Section 1.2: Medical Biochemistry & Enzymology

Medical Biochemistry & Enzymology

A solid understanding of cellular biochemistry and enzyme kinetics is critical for the SPLE. Many therapeutic agents target metabolic pathways or act as enzyme inhibitors. Pharmacists must understand these molecular mechanisms to anticipate drug actions, toxicities, and drug-drug interactions.

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1. Major Metabolic Pathways

Metabolism consists of interconnected catabolic (breakdown) and anabolic (synthesis) pathways that sustain cellular energy and generate critical biomolecules.

Carbohydrate Metabolism

• Glycolysis: The anaerobic breakdown of one glucose molecule into two molecules of pyruvate, generating a net of 2 ATP and 2 NADH.
  • Hexokinase vs. Glucokinase: Hexokinase is present in most tissues, has a high affinity (low Km), and is feedback-inhibited by glucose-6-phosphate. Glucokinase is located in the liver and pancreatic beta cells, has a low affinity (high Km), and is not feedback-inhibited by glucose-6-phosphate, acting as a glucose sensor.
  • Phosphofructokinase-1 (PFK-1): The rate-limiting and major regulatory enzyme of glycolysis. It is allosterically activated by fructose-2,6-bisphosphate and AMP, and inhibited by ATP and citrate.
  • Pyruvate Kinase: The final step of glycolysis, converting phosphoenolpyruvate to pyruvate.
• Pyruvate Dehydrogenase (PDH) Complex: Catalyzes the oxidative decarboxylation of pyruvate to acetyl-CoA, linking glycolysis to the tricarboxylic acid (TCA) cycle. The PDH complex requires five coenzymes: thiamine pyrophosphate (Vitamin B1), FAD (Vitamin B2), NAD (Vitamin B3), Coenzyme A (Vitamin B5), and lipoic acid. A deficiency in thiamine (common in chronic alcoholism) impairs this step, leading to Wernicke-Korsakoff syndrome.
• Tricarboxylic Acid (TCA) Cycle: Occurs in the mitochondrial matrix. Acetyl-CoA condenses with oxaloacetate to form citrate. For each turn, the cycle produces 3 NADH, 1 FADH2, 1 GTP, and 2 CO2. The rate-limiting enzyme is isocitrate dehydrogenase, which is activated by ADP and inhibited by ATP and NADH.
• Gluconeogenesis: The synthesis of glucose from non-carbohydrate precursors (lactate, glycerol, glucogenic amino acids) during fasting. It bypasses the three irreversible steps of glycolysis using pyruvate carboxylase, phosphoenolpyruvate carboxykinase (PEPCK), fructose-1,6-bisphosphatase, and glucose-6-phosphatase.
• Glycogen Metabolism:
  • Glycogen Synthase: The rate-limiting enzyme for glycogenesis (synthesis), activated by insulin (via dephosphorylation).
  • Glycogen Phosphorylase: The rate-limiting enzyme for glycogenolysis (breakdown), activated by glucagon and epinephrine (via phosphorylation).
• Pentose Phosphate Pathway (PPP): Occurs in the cytosol and generates NADPH and ribose-5-phosphate.
  • Glucose-6-Phosphate Dehydrogenase (G6PD): The rate-limiting step of the PPP. G6PD is essential for maintaining reduced glutathione in erythrocytes, which neutralizes reactive oxygen species (ROS).
  • Clinical Pearl: Patients with G6PD deficiency are susceptible to acute intravascular hemolysis when exposed to oxidant drugs (e.g., rasburicase, primaquine, sulfonamides, nitrofurantoin, dapsone) or fava beans, because their red blood cells cannot generate sufficient NADPH to combat oxidative stress.

Lipid Metabolism

• Beta-Oxidation: The mitochondrial breakdown of fatty acids to acetyl-CoA. Long-chain fatty acids must be transported across the inner mitochondrial membrane via the carnitine shuttle (carnitine palmitoyltransferase I and II).
• Fatty Acid Synthesis: Occurs in the cytosol. The rate-limiting step is catalyzed by acetyl-CoA carboxylase, which converts acetyl-CoA to malonyl-CoA in a biotin-dependent reaction.
• Cholesterol Synthesis: Occurs in the liver. The rate-limiting step is catalyzed by HMG-CoA reductase, which converts HMG-CoA to mevalonate. HMG-CoA reductase is the target of statins (competitive inhibitors), which mimic the substrate structure and block the active site.
• Ketogenesis: During prolonged starvation or uncontrolled diabetes (DKA), the liver uses excess acetyl-CoA to synthesize ketone bodies (acetoacetate, beta-hydroxybutyrate, and acetone) as alternative fuel sources.

Amino Acid & Nitrogen Metabolism

• Transamination: The transfer of an amino group from an amino acid to a ketoacid, catalyzed by aminotransferases (e.g., ALT, AST) requiring pyridoxal phosphate (Vitamin B6).
• Urea Cycle: Detoxifies ammonia into urea in the liver. The rate-limiting step is carbamoyl phosphate synthetase I (CPS I). Deficiencies in urea cycle enzymes lead to hyperammonemia, causing encephalopathy. Treatment includes nitrogen scavengers (e.g., sodium phenylbutyrate) and lactulose.
• Catecholamine Synthesis: Synthesized from the aromatic amino acid tyrosine:

$\text{Tyrosine} \xrightarrow{\text{Tyrosine Hydroxylase}} \text{L-DOPA} \rightarrow \text{Dopamine} \rightarrow \text{Norepinephrine} \rightarrow \text{Epinephrine}$

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2. Enzyme Kinetics

Enzymes are biological catalysts that accelerate reaction rates by lowering the activation energy. Enzyme-catalyzed reactions typically follow Michaelis-Menten kinetics.

Michaelis-Menten Parameters

• Maximum Velocity (Vmax): The rate of reaction when the enzyme is fully saturated with substrate.
• Michaelis Constant (Km): The substrate concentration at which the reaction rate is exactly half of Vmax. Km is an indicator of the enzyme's affinity for its substrate (an inverse relationship: a lower Km means higher affinity).

Lineweaver-Burk (Double Reciprocal) Plot

To linearize the Michaelis-Menten equation, a double reciprocal plot is used:

$\frac{1}{v_0} = \frac{K_m}{V_{max}} \left(\frac{1}{[S]}\right) + \frac{1}{V_{max}}$

• Y-intercept: Represented by $1/V_{max}$
• X-intercept: Represented by $-1/K_m$
• Slope: Represented by $K_m/V_{max}$

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3. Mechanisms of Enzyme Inhibition

Pharmacology leverages enzyme inhibitors to modulate physiological pathways. Inhibitors are categorized based on their binding characteristics and kinetic impact.

Reversible Inhibition

1. Competitive Inhibition:
   • The inhibitor binds reversibly to the active site of the free enzyme, competing directly with the substrate.
   • Kinetics: Vmax remains unchanged (can be overcome by increasing substrate concentration), while the apparent Km increases (requires more substrate to reach half-saturation).
   • Examples: Statins competing with HMG-CoA; methotrexate competing with dihydrofolate for dihydrofolate reductase.
2. Non-competitive Inhibition:
   • The inhibitor binds reversibly to an allosteric site on either the free enzyme or the enzyme-substrate complex.
   • Kinetics: Vmax decreases (cannot be overcome by substrate), while Km remains unchanged (affinity for substrate is unaffected).
   • Examples: Non-nucleoside reverse transcriptase inhibitors (NNRTIs) like efavirenz; allosteric regulation.
3. Uncompetitive Inhibition:
   • The inhibitor binds only to the enzyme-substrate complex (ES), lock-and-key fashion.
   • Kinetics: Both Vmax and Km decrease proportionally. The ratio of Km/Vmax remains constant (parallel lines on Lineweaver-Burk).

Irreversible Inhibition

Irreversible inhibitors bind covalently or extremely tightly to the enzyme, permanently inactivating it. Cells must synthesize new enzyme molecules to restore activity.

• Examples:
  • Aspirin: Covalently acetylates serine residues on COX-1 and COX-2 enzymes.
  • Proton Pump Inhibitors (PPIs): Form a covalent disulfide bond with the H+/K+-ATPase pump in gastric parietal cells.
  • Organophosphates: Covalently bind and phosphorylate the active site of acetylcholinesterase.

Enzyme Inhibition Types and Kinetics Summary