH2. So, at a highly basic pH, glycine is now negatively charged.

Titration Curve for Glycine - pKa1

- At low pH values, glycine exists predominantly as +NH3CH2COOH; it is fully protonated with a positive charge. - As the solution is titrated with NaOH, the carboxyl group will deprotonate first because it is more acidic than the amino group. When 0.5 equivalents of base have been added to the solution the concentrations of the fully protonated glycine and its zwitterion, +NH3CH2COO-, are equal. At this point, the pH equals pKa1. When the pH is close to the pKa value of a solute, a solution is acting as a buffer, and the titration curve is relatively flat.

Positively Charged under Acidic Conditions

- At pH 1, there are plenty of protons in solution. Because we're far below the pKa of the amino group, the amino group will be fully protonated (-NH3+), and thus positively charged. - Because we're also below the pKa of the carboxylic acid group, it too will be fully protonated (-COOH) and thus neutral. - Therefore, at very acidic pH values, amino acids tend to be positively charged.

Zwitterions at Intermediate pH

- At physiological pH (7.4), we've moved far above the pKa of the carboxylic acid group. The carboxyl group will be in its conjugate base form and be deprotonated, becoming COO-. - Conversely, we're still well below the pKa of the basic amino group, so it will remain fully protonated and in its conjugate acid form (NH3+). Thus we have a molecule that has both a positive and negative charge, but overall, the molecule is electrically neutral. - We call such molecules dipolar ions, or zwitterions. The two charges neutralize one another, and zwitterions exist in water as internal salts.

pKa of Amino Acids

- Because all amino acids have at least two groups that can be deprotonated, they all have at least two pKa values. - The first one, pKa1, is the pKa for the carboxyl group, and is usually around 2. - For most amino acids, pKa2 is the pKa for the amino group, which is usually between 9 and 10. - For amino acids with an ionizable side chain, there will be three pKa values.

Resonance in Peptide Bond

- Because amide groups have delocalizable pi bond electrons in the carbonyl and in the lone pair on the amino nitrogen, they can exhibit resonance. Thus, the C-N bond in the amide has partial double bond character. - As a result, rotation of the protein backbone around its C-N amide bonds is restricted, which makes the protein more rigid. Rotation around the remaining bonds in the backbone, however, is not restricted as those remain single bonds.

Secondary Structures and Proline

- Because of its rigid cyclic structure, proline will introduce a kink in the peptide chain when it is found in the middle of an alpha-helix. - Proline residues are thus rarely found in alpha-helices, except in helices that cross the cell membrane. - Similarly, it is rarely found in the middle of pleated sheets. - On the other hand, proline is often found in the turns between the chains of a beta-pleated sheet, and it is often found as the residue at the start of an alpha-helix.

Beta-Pleated Sheets

- Can be parallel or antiparallel. - The peptide chains lie alongside one another, forming rows or strands held together by intramolecular hydrogen bonds between carbonyl oxygen atoms on one chain and amide hydrogen atoms in an adjacent chain. - To accommodate as many hydrogen bonds as possible, the beta-pleated sheets assume a pleated or rippled shape. The R groups of amino residues point above and below the plane of the beta-pleated sheet.

Fibrous Proteins

- Collagen - Have structures that resemble sheets or long strands.

Peptides - Oligopeptide - Polypeptide

- Composed of amino acid subunits, sometimes called residues. - The term oligopeptide is used for relatively small peptides, up to about 20 residues. - Longer chains are called polypeptides.

Conjugated Proteins

- Derive part of their function from covalently attached molecules called prosthetic groups. These prosthetic groups can be organic molecules, such as vitamins, or even metal ions, such as iron. - Proteins with lipid, carbohydrate, and nucleic acid prosthetic groups are referred to as lipoproteins, glycoproteins and nucleoproteins respectively. - These prosthetic groups have major roles in determining the function of their respective proteins. For example, each of hemoglobin's subunits contains a prosthetic group called heme. The heme group, which contains an iron atom in its core, binds to and carries oxygen; as such, hemoglobin is inactive without the heme group. These groups can also direct the protein to be delivered to a certain location, such as the cell membrane, nucleus, lysosome or ER.

Amino Acid Structures - Polar Side Chains

- Five amino acids have side chains that are polar, but not aromatic: serine, threonine, asparagine, glutamine and cysteine.

Negatively Charged (Acidic) Side Chains

- Only two of the 20 amino acids have negative charges on their side chains at physiological pH (7.4). - Those two are aspartic acid (aspartate), which is related to asparagine, and glutamic acid (glutamate), which is related to glutamine.

Peptide Bond Formation

- Peptide bond formation is an example of a condensation or dehydration reaction because it results in the removal of a water molecule. - Can also be viewed as an acyl substitution reaction, which can occur with all carboxylic acid derivatives. - When a peptide bond forms, the electrophilic carbonyl carbon on the first amino acid is attacked by the nucleophilic amino group on the second amino acid. After that attack, the hydroxyl group of the carboxylic acid is kicked off. The result is the formation of a peptide bond.

Proteins

- Polypeptides that range from just a few amino acids in length up to thousands. They serve many functions in biological systems, such as enzymes, hormones, membrane pores and receptors, and elements of cell structure. - The main actors in cells.

Aspartic Acid

- Related to asparagine. - Has a carboxylate (-COO-) group in its side chain, rather than an amide. - Three letter abbreviation: Asp - One letter abbreviation: D

Glutamic Acid

- Related to glutamine. - Has a carboxylate (-COO-) group in its side chain, rather than an amide. - Three letter abbreviation: Glu - One letter abbreviation: E

Amino Acid Structures - Nonpolar, Nonaromatic Side Chains

- Seven amino acids: glycine, alanine, valine, leucine, isoleucine, methionine, proline.

Denaturation - Solutes

- Solutes such as urea denature proteins by directly interfering with the forces that hold the protein together. They can disrupt tertiary and quaternary structures by breaking disulfide bridges. They can even overcome the hydrogen bonds and other side chain interactions that hold alpha helices and beta-pleated sheets intact. - Similarly, detergents such as SDS can solubilize proteins, resulting in a hydrophobic core that promotes denaturation of the protein.

Tertiary Structure of Proteins - Bonding

- The 3D structure can also be determined by hydrogen bonding, as well as acid-base interactions between amino acids with charged R groups, creating salt bridges. - A particularly important component of tertiary structure is the presence of disulfide bonds. Disulfide bonds create loops in the protein chain. - Forming a disulfide bond requires the loss of two protons and two electrons (oxidation).

Hydrophobic and Hydrophilic Amino Acids

- The amino acids with long alkyl side chains - alanine, isoleucine, leucine, valine and phenylalanine - are all strongly hydrophobic and thus more likely to be found in the interior of proteins, away from water on the surface of the protein. - All the amino acids with charged side chains - positively charged histidine, arginine and lysine, plus negatively charged glutamate and aspartate - are hydrophilic as are the amides asparagine and glutamine. The surface of a protein tends to be rich in amino acids with charged side chains. - The remaining amino acids lie somewhere in the middle are are neither particularly hydrophilic nor hydrophobic.

Alpha Amino Acids

- The amino group and the carboxyl group are bonded to the same carbon, the alpha carbon of the carboxylic acid. - The alpha carbon is the carbon adjacent to the carboxyl carbon. - The alpha carbon has two other groups attached to it: a hydrogen atoms and a side chain, also called an R group, which is specific to each amino acid. The side chains determine the properties of amino acids, and therefore their functions.

Aspartate

- The deprotonated form of aspartic acid.

Glutamate

- The deprotonated form of glutamic acid.

N-terminus & C-terminus

- The free amino end is known as the amino terminus or N-terminus, while the free carboxyl end is the carboxy terminus or C-terminus. - By convention, peptides are drawn with the N-terminus on the left and the C-terminus on the right. - They are read from N-terminus to C-terminus.

Trytophan

- The largest of the amino acids with an aromatic side chain. - Has a double ring system that contains a nitrogen atom. - Three letter abbreviation: Trp - One letter abbreviation: W

Protein Primary Structure

- The linear arrangement of amino acids coded in an organism's DNA. - The sequence of amino acids, listed from the N-terminus or amino end, to the C-terminus, or carboxyl end. - Stabilized by the formation of covalent peptide bonds between adjacent amino acids. - Encodes all of the information needed for folding at all of the higher structural levels; the secondary, tertiary and quaternary structures a protein adopts are the most energetically favorable arrangements of the primary structure in a given environment. - Can be determined by a lab technique called sequencing.

Protein Secondary Structure

- The local structure of neighboring amino acids. - Primarily the result of hydrogen bonding between nearby amino acids. - Two most common secondary structures are alpha-helices and beta pleated sheets. - The key to the stability of both structures is the formation of intramolecular hydrogen bonds between different residues.

Isoelectric Point

- The pH at which the molecule is electrically neutral. - For neutral amino acids, it can be calculated by averaging the two pKa values for the amino and carboxyl groups: - pI neutral amino acid = (pKa NH3+ group + pKa COOH group)/2. - For amino acids with non-ionizable side chains, the pI is usually around 6.

Peptide Bonds

- The residues in peptides are joined together through peptide bonds, a specialized form of an amide bond, that forms between the -COO- group of one amino acid and the NH3+ group of another amino acid. This forms the functional group -C(O)NH-.

Roles of the Formation of Quaternary Structures

- They can be more stable, by further reducing the surface area of the protein complex. - They can reduce the amount of DNA needed to encode the protein complex. - They can bring catalytic sites close together, allowing intermediates from one reaction to be directly shuttled to a second reaction. - Most importantly, they can induce cooperativity or allosteric effects. One subunit can undergo conformational or structural changes, which either enhance or reduce the activity of the other subunits.

Amino Acids as Amphoteric Species

- They have both an acidic carboxylic acid group and a basic amino group. - They can either accept a proton or donate a proton; how they react depends on the pH of their environment. - Ionizable groups tend to gain protons under acidic conditions and lose them under basic conditions. So, in general, at low pH, ionizable groups tend to be protonated; at high pH, they tend to be deprotonated. - The pKa of a group is the pH, at which, on average, half of the molecules of that species are deprotonated. If the pH is less than the pKa, a majority of the species will be protonated. If the pH is higher than the pKa, a majority of the species will be deprotonated.

Amino Acid Structures - Aromatic Side Chains

- Three amino acids: tryptophan, phenylalanine and tyrosine.

Tertiary Structures of Proteins

- Three-dimensional shape. - Mostly determined by hydrophilic and hydrophobic interactions between R groups of amino acids. - Hydrophobic residues prefer to be on the interior of proteins, which reduces their proximity to water. Hydrophilic N-H and C=O bonds found in the polypeptide chain get pulled in by these hydrophobic residues. These hydrophilic bonds can then form electrostatic interactions and hydrogen bonds that further stabilize the protein from the inside. As a result of these hydrophobic interactions, most of the amino acids on the surface of proteins have hydrophilic (polar or charged) R groups; highly hydrophobic R groups, such as phenylalanine, are almost never found on the surface of a protein.

Proline

- Unique in that it forms a cyclic amino acid. In all the other amino acids, the amino group is attached only to the alpha-carbon. - In proline, however, the amino nitrogen becomes a part of the side chain, forming a five-membered ring. - That ring places notable constraints on the flexibility of proline, which limits where it can appear in a protein and can have significant effects on proline's role in secondary structure. - Three letter abbreviation: Pro - One letter abbreviation: P

Titration of Amino Acids

- We assume that the titration of each proton occurs as a distinct step, resembling that of a simple monoprotic acid. Thus, the titration curve looks like a combination of two monoprotic acid titration curves (or three curves if the side chain is charged).

Denaturation - Temperature

- When the temperature of a protein increases, its average kinetic energy increases. When the temperature gets high enough, this extra energy can be enough to overcome the hydrophobic interactions that hold a protein together, causing the protein to unfold.

Titration - Acidic Amino Acids

- For amino acids with charged side chains, such as glutamic acid and lysine, the titration curve has an extra step, but works along the same principles. - Because glutamic acid has two carboxyl groups and one amino group, its charge in its fully protonated state is +1. It undergoes the first deprotonation, losing the proton from its main carboxyl group, just as glycine does. At that point, it is electrically neutral. When it loses its second proton, just as with glycine, its overall charge will be -1. - However, the second proton that is removed in this case comes from the side chain carboxyl group, not the amino group. This is a relatively acidic group, with a pKa of around 4.2. The result is that the pI of glutamic acid is much lower than that of glycine, around 3.2. - The isoelectric point for an acidic amino acid can be calculated as follows: pI = (pKa R group + pKa COOH group)/2. - Amino acids with acidic side chains have relatively low isoelectric points (below 6).

Stereochemistry of Amino Acids

- For most amino acids, the alpha-carbon is a chiral (or stereogenic) center. Thus, most amino acids are optically active. - The one exception is glycine, which has a hydrogen atom as its R group, making it achiral. - All chiral amino acids used in eukaryotes are L-amino acids, so the amino group is drawn on the left in a Fischer projection. - In the Cahn-Ingold-Prelog system, this translates to an (S) absolute configuration for almost all chiral amino acids. The only exception is cysteine, which, while still being an an L-amino acid, has an (R) absolute configuration because the -CH2SH group has priority over the -COOH group.

Phenylalanine

- Has a benzyl side chain ( a benzene ring plus a -CH2 group). - Relatively nonpolar. - Three letter abbreviation: Phe - One letter abbreviation: F

Tyrosine

- Has a benzyl side chain with an OH group. The OH group makes tyrosine relatively polar. - Three letter abbreviation: Tyr - One letter abbreviation: Y

Isoleucine

- Has a hydrocarbon side chain with four carbons. - Three letter abbreviation: Ile - One letter abbreviation: I

Alanine

- Has a methyl side chain. - Three letter abbreviation: Ala - One letter abbreviation: A

Glycine

- Has a single hydrogen atom as its side chain, and is therefore achiral. - The smallest amino acid. - Three letter abbreviation: Gly - One letter abbreviation: G

Lysine

- Has a terminal primary amino group. - Three letter abbreviation: Lys - One letter abbreviation: K

Cysteine

- Has a thiol (-SH) group in its side chain. Because sulfur is larger and less electronegative than oxygen, the S-H bond is weaker than the O-H bond. This leaves the thiol group in cysteine prone to oxidation. - Three letter abbreviation: Cys - One letter abbreviation: C

Serine

- Has an -OH group in its side chain, which makes it highly polar and able to participate in hydrogen bonding. - Three letter abbreviation: Ser - One letter abbreviation: S

Threonine

- Has an -OH group in its side chain, which makes it highly polar and able to participate in hydrogen bonding. - Three letter abbreviation: Thr - One letter abbreviation: T

Asparagine

- Has an amide side chain. Unlike the amino group common to all amino acids, the amide nitrogen does not gain or lose protons with changes in pH; it does not become charged. - Three letter abbreviation: Asn - One letter abbreviation: N

Glutamine

- Has an amide side chain. Unlike the amino group common to all amino acids, the amide nitrogen does not gain or lose protons with changes in pH; it does not become charged. - Three letter abbreviation: Gln - One letter abbreviation: Q

Histidine

- Has an aromatic ring with two nitrogen atoms (called an imidazole). - The pKa of the side chain is relatively close to 7.4 - it's about 6 - so, at physiologic pH, one nitrogen atom is protonated and the other isn't. Under more acidic conditions, the second nitrogen atom can become protonated, giving the side chain a positive charge. - Three letter abbreviation: His - One letter abbreviation: H

Leucine

- Has an isobutyl side chain. - Three letter abbreviation: Leu - One letter abbreviation: L

Valine

- Has an isopropyl side chain. - Three letter abbreviation: Val - One letter abbreviation: V

Arginine

- Has three nitrogen atoms in its side chain; the positive charge is delocalized over all three nitrogen atoms. - Three letter abbreviation: Arg - One letter abbreviation: R

Positively Charged (Basic) Side Chains

- Have side chains with positively charged nitrogen atoms: lysine, arginine and histidine.

Molten Globules

- Intermediate states of protein folding.

Titration - Basic Amino Acids

- Lysine has two amino groups and one carboxyl group. Thus its charge in its fully protonated state is +2, not +1. Losing the carboxyl proton, which still happens around pH 2, brings the charge down to +1. - Lysine does not become electrically neutral until it loses the proton from its main amino group, which happens around pH 9. It gets a negative charge when it loses the proton on the amino group in its side chain, which happens around pH 10.5. - Thus, the isoelectric point of lysine is the average of the pKa values for the amino group and side chain. - The isoelectric point for a basic amino acid can be calculated as: pI = (pKa NH3+ group + pKa R group)/2 - Amino acids with basic side chains have relatively high isoelectric points (above 6).

Amino Acids

- Molecules that contain two functional groups: an amino group (NH2) and a carboxyl group (COOH).

Globular Proteins

- Myoglobin - Tend to be spherical.

Quaternary Structure of Proteins

- Not all proteins have quaternary structure. They only exist for proteins that contain more than one polypeptide chain. For these proteins, the quaternary structure is an aggregate of smaller globular peptides, or subunits, and represents the functional form of the protein.

Methionine

- One of the only two amino acids that contains a sulfur atom in its side chain. - Because the sulfur has a methyl group attached, it is considered relatively nonpolar. - Three letter abbreviation: Met - One letter abbreviation: M

Folding and the Solvation Layer

- Whenever a solute dissolves in a solvent, the nearby solvent molecules form a solvation layer around that solute. From an enthalpy standpoint, even hydrocarbons are more stable in aqueous solution than in organic ones (H<0). However, when a hydrophobic side chain is placed in aqueous solution, the water molecules in the solvation layer cannot form hydrogen bonds with the side chain. This forces the nearby water molecules to rearrange themselves into specific arrangements to maximize hydrogen bonding - which means a negative change in entropy. Negative changes in entropy represent increasing order and thus are unfavorable. This entropy change makes the overall process non spontaneous (G>0). - On the other hand, putting hydrophilic residues on the exterior of the protein allows the nearby water molecules more latitude in their positioning, thus increasing their entropy, and making the overall solvation process spontaneous. Thus, by moving hydrophobic residues away from water molecules and hydrophilic residues toward water molecules, a protein achieves maximum stability.