Comprehensive Biochemistry Study Notes: Amino Acid Properties, Secondary Structures, and Enzymatic Mechanisms

Student Consultation and Engagement Observations

• Enrollment and Consultation Statistics:

  • In the Midrand location, there are approximately $260$ to $270$ students enrolled.

  • Despite this high enrollment, only approximately $2$ to $3$ students have booked official consultations.

• Purpose of Q&A Sessions:

  • Because students are not booking consultations, Q&A sessions are utilized to gauge whether students actually understand the material or if they are simply not seeking help despite being confused.

  • The sessions aim to identify if the current "history" of the course (previous student performance or course reputation) is causing undue panic.

Fundamental Amino Acid Properties in Catalytic Mechanisms

• General Requirement: Students must know the properties of all amino acid side chains (R-groups) to understand enzymatic mechanisms.

• Backbone Structure:

  • The protein backbone follows a repeating $NCC$ sequence ($N-C_{\alpha}-C$).

  • The backbone consists of the amino group and carboxylic group bonded via peptide bonds.

  • Hydrogen bonds form between the backbone amide hydrogens and carbonyl oxygens to stabilize the structure.

• R-Group Identification:

  • Mechanistic slides (e.g., Lysozyme, RNAs, Chymotrypsin) often only show the R-groups, not the backbone, to simplify the diagrams.

  • Students must be able to identify the specific residue (e.g., Valine, Serine, Histidine, Glutamic acid, Aspartate) solely based on the chemical structure of the side chain, even if the name is not explicitly labeled.

• Residue Examples in Mechanisms:

  • Glutamic Acid: Identified by its ability to exist in a protonated state ($NH$ or $COOH$ depending on context) to donate a proton.

  • Aspartate 52 ($Asp52$): Identified by lone pairs that allow it to accept protons or act as a nucleophile/stabilizer.

  • Histidine 12 ($His12$) and Histidine 119 ($His119$): Involved in RNAs mechanisms. One acts to stabilize the leaving group by donating a hydrogen, while the other waits to receive.

Effects of Mutations on Enzyme Function

• Substitution Scenarios:

  • If a catalytic residue like $His12$ is mutated and replaced by Valine ($Val$) or Lysine ($Lys$), the function is typically lost.

  • Property vs. Function: Even if a substitute amino acid shares a similar property (e.g., both being acidic or basic), the specific catalytic function is rarely maintained.

  • Every active site has a specific "core catalytic residue."

• The Service Provider Analogy:

  • Changing a catalytic residue is compared to buying airtime: Buying Telkom airtime to use on a Vodacom network. While both are "airtime" (sharing a general property), they are not compatible with the specific service provider (the catalytic mechanism). The airtime (function) will not work.

• Consequences of Mutation:

  • Disruption of substrate binding.

  • Total blockage of the catalytic activity.

  • Potential introduces of unnecessary interactions (e.g., changing Serine to Cysteine introduces potential disulfide bonds that could distort the structure).

Protein Secondary Structure: Alpha Helices vs. Beta Sheets

• Alpha Helices ($\alpha$-helices):

  • Architecture: It takes $3.6$ amino acid residues to complete one full turn of the helix.

  • Stability: Stabilized by internal hydrogen bonds between the backbone components.

  • Residue Preferences: Requires small, flexible residues that do not disrupt the spiral symmetry.

  • Common Residues: Alanine ($Ala$), Leucine ($Leu$), Methionine ($Met$), Glutamate ($Glu$), and Lysine ($Lys$).

  • Incompatible Residues: Bulky or branched aromatic residues cause "bulging" or distortion, preventing the seamless $3.6$ turn.

• Beta Sheets ($\beta$-sheets):

  • Architecture: Two or more strands joined together with a gap between hydrogen-bonded backbones.

  • Residue Preferences: Bulky, branched, or aromatic residues are preferred because the gap between strands accommodates larger side groups without disrupting the overall structure.

  • Common Residues: Tyrosine ($Tyr$), Tryptophan ($Trp$), Threonine ($Thr$), Phenylalanine ($Phe$), Valine ($Val$), and Isoleucine ($Ile$).

• Helix Breakers (Proline and Glycine):

  • Proline ($Pro$) and Glycine ($Gly$) are typically found in alpha-helix breaks, loops, or beta-turns.

  • In Glycine, the side chain is just a Hydrogen ($H$), making it too small/flexible to maintain the rigid $3.6$ turn, thereby destabilizing the helix.

• Ramachandran Plot Distribution:

  • Residues cluster in specific quadrants based on their phi ($\phi$) and psi ($\psi$) angles.

  • Beta sheets generally appear in the top-left quadrant of the plot.

• Hydrophobic Core:

  • Hydrophobic residues (like Leucine, Valine, Isoleucine, Methionine, Phenylalanine) tend to cluster in the center of the protein (often as beta sheets) to be shielded from the aqueous environment.

Reaction Kinetics and Two-Step Mechanisms

• Energy Diagrams:

  • X-axis: Reaction progress (Reaction coordinate).

  • Y-axis: Free energy ($G$).

• Two-Step Reaction Components:

  • Contains two transition states ($TS_1$ and $TS_2$) and one intermediate state.

  • $TS_1$: Formation of the Enzyme-Substrate ($ES$) complex.

  • Intermediate: A temporary species formed after the first transition state but before the final product.

  • $TS_2$: Breakdown of the intermediate to form the product.

• Rate-Limiting Step:

  • The step with the highest energy peak (highest barrier) is the rate-determining (rate-limiting) step.

  • If $TS_1$ is higher than $TS_2$, the first step is rate-limiting. If $TS_2$ is higher, the second step is rate-limiting.

• Thermodynamics ($K_{eq}$ and $\Delta G$):

  • K_{eq} > 1: Favors the products; typically exergonic (releases energy); negative $\Delta G$. The peak is often lower on the product side.

  • K_{eq} < 1: Favors the reactants; endergonic (requires energy); positive $\Delta G$. The highest peak is often on the product side because more energy is needed to push the reaction forward.

• Enzyme Catalysis Role:

  • Enzymes lower the activation energy barrier ($\Delta G^\ddagger$) compared to uncatalyzed reactions.

  • They allow reactions to occur rapidly at body temperature ($37\,^\circ\text{C}$) without the need for extreme external heat (which would denature proteins/kill the organism).

Specific Enzyme Mechanism: Lysozyme

• Function: Cleaves glycosidic bonds ($\beta(1 \rightarrow 4)$) in the peptidoglycan cell walls of bacteria.

• Substrate: Alternating units of N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG). The bond cleavage occurs between the D site (NAM) and E site (NAG).

• Catalytic Residues:

  • Glutamic Acid 35 ($Glu35$): Acts as a general acid by donating a proton to the oxygen of the glycosidic bond.

  • Aspartate 52 ($Asp52$): Acts as a nucleophile/base to stabilize the resulting carbocation intermediate.

• Mechanism Steps:

  1. Acid Catalysis: $Glu35$ donates a proton to the glycosidic oxygen ($O_1$) between carbon 1 ($C_1$) of NAM and carbon 4 ($C_4$) of NAG.

  2. Bond Cleavage: The bond breaks, releasing the "E" sugar (NAG) as the first leaving group.

  3. Intermediate Stabilization: A covalent or electrostatic intermediate is formed at the "D" sugar (NAM).

  4. Water Involvement: A water molecule enters; $Glu35$ (now deprotonated) acts as a base to deprotonate the water, allowing the resulting $OH^-$ to attack the intermediate to release the final product and regenerate the enzyme.

Questions & Discussion

• Question regarding the Schiff Base:

  • Student Inquiry: Can you go through the Schiff base (shift base)?

  • Response: The lecturer will provide a detailed recording specifically explaining the Schiff base mechanism and the associated calculations.

• Question regarding Graph Labeling:

  • Point of Clarification: When drawing kinetics graphs (Michaelis-Menten or Lineweaver-Burk), students must include a title, correctly labeled axes (with units), and precise intercepts ($V_{max}$, $K_m$, $-1/K_m$, etc.). Failure to label correctly results in a "markdown" (loss of marks).

• Question regarding Amino Acid Size:

  • Student Inquiry: How do we identify if an amino acid is large or small?

  • Response: By looking at the R-group. Glycine consists of only a hydrogen (very small). Alanine has a methyl group ($CH_3$). Aromatic amino acids (Phenylalanine, Tryptophan, Tyrosine) have rings that make them "bulky." The lecturer compares it to a handshake: you can shake a normal hand (flexible/small), but if someone holds a fist (bulky/rigid), your hand just slides over the wrist; it disrupts the expected interaction.