Amino Acids, Hemoglobin Function, and Enzyme Kinetics

Posted on May 2, 2025 in Biotechnology

Amino Acid Classification

Nonpolar Amino Acids

Glycine (G), Alanine (A), Valine (V), Leucine (L), Isoleucine (I), Proline (P), Phenylalanine (F), Methionine (M), Tryptophan (W)

Polar (Uncharged) Amino Acids

Serine (S), Threonine (T), Cysteine (C), Asparagine (N), Glutamine (Q), Tyrosine (Y)

Polar (Charged) Amino Acids

Aspartic Acid (D), Glutamic Acid (E), Lysine (K), Arginine (R), Histidine (H)

Protein Structure Notes

Beta-Sheet Constraints

Amino acids with large side groups (R groups) cannot easily fit into a beta-sheet. Alanine (–CH₃), Serine (–CH₂–OH), and Glycine (–H) are common in beta-sheets due to their smaller R groups.

Note: The phrase “not affected means C is present” is unclear but has been retained with minor correction. It might refer to Cysteine’s role or disulfide bonds, but context is needed for accurate interpretation.

Beta Turns

Beta turns often connect strands of antiparallel beta-sheets. They are stabilized by a hydrogen bond from a carbonyl oxygen to an amide proton three residues down the sequence. Proline in position 2 or Glycine in position 3 are common in beta turns.

Hemoglobin and Oxygen Transport

Role of 2,3-BPG in Oxygen Affinity

• More 2,3-BPG = Lower O₂ affinity → Right shift (enhances O₂ unloading in tissues).
• Less 2,3-BPG = Higher O₂ affinity → Left shift (oxygen remains bound more tightly).

BPG Binding Site on Hemoglobin

2,3-BPG binds to a pocket in the center of the tetramer, interacting with positively charged residues: Lysine (Lys), Arginine (Arg), Histidine (His), and the N-Terminus of the beta chains. Low pH increases the positive charge (protonation), strengthening BPG binding.

Hemoglobin States: Taut (T) and Relaxed (R)

• The deoxy form (Taut or T state) is stabilized by salt bridges, including those involving BPG.
• Binding of O₂ breaks these salt bridges, leading to the Relaxed (R) state.
• Low pH (more H⁺) stabilizes additional salt bridges (e.g., involving Histidine), favoring the T state and the release of O₂.

Oxygen Binding Curve Characteristics

• As [O₂] approaches 0, the fractional saturation (Y) equation simplifies: 1 + K_f [O₂] approaches 1, and Y ≈ K_f [O₂]. This shows a linear relationship at very low oxygen concentrations.
• As [O₂] approaches infinity, Y approaches 1 (saturation). The overall binding curve is sigmoidal for hemoglobin (cooperative binding), though the description here mentions hyperbolic, which is typical for myoglobin or single subunits.

Levels of Protein Structure

• Primary structure: The sequence of amino acids.
• Secondary structure: Local arrangements like alpha-helices and beta-sheets, formed by hydrogen bonds between backbone atoms.
• Tertiary structure: The final 3D conformation of a single polypeptide chain.
• Quaternary structure: The arrangement of multiple polypeptide subunits (like in hemoglobin).

The Bohr Effect Explained

• CO₂ reacts with the N-terminal amino groups of the hemoglobin beta-subunits to form carbamates:
  • CO₂ + R-NH₂ ↔ R-NH-COO^– + H⁺
• The H⁺ generated by this reaction in the tissues further promotes the release of O₂ (stabilizes the T state).
• Approximately 20% of CO₂ is transported by hemoglobin to the lungs. In the lungs, high O₂ concentration reverses the reaction, releasing CO₂ and raising the pH, which favors the R state and O₂ binding.

pH and Oxygen Binding Equilibrium

• When O₂ is high (lungs), Hb binds O₂ and releases H⁺ (favors R state).
• When O₂ is low and H⁺ is high (tissues), H⁺ binds to Hb, promoting O₂ release (favors T state).

H-Hb⁺ + O₂ ↔ Hb-O₂ + H⁺

Enzyme Kinetics Fundamentals

Reaction Order Basics

• First Order: Reaction rate (V) is dependent on substrate concentration [S].
• Zero Order: Reaction rate (V) is independent of substrate concentration [S] (e.g., when enzyme is saturated).
• (Second order reactions involve two reactant concentrations).

Michaelis-Menten Kinetics

Initially, the reaction rate (V) increases linearly as a function of [S] (approximating first-order kinetics). However, the rate begins to approach a limit (V_max) as the enzyme becomes saturated with substrate. This relationship produces a hyperbolic curve when plotting V versus [S].

Michaelis-Menten Assumptions

The derivation of the Michaelis-Menten equation relies on these assumptions:

• The formation of an enzyme-substrate (ES) complex is a necessary intermediate step (E + S ↔ ES → E + P).
• Steady-state approximation: The concentration of the ES complex remains relatively constant over the measured time period (d[ES]/dt = 0). This holds when [S] >> [E], ensuring that as ES breaks down to E + P, sufficient S is available to immediately reform ES.
• Only the initial velocity (V₀) is measured. This means measurements are taken before significant product (P) accumulates, so the reverse reaction (E + P → ES, rate constant k_-2) is negligible. Thus, k_-2[E][P] ≈ 0.

Under these conditions, the velocity of product formation is primarily determined by the breakdown of the ES complex: V = k₂[ES] (where k₂ is often called k_cat).

Understanding Km (Michaelis Constant)

• Definition: K_m is the substrate concentration [S] at which the reaction velocity is half of V_max (V = ½ V_max).
• Affinity Interpretation:
  • High K_m = Lower apparent affinity (a higher [S] is needed to reach ½ V_max).
  • Low K_m = Higher apparent affinity (a lower [S] is needed to reach ½ V_max).
• Note: K_m strictly represents affinity (as the dissociation constant K_d of the ES complex) only when the rate of ES dissociation back to E + S (k_-1) is much greater than the rate of product formation (k₂). However, K_m is always the [S] required for ½ V_max and is widely used as a practical indicator of the enzyme’s interaction with its substrate.

Catalytic Efficiency (kcat/Km)

• Definition: The ratio (k_cat / K_m) is known as the catalytic efficiency or specificity constant. It reflects how efficiently an enzyme converts substrate to product at low substrate concentrations.
  • k_cat: The turnover number (k₂), representing the maximum number of substrate molecules converted to product per enzyme molecule per unit time (e.g., turnovers/sec).
  • K_m: Represents the substrate concentration needed for effective catalysis to occur (½ V_max), related to affinity.
• Highly Efficient Enzymes: These enzymes are often described as “diffusion-controlled.” They typically have:
  • High k_cat (fast catalytic rate).
  • Low K_m (high affinity for the substrate).
• For such enzymes, the rate of the reaction is limited primarily by the rate at which the enzyme encounters the substrate through diffusion in the solution.