Enzyme Regulation Notes

Enzyme Regulation

Enzyme regulation is crucial for proper timing and location of enzyme function, coordinating biochemical processes within an organism. Metabolic processes in living cells involve sequential steps, each catalyzed by a different enzyme.

Metabolic Pathways

• Molecule A is converted to molecule F through a series of enzyme-catalyzed steps.
• Intermediates: Products of one reaction serve as substrates for the next (e.g., molecule B).
• Regulation: Usually occurs at a single, key enzymatic step. Inhibiting enzyme 1 shuts down the entire pathway.
• Example: Glycolytic pathway (oxidizes sugars).

Feedback Inhibition

• Final product (e.g., molecule Z) inhibits the initial, unique step of its synthesis.
• Molecule Z is neither a substrate nor a product of the inhibited enzyme (e.g., the enzyme that converts molecule B to molecule X).
• Doesn't inhibit other uses of intermediate molecules(e.g., synthesis of molecule C from molecule B).
• Physiological importance: Prevents wasted resources and accumulation of unneeded intermediates by only initiating the pathway when needed.

Mechanisms of Enzyme Regulation

• Competitive Inhibition: Many drugs are substrate analogs, binding to the active site.
• Allosteric Control: Regulatory molecule binds to a site distinct from the active site.
• Reversible Covalent Modification: Phosphorylation/dephosphorylation (hormonal regulation).
• Proteolytic Activation: Extracellular mechanism to keep enzymes inactive until needed (e.g., digestive enzymes in the gut, blood clotting cascade).

Allosteric Regulation

• Regulatory molecule binds to a site other than the active site.
• This binding alters the enzyme's tertiary and quaternary structure, changing the active site conformation.
• Allosteric proteins often have multiple active and regulatory sites.
• Cooperativity: Substrate binding to one active site affects the binding/activity of other active sites.

Examples of Allosteric Proteins

1. Aspartate Transcarbamylase (ATCase):

   • Bacterial enzyme that synthesizes N-carbamoyl aspartate (pyrimidine synthesis).
   • Inhibited by CTP (cytosine triphosphate, an end product) via feedback inhibition.
   • Catalytic and regulatory sites are on different polypeptides.
   • Complex structure: catalytic trimers and regulatory dimers.
   • Complete molecule: Two catalytic trimers and three regulatory dimers.

2. Hemoglobin:

   • Transport protein (not an enzyme) that illustrates allosteric principles.
   • Oxygen binding to one site affects the affinity of other sites for oxygen.
   • Binding of regulatory molecules affects oxygen binding.

Kinetics of Allosteric Enzymes

• Do not follow Michaelis-Menten kinetics.
• Exhibit sigmoidal (S-shaped) curves.
• At low substrate concentrations, the relationship between substrate concentration and product formation is nonlinear.
• Cooperativity: Binding of one substrate molecule enhances binding of others.
  • Requires regulatory subunits.
  • Purified catalytic subunits do not generate sigmoidal kinetics.

Allosteric Effects Involve Shape Changes

• Two conformations: T (Tense) and R (Relaxed).
  • T state: relatively inactive.
  • R state: active.
• Ligand (substrate) binding converts T to R form.
• This modifies the shape of adjacent subunits, making them more receptive to ligand binding.
• ATCase: Aspartate enhances the stability of the R state.
• CTP enhances the stability of the T state, inhibiting activity.

Hemoglobin and Cooperativity

• Tetrameric protein transporting oxygen.
• Oxygen binds to a heme prosthetic group on each subunit.
• Sigmoidal curve: Cooperativity in oxygen binding.

Significance of Cooperativity for Hemoglobin

• Allows efficient oxygen uptake in lungs (high concentration) and release in tissues (low concentration).
• Adult humans: Accepts oxygen at 100 torr and releases it at 20 torr.
• Cooperativity increases the amount of oxygen delivered to tissues.

Allosteric Modulators of Hemoglobin

1. 2,3-Bisphosphoglycerate (2,3-BPG):

   • Produced in red blood cells.
   • Fits in a binding pocket of deoxyhemoglobin, reducing its oxygen-binding ability.
   • This promotes oxygen release in tissues.

2. Hydrogen Ions (Bohr Effect):

   • Increased carbon dioxide concentrations lead to carbonic acid production, which dissociates into hydrogen ions and bicarbonate.
   • This promotes oxygen release in metabolically active tissues.

Fetal Hemoglobin

• Composed of two alpha and two gamma subunits (instead of two alpha and two beta in adults).
• Doesn't bind 2,3-BPG as readily.
• Higher oxygen affinity than adult hemoglobin at low oxygen tensions.
• Facilitates oxygen transfer from maternal circulation to the fetus across the placenta.

Reversible Covalent Modification

• Different from allosteric regulation (non-covalent binding).
• Examples:
  • Phosphorylation/dephosphorylation.
  • Acetylation/deacetylation.
  • Myristoylation/farnesylation (lipid chain attachment for membrane anchoring).

Protein Kinases

• Enzymes that catalyze phosphorylation.
• Two classes:
  • Serine/threonine kinases.
  • Tyrosine kinases.
• Some are specific to one protein; others are multifunctional.
• Obtain phosphate group and energy from ATP.
• \text{ATP} + \text{Protein} \rightarrow \text{ADP} + \text{Phosphorylated Protein}
• Over 500 human protein kinases.

Protein Phosphatases

• Enzymes that remove phosphate groups.
• Hydrolysis reaction (not the reverse of kinase reaction; ATP not generated).
• \text{Phosphorylated Protein} + \text{H}2\text{O} \rightarrow \text{Protein} + \text{P}i
• Phosphorylation status depends on relative activities of kinases and phosphatases.

Effects of Phosphorylation

• Adds a large, negatively charged phosphoryl group.
• Alters electrostatic interactions.
• Can form multiple hydrogen bonds.
• Markedly alters substrate binding and catalytic activity.

Regulation by Phosphorylation

• Many metabolic enzymes are regulated.
• Examples:
  • Glycogen synthase: Active when dephosphorylated, inactive when phosphorylated.
  • Glycogen phosphorylase: Inactive when dephosphorylated, active when phosphorylated.

Protein Kinase A (PKA) Activation

• Regulated by hormones (epinephrine, glucagon).
• Hormones stimulate synthesis of cyclic AMP (cAMP) from ATP.
• cAMP activates PKA, which phosphorylates various cellular proteins.

Mechanism of PKA Activation

• PKA is a multimeric protein with catalytic and regulatory subunits.
• Separate catalytic subunits are active.
• Regulatory subunits inhibit catalytic subunits.
• Regulatory subunits have a pseudosubstrate sequence that binds to the active site.
• cAMP binding to regulatory subunits releases the pseudosubstrate.
• This activates the catalytic subunits.

Enzyme Activation by Proteolysis

• Irreversible (unlike allosteric effects and phosphorylation/dephosphorylation).
• Enzyme synthesized as an inactive proenzyme (zymogen).
• Protease cleavage activates the enzyme.
• Prevents enzymes (e.g., digestive proteases) from damaging the cells where they're made.

Example: Chymotrypsinogen Activation

• Chymotrypsinogen is activated to chymotrypsin by trypsin in the small intestine.
• Hydrolysis splits the initial polypeptide into two pieces (15 and 230 amino acids) linked by disulfide bonds.
• Chymotrypsin can further hydrolyze itself into three covalently bonded polypeptides (A, B, and C chains).

Structural Changes During Chymotrypsinogen Activation

• Trypsin converts one peptide bond into a free carboxyl group and a free amino group.
• The newly free amino group of isoleucine 16 forms an electrostatic bond with aspartate 194.
• This initiates conformational changes, including formation of the substrate-binding cavity.
• The catalytic triad is brought into the appropriate physical relationship for activity.

Cascade of Zymogen Activation in Digestion

• Zymogens produced by the pancreas protect it from auto-digestion.
• Enteropeptidase (produced by duodenal cells) activates trypsinogen to trypsin.
• Trypsin then activates other zymogens (chymotrypsinogen, proelastase).
• Pro lipase is not a zymogen. It should say phospholipase A2.