Bio385 - SG1 Amino acids

BIO 385 |  Amino Acids & Buffers READING ASSIGNMENT LPoB8 – pgs 70-80, LibreTexts Ch 26-1,2,3 PREPARATION & PONDERING 1. *List the 20 common proteinogenic amino acids with their one letter code and list whether they are aliphatic, aromatic, polar, acidic or basic. Then identify the following special amino acids a. Smallest R group i. b. Largest R group i. c. Alcohol R-groups (Why? because these are sites for phosphorylation) i. d. Thiol R-group i. e. α-amino group forms part of a ring, also disrupts α-helix formation i. 2. Describe both the Absolute D,L- and the R,S-configurations used by chemists to communicate stereochemistry variations. All biological amino acids are found with which rotation? a. All biological amino acids are found on a clockwise rotation. b. All amino acids have L configuration c. L-Glyceraldehyde and the natural amino acids all have the S absolute configuration. The two exceptions are glycine and cysteine 3. Describe the ranking of groups used by chemists to determine R or S configuration for a given chiral carbon. Be able to apply these rules to assign R or S to a chiral carbon. 4. What functional groups commonly act as weak acids and weak bases? Use chemical logic to describe why these groups behave that way. Functional groups in organic chemistry can often act as weak acids or weak bases 1 1

BIO 385 |  depending on their chemical properties. Two common functional groups that exhibit this behavior are the carboxylic acid group (-COOH) and the amino group (-NH2). 1. Carboxylic Acid Group (-COOH): - Weak Acid Behavior: The carboxylic acid group consists of a carbonyl group (C=O) and a hydroxyl group (-OH) bonded to the same carbon atom. The oxygen atom in the hydroxyl group is electronegative and can pull electron density away from the hydrogen atom in the -OH group. This results in the formation of a polar covalent bond, where the oxygen is partially negatively charged and the hydrogen is partially positively charged. - When dissolved in water, the hydrogen atom in the -OH group can ionize by donating a proton (H+) to water, forming the hydronium ion (H3O+) and the carboxylate ion (-COO-): ``` -COOH ⇌ -COO- + H+ ``` This reversible reaction represents the weak acid behavior of the carboxylic acid group. 2. Amino Group (-NH2): - Weak Base Behavior: The amino group contains a nitrogen atom with a lone pair of electrons. Nitrogen is less electronegative than oxygen, so it is more likely to share its lone pair of electrons. When dissolved in water, the lone pair on the nitrogen atom can accept a proton (H+) from water, forming the ammonium ion (NH4+) and hydroxide ion (OH-): ``` -NH2 + H2O ⇌ NH4+ + OH- ``` This reversible reaction represents the weak base behavior of the amino group. In summary, the weak acid behavior of the carboxylic acid group is due to the ability of the -OH group to donate a proton (H+) to water, while the weak base behavior of the amino group is due to the lone pair of electrons on the nitrogen atom's ability to accept a proton from water. These behaviors are determined by the electron distribution and electronegativity differences between the atoms in these functional groups. 5. Using amino acids as case, what is the common pKa range for amines? Carboxyl groups? Amino acids contain both amino groups (-NH2) and carboxyl groups (-COOH). These functional groups can act as weak acids and weak bases, and their pKa values can vary depending on the specific amino acid and its local environment. However, there is a common pKa range for amines and carboxyl groups in amino acids. 2

BIO 385 |  NH3+-CH(R)-COOH. 2. **pKa1 < pH < pKa2 (Buffering Region 1):** - In this pH range, the amino group (-NH2) starts to deprotonate and become neutral, while the carboxyl group (-COOH) remains protonated. - The amino acid acts as a buffer because it can accept or donate protons. - The predominant form is a zwitterion, with the amino group (-NH3+) carrying a positive charge, and the carboxyl group (-COO-) carrying a negative charge: NH3+- CH(R)-COO-. 3. **pKa2 < pH < pKa3 (Buffering Region 2):** - In this pH range, the carboxyl group (-COO-) continues to deprotonate and become more negatively charged. - The amino group (-NH2) remains deprotonated. - The predominant form is still the zwitterion, NH3+-CH(R)-COO-. 4. **pH > pKa3 (Second Basic Equivalence Point):** - At very high pH, the solution is strongly basic. - Both the amino group (-NH2) and the carboxyl group (-COO-) are fully deprotonated. - The predominant form is the fully deprotonated amino acid with a net negative charge: NH2-CH(R)-COO-. **Drawing the Titration Curve:** To draw the titration curve of an amino acid, plot pH on the vertical axis and volume of added strong acid or base on the horizontal axis. The curve will show two buffering regions, corresponding to the zwitterionic form of the amino acid, and two equivalence points, where the amino acid becomes fully protonated or fully deprotonated. In summary, the titration curve of an amino acid displays changes in pH as protons are added or removed. At specific pH ranges, the amino acid exists primarily as a zwitterion, with both a positive and a negative charge, making it an effective buffer. The pKa values correspond to the pH values at which specific protonation or deprotonation events occur. 8. Explain why is the pH of a system so important to protein folding and binding? The pH of a system is crucial to protein folding and binding because it directly affects the charge distribution and ionization states of amino acid residues in proteins. The three main reasons why pH is important in these processes are electrostatic interactions, protonation state of ionizable groups, and the stability of protein structures: 1. **Electrostatic Interactions:** - Proteins are composed of amino acids, and many of these amino acids contain ionizable groups, such as carboxyl (-COOH) and amino (-NH2) groups. 4

BIO 385 |  - pH determines the charge on these groups. At low pH (acidic conditions), these groups tend to be protonated (carry a positive charge). At high pH (basic conditions), they tend to be deprotonated (carry a negative charge). - Electrostatic interactions, such as attractive forces between oppositely charged groups (ionic bonds and salt bridges), play a critical role in protein folding and binding. The pH-dependent charge on amino acids influences these interactions. 2. **Protonation State of Ionizable Groups:** - The protonation state of specific amino acid side chains can affect their chemical properties, including their ability to form hydrogen bonds and participate in hydrophobic interactions. - For example, the pH-dependent protonation of histidine (His) residues can significantly impact the stability and function of proteins. Histidine acts as a pH- sensitive switch in many enzymes and proteins. - The availability of charged and uncharged groups can influence the three- dimensional structure of a protein. 3. **Stability of Protein Structures:** - Protein folding and binding processes are highly dependent on the stability of protein structures, including secondary structures like alpha-helices and beta-sheets. - pH can affect the stability of these structures by altering the charge distribution along the protein backbone and at key interaction sites. - For example, in an environment with a pH close to the isoelectric point (pI) of a protein (where the net charge is zero), the protein may be less stable and more prone to denaturation. 4. **Binding Affinity and Specificity:** - For proteins involved in binding interactions, such as enzymes, receptors, and antibodies, the pH can influence the binding affinity and specificity. - pH-sensitive groups in the binding sites of proteins can modulate their ability to recognize and bind to specific ligands or substrates. - Changes in pH can disrupt or enhance binding interactions depending on the protonation states of key residues involved in the binding site. In summary, pH plays a fundamental role in modulating the charge distribution and chemical properties of amino acid residues in proteins. This, in turn, affects the electrostatic interactions, stability, and binding characteristics of proteins. Understanding and controlling pH conditions are essential for studying and manipulating protein folding, stability, and binding in various biological and biochemical processes. VOCABULARY You should have both a formal and working definition of the following terms. Many of these are found in the book or in the outline below. 5

BIO 385 |  i. Glycine, alanine, methionine, proline (helix breaker) leucine, isoleucine, valine (branched chains) b. Aromatics (3) – aromatic group = means double/single bond ring pattern. Flat bulky i. Tryptophan, two rings (hex + pent) ii. tyrosine, phenylalanine (hex ring), tyrosine has -OH c. Polar (5) i. Serine, Threonine (hydroxyl groups) – O linked substate ii. Asparagine, Glutamine (amide groups) N linked substrate iii. Cysteine – (sulfhydryl group) actually quite non-polar but potentially acidic, forms disulfide bonds in protein structure d. Acidic (2) - carboxyl groups that donate protons to solution i. Typically shown with negative (-) charge at physiological pH ii. Aspartate (4C), glutamate (5C) e. Basic (3) – amine group that accepts protons (base) i. Typically shown with a net positive charge at physiological pH ii. Lysine (6C + amino) iii. Arginine (5C + Guanidinium) iv. Histidine (1C + Imidazole Penta Ring) 4. pKa’s, titration curves and zwitterions a. weak acids – incompletely donate protons to solution and form conjugate base i. carboxyl groups and phosphoryl/phosphate groups are predominant weak acids ii. all amino acids have at least 1 carboxyl group, Acidics have 2. iii. Smaller pKa = better H + donor, stronger acid. Larger pKa weaker acid/better base b. weak bases – incompletely accept protons from solution and form conjugate acids i. Amino, guanidinium, Imidazole groups are common biological bases ii. All amino acids have 1 amino group, Basics have 2 of the above. c. pK a = -log K a for acid dissociation reaction. i. The lower the pK a the stronger the acid (proton comes off easier) ii. The lower the pK a the weaker the conjugate base (doesn’t grab protons well) iii. In systems with very low pK a , the conjugate base dominates. d. Henderson-Hasselbalch formula plots the relationship between pH and pK a . i. pH = pK a + log[A - ]/[HA] ii. Titration curve – y axis = pH, x axis = % of weak acid or conjugate base iii. pKa is inflection point, steep up, log down, buffer zone e. pH plays a big role in side-chain charge and enzyme binding and activity f. Phosphate is a polyprotic acids with multiple pKa’s g. Amino acids are Zwitterions that have both a weak acid and weak base in same molecule II. Buffers maintain pH in biological systems A. Buffers contain a mix of weak acid and conjugate base. 1. Buffers resist (don’t prevent) pH changes in a system 2. Weak acid reacts/sponges excess base (OH - ) in system 3. Conjugate base absorbs/sponges excess acid (H + ) added to system. B. Titration Plots show relationship between buffer and pH 1. pH changes based on percentage of HA (or A - ) 7

BIO 385 |  2. Buffering region exists (large change in %HA, little change in pH 3. pK a occurs at inflection point in curve. 4. Each buffer has unique buffering range (±1 pK a ) 5. Buffer is most stable when desired pH = pK a C. Carbonic Acid is the primary physiological buffer 1. CO 2 + H 2 O 🡪 H 2 CO 3 🡪 HCO 3 - + H + III. pH, Buffers, and protein folding/binding A. The acidic and basic side amino acids side chains in a protein can be protonated or unprotonated B. Creates on overall charge on proteins that serves as a basis for native electrophoresis C. Protonation/charge is critical to protein structure and protein binding D. Protonation depends on the pH and the pKa of the side chain. 1. Amino acid electrophoresis at different pH levels 2. Isoelectric point – pH that results in net neutral charge for amino acids a. Polars and non-polars isoeletric point is average of pK a ’s b. Acidics and basics isoelectric point is average of two proximal pK a ’s E. pK a of the side chain can be modified by the micro-environment. Practice problems 1. (LPoB8 3-1) Amino Acid Constituents of Glutathione Glutathione is an important peptide antioxidant found in cells from bacteria to humans. Glutathione Identify the three amino acid constituents of glutathione. What is unusual about glutathione’s structure? 2. (LPoB8 3-2) Absolute Configuration of Ornithine. Ornithine is an amino acid that is not a building block of proteins. Instead, ornithine is an important intermediate in the urea cycle, the metabolic process that facilitates the excretion of ammonia waste products in animals. 8

BIO 385 |  a) Why is alanine predominantly zwitterionic at its pI? b) What fraction of alanine is in the completely uncharged form at its pI? a. 100% 5. (LPoB8 3-5) Ionization State of Histidine. Each ionizable group of an amino acid can exist in one of two states, charged or neutral. The electric charge on the functional group is determined by the relationship between its pK a and the pH of the solution. This relationship is described by the Henderson-Hasselbalch equation. a) Histidine has three ionizable functional groups. Write the equilibrium equations for its three ionizations, and assign the proper pK a  for each ionization. Draw the structure of histidine in each ionization state. What is the net charge on the histidine molecule in each ionization state? b) Draw the structures of the predominant ionization state of histidine at pH 1, 4, 8, and 12. Note that you can approximate the ionization state by treating each ionizable group independently. c) What is the net charge of histidine at pH 1, 4, 8, and 12? For each pH, will histidine migrate toward the anode (+) or toward the cathode (−) when placed in an electric field? 6. (LPoB8 3-6) Separation of Amino Acids by Ion-Exchange Chromatography We can analyze mixtures of amino acids by first separating the mixture into its components through ion-exchange chromatography. Amino acids placed on a cation-exchange resin (see Fig. 3-17a) containing sulfonate (−SO 3 −) groups flow down the column at different rates because of two factors that influence their movement: (1) ionic attraction between the sulfonate residues on the column and positively charged functional groups on the amino acids, and (2) aggregation of nonpolar amino acid side chains with the hydrophobic backbone of the polystyrene resin. Note that the ionic attraction is more significant than hydrophobicity for this column media. For each pair of amino acids listed, determine which will be eluted first from the cation-exchange column by a pH 7.0 buffer. a. Glutamate and lysine b. Arginine and methionine c. Aspartate and valine d. Glycine and leucine e. Serine and alanine 7. (LPoB8 3-8) Comparing the pKa Values of Alanine and Polyalanine The titration curve of alanine shows the ionization of two functional groups with pKa values of 2.34 and 9.69, corresponding to the ionization of the carboxyl and the protonated amino groups, respectively. The titration of di-, tri-, and larger oligopeptides of alanine also shows the ionization of only two functional groups, although the experimental pKa values are different. The table summarizes the trend in pKa values. Amino acid or peptide pK1 pK2 Ala 2.34 9.69 10

BIO 385 |  Ala–Ala 3.12 8.30 Ala–Ala–Ala 3.39 8.03 Ala–(Ala)n–Ala, n ≥ 4 3.42 7.94 a. Draw the structure of Ala–Ala–Ala. Identify the functional groups associated with pK 1 and pK 2 . b. Why does the value of pK 1 increase with each additional Ala residue in the oligopeptide? c. Why does the value of pK 2 decrease with each additional Ala residue in the oligopeptide? 11