Biochemistry 601

September 1, 1999 Amino Acids of Naturally Occurring Proteins

Last modified 9/1/99.
Dr. Landry

Rm. 6055
landry@mailhost.tcs.tulane.edu
 

Reading Assignment

Resources

Topics for discussion

Proteins have diverse functions: structural, enzymatic, regulatory. For example consider the following diseases and their associated defective proteins: 
DISEASE                       DEFECTIVE PROTEIN                 VVP, p.

Genetic Defects

cystic fibrosis                 Cl- channel                        
familial hypercholesterolemia   LDL receptor                       264   
hemolytic anemia                G-6-P dehydrogenase                
hemophilia (VIII)               factor X protease activator        318
I-cell disease                  UDP-GlcNAc phosphotransferase      260
sickle cell anemia              hemoglobin                         176
xeroderma pigmentosum           DNA exinuclease                    799
cancer                          ras, tyrosine kinase, etc.         682
Marfan syndrome                 fibrilin


Dietary Deficiencies

scurvy (ascorbate)          collagen                               135
beriberi (thiamine)         pyruvate dehydrogenase,                404
                                a-ketogluterate dehydrogenase,
                                pyruvate dehydrogenase
Amyloidoses
Alzheimer's disease         amyloid protein precursor (APP)        156
Prion diseases              prion protein precursor (PrP)          156


Diverse functions are expressed in the 3-dimensional structure of proteins in the form of catalytic sites, regulated ligand binding sites, fiber-like properties, etc.

All proteins are linear chains of amino acids linked together by peptide bonds to form polypeptides. Protein 3-dimensional structure is specified by the linear sequence of amino acids. Noncovalent interactions within the polypeptide and between the polypeptide and solvent (water) direct the formation and stability of the native protein fold.

Amino Acids - Structure and Properties

Reading assignment:

Objectives

Ionization of Weak Acids and Bases (VVP, pp. 31-36)

Given an acid (HA) that dissociates into a proton (H+) and the conjugate base (A-), we express the equilibrium dissociation as

Ka = [H+][A-]/[HA] ; and rearrange as

1/[H+] = (1/Ka)([A-]/[HA])

Assume:

pH = - log [H+] = log (1/[H+])
pK = - log [Ka] = log (1/Ka)

substitute to obtain the Henderson-Hasselbalch equation:

pH = pKa + log ([A-]/[HA]) [1]

Example 1

Determine the carboxylate/carboxylic acid ratio for the alpha-carboxyl group of alanine (pKa = 2.3) in solution at neutral pH.

Substituting into equation 1:

7 = 2.3 + log ([A-]/[HA]) ; subtract 2.3 from both sides...

[A-]/[HA] = 10**4.6 , which is more than 10,000/1

Example 2

At pH =9.4, what percentage of the lysine sidechain amino group (pK = 10.0) is uncharged?

9.4 = 10.0 + log (uncharged/cation)
log (uncharged/cation) = -0.6
(uncharged/cation) = 10**(-0.6) = 0.25

Thus, the fraction uncharged is 0.25/(1+0.25) = 0.2 or 20%.

Isoelectric Point (pI)

The Isoelectric Point (pI) is the pH at which a molecule has no net charge.

Amino acids form dipolar ions or zwitterions: 

For example, the pI for alanine is evaluated as follows:

pI = 1/2 (pK1 + pK2)

pIAla = 1/2(2.3 + 9.9) = 6.1

The pI of molecules with more than two pK's

For a molecule having more than two ionizable groups, the pI is approximately equal to the average of the relevant pair of pK's. For example, aspartic acid has three pK's with the following values:

corresponding to the equilibria shown 

The relevant pair of pK's are pK1 and pK2. At the pH corresponding to the average of these two pK's, the amino group is fully ionized, and the two carboxylates are partially ionized. Thus, the pI of aspartate is as follows:

pIAsp = 1/2(2.0 + 3.9) = 3.0

Sample Problems

  1. What is the molarity of pure water? Show that a change in the concentration of water by ionization does not appreciably affect the molarity of the solution.
  2. When sufficient acid is added to lower the pH by one unit, what is the corresponding increase in hydrogen ion concentration?
  3. You have a solution of HCl that has a pH of 2.1. what is the concentration of HCl need to make this solution?
  4. The charged form of the imidazole ring of histidine is believed to participate in a reaction catalyzed by an enzyme. At pH 7.0, what is the probability that the imidazole ring will be charged?
  5. Calculate the pH at which a solution of cysteine would have no net charge.

Answers:

1. Water molarity=1000/18=55.6M. Since Kw=1.0x10**(-14), the concentration of OH(-) and H(+) are 10**(-7) each. Thus, the actual concentration of water is 55.6-0.0000001. This difference is negligible.

2. Tenfold change in H(+) concentration.

3. Assume that HCl in solution is completely ionized to H(+) and Cl(-).
pH=-log[H(+)]=2.1
[H(+)]=10**(-2.1)=7.94x10**(-3)M

4. For the histidine ring, the pK=6.0.
pH=pK + log ([His]/[His(+)])
7.0=6.0+ log ([His]/[His(+)])
log ([His]/[His(+)])=1.0
([His]/[His(+)])=10
Thus, the ratio is 10:1 and the probability is 1/(10+1)=0.09 or 9%.

5. Average the pK's of the carboxyl (pK=1.8) and sidechain (pK=8.3). The pI=5.1.

The Twenty Amino Acids

-occuring in proteins translated on ribosomes

View the structures in a kinemage.

Amino acids are important as:

General form of an L-Amino Acid (zwitterionic form) 

Alternate absolute configurations of amino acids can be distinguished using the "corncrib" mnemonic. Note the order of alpha-carbon substituents, CO-R-N, is clockwise in the L configuration. 

D-amino acids are used by bacteria and plants. They are incorporated into polypeptides by specialized ligases.

Amino Acid Classification

Amino acids are grouped by the chemical properties of the sidechain. The same amino acid can fall into multiple groups.

size - for example, affecting how well the sidechain fits in a binding site

hydrophobicity- for example, determining whether the sidechain will be buried in a protein interior or membrane as opposed to interacting with the solvent (water)

H-bonding potential - for example, providing geometrically specific interactions or chemical catalysis

charge - for example, providing for long-range electrostatic interactions

polarity - providing water solubility

chemical structure - i.e., aliphatic, aromatic, sulfur-containing, aliphatic hydroxyl-containing, basic, acidic, amide derivative

Aliphatic Amino Acids

glycine
alanine

hydrophobic
valine - beta-branched
leucine
isoleucine - beta-branched, second chiral center

a-imino acid
proline - containing a pyrrolidine ring

Aromatic Amino Acids

- all are considered hydrophobic. However, tyrosine and tryptophan also can form hydrogen bonds.

phenylalanine
tyrosine
tryptophan - containing an indole group

Sulfur-containing

- both are considered hydrophobic. Cysteine can form disulfide bonds with another cysteine.

cysteine - containing a thiol group
methionine - containing a thioether group

Aliphatic hydroxyl-containing

serine
threonine - beta-branched, second chiral center

Basic

lysine - containing a primary amine
arginine - containing a guanidino group
histidine - containing an imidazole group

Acidic

aspartate
glutamate

Amide Derivatives

(of the acidic amino acids)

asparagine
glutamine

Peptide Bond Formation

attends the condensation of two amino acids with release of one molecule of H2O. The peptide bond is a specialized form of amide bond. The reaction is thermodynamically unfavorable. Both in nature and in the laboratory, the reaction is driven by using activated derivatives of the amino acids whose reactivity is much stronger.

Post-translational Modification

After synthesis, proteins undergo important and often extensive covalent modification, sometimes the removal of a polypeptide segment, sometimes addition of a new chemical moiety (VVP, pp. 90, 256-258, 881-882). Often such proteins will not be "active" until a modification has occurred (e.g. phosphorylation). A modification can prolong the life of a protein (acetylation), or shorten it (ubiquitination). Some modifications are reversible and others are permanent. Many modifications are listed here, but they shall not be covered in detail until later in the course. The structures of a few key modifications are shown in VVP, p.90.

Proteolytic cleavage associated with protein sorting.

Proteins destined for transport into membrane-bound compartments such as the mitochondrion and the endoplasmic reticulum typically are synthesized with an amino-terminal targeting sequence that directs the protein to the target organelle. The targeting sequences are usually removed by proteolytic cleavage within the organelle.

In the Cytosol

In the Secretory Pathway

Homework

Examine the four amino acids given below:

  1. Name the four amino acids.
  2. Name the other amino acids of the same class as D.
  3. Draw the structure of cysteine at pH 1.
  4. Name an example of a protein for each post-translational modification specified above.
  5. Compare and contrast acquisition of amino-terminal acetyl and formyl groups.
  6. Name and draw the structure of an amino acid which is an example of the following groups: (a) nonpolar aliphatic, (b) nonpolar aromatic, (c) basic, (d) acidic, (e) sulfur-containing, (f) hydroxyl-containing.
  7. List the twenty amino acids in order of hydrophobicity (use your own judgment).
  8. Find a hydrophobicity ranking in the literature (textbook, Web site, etc.) and compare it with your own ranking. Rationalize deviant amino acids.
  9. How did earth-bound biological materials come to be dominated by L-amino acids? Could it have gone the other way?
End of document