Enzyme Kinetics & Inhibition Continue

🧬 Advanced Enzymology & Regulation

Bi-substrate Kinetics, Allostery & Protein Purification Notes

1. Bi-Substrate Reactions (Cleland Kinetics)

Over 60% of known biochemical reactions involve two substrates and two products (Bi-Bi reactions). They are broadly classified into two mechanisms:

A. Sequential (Single-Displacement)

• Mechanism: All substrates must bind to the enzyme before any product is released. Forms a ternary complex (E-A-B).
• Ordered Sequential: Substrates bind in a strict, specific order (e.g., NAD⁺ binding before the target molecule in Dehydrogenases).
• Random Sequential: Substrates can bind in any order (e.g., Creatine kinase).

B. Ping-Pong (Double-Displacement)

• Mechanism: One substrate binds and one product is released before the second substrate binds.
• Key Feature: The enzyme is temporarily covalently modified (substituted enzyme intermediate, E'). No ternary complex is formed.
• Example: Aminotransferases (Transaminases) requiring PLP cofactor.

2. Allosteric Enzymes

Allosteric enzymes do not follow Michaelis-Menten kinetics. They consist of multiple subunits and exhibit cooperativity, where the binding of a substrate to one active site affects the affinity of other active sites.

• Sigmoidal Kinetics: Instead of a hyperbola, they plot an S-shaped (sigmoidal) curve of Velocity vs. Substrate Concentration.
• T and R States: They exist in two conformational states. The T (Tense) state has low affinity for substrate. The R (Relaxed) state has high affinity.
• MWC (Concerted) Model: All subunits undergo the T → R transition simultaneously. No hybrid states exist.
• KNF (Sequential) Model: Subunit changes conformation one by one upon substrate binding.

3. Isoenzymes & Ribozymes

Isoenzymes (Isozymes)

Enzymes that catalyze the same chemical reaction but have different amino acid sequences and kinetic properties (different Km/Vmax). They are coded by different genes or arise from alternative splicing.

Example: Lactate Dehydrogenase (LDH)
LDH is a tetramer made of H (Heart) and M (Muscle) subunits.

• LDH-1 (H₄): High affinity for lactate, found in heart.
• LDH-5 (M₄): Low affinity, works best anaerobically, found in skeletal muscle.

Ribozymes

RNA molecules that possess true enzymatic catalytic activity. They proved that proteins are not the only biological catalysts.

• RNase P: Cleaves the 5' end of pre-tRNAs.
• Peptidyl Transferase: The 23S rRNA in the bacterial ribosome catalyzes peptide bond formation during translation.
• Self-splicing Introns: Group I and II introns that splice themselves out of mRNA without protein assistance.

4. Enzyme Regulation Strategies

• Covalent Modification: The reversible addition of chemical groups. The most common is Phosphorylation (catalyzed by Kinases) and Dephosphorylation (by Phosphatases). Usually occurs on Serine, Threonine, or Tyrosine residues (-OH groups).
• Proteolytic Cleavage (Zymogens): Many enzymes are synthesized as inactive precursors (zymogens/proenzymes). They are activated by specific irreversible peptide bond cleavage. Example: Trypsinogen → Trypsin (activated by Enteropeptidase).
• Feedback Inhibition: The end product of a metabolic pathway acts as an allosteric inhibitor of the first committed step (the pacemaker enzyme) of that pathway.

5. Enzyme Purification & Assays

The goal of purification is to isolate a specific enzyme while maintaining its catalytic activity. Success is tracked using a purification table.

🔥 Crucial Exam Formulas:

• Total Activity: Enzyme units (U) = μmol of product formed per minute.
• Specific Activity: = Total Activity (U) / Total Protein (mg). This value MUST increase as the enzyme becomes more pure!
• Fold Purification: = Specific Activity of current step / Specific Activity of crude extract.
• Yield (%): = (Total Activity of current step / Total Activity of crude extract) × 100.

Common Chromatographic Techniques:

• Gel Filtration (Size Exclusion): Separates by size. Rule: Large proteins elute FIRST, small proteins get trapped in bead pores and elute LAST.
• Ion-Exchange Chromatography: Separates by net charge. Cation exchangers (e.g., CM-cellulose) bind positively charged proteins. Anion exchangers (e.g., DEAE-cellulose) bind negatively charged proteins. Elution is done by increasing salt (NaCl) concentration.
• Affinity Chromatography: Most specific method. Uses a covalently attached ligand (e.g., Glucose for a Glucokinase). Eluted by adding free ligand to outcompete the bead.