Enzyme Regulation Notes

Enzyme Regulation

Enzymes and Regulation

• Enzyme regulation, Hemoglobin's oxygen transport, covalent modification, proteolytic cleavage, and allosteric inhibition of aspartate transcarbamoylase (ATCase) are key topics.

Metabolic Pathways

• Metabolic pathways involve multiple enzyme steps within a single pathway.
• The glycolytic pathway oxidizes sugars.
• Regulation of a pathway is achieved by regulating one key enzyme.
• Inhibition of the first enzyme in a pathway can shut down the entire pathway.

Feedback Inhibition

• Feedback inhibition: the end-product of a pathway inhibits the first unique step in its synthesis.
• This regulates the product's concentration within the cell.
• Example: Molecule Z inhibits the conversion of molecule B to molecule X.

Physiological Regulation of Enzyme Activity

• Mechanisms:
  • Allosteric control: using non-substrate small molecules as modulators.
  • Reversible covalent modification: e.g., phosphorylation/dephosphorylation for hormonal regulation.
  • Proteolytic activation: activating digestive enzymes in the gut and blood-clotting cascade after injury.

Allosteric Regulation

• Allosteric proteins have regulatory and active sites.
• Signal molecules bind to sites distant from the active site, causing conformational changes that affect the active site.
• Allosteric proteins show cooperativity: activity at one site affects others.
• Examples: ATCase and hemoglobin.

ATCase: End-Product Inhibition

• ATCase catalyzes the first step in pyrimidine synthesis.
• It is inhibited by CTP, an end-product of the pathway, demonstrating end-product or feedback inhibition.

Allosteric Inhibition of ATCase

• CTP binds to a regulatory site separate from the catalytic site.
• ATCase has catalytic trimers linked by regulatory chain dimers.

Kinetics of Allosteric Enzymes

• Allosteric enzymes do not follow Michaelis-Menten kinetics; they show sigmoidal kinetics.
• Cooperativity: Substrate binding increases binding properties in other subunits.
• Isolated catalytic subunits follow normal kinetics; regulatory subunits cause the sigmoidal curve.

Allosteric Effects and Shape Changes

• ATCase exists in two conformations:
  • Tense (T) state: compact and less active.
  • Relaxed (R) state: expanded and more active.
• Substrate binding favors the R state, while CTP binding favors the T state.
• Cooperativity: Ligand binding to one subunit affects the shape of others.

Hemoglobin and Cooperativity

• Hemoglobin: a tetrameric protein transporting oxygen.
• Cooperativity allows hemoglobin to deliver more oxygen to tissues.
• Partial pressure of oxygen (pO_2) in the lungs is ~100 torr, and in tissues, it is ~20 torr.
• Oxygen delivery depends on the difference in hemoglobin saturation at these pO_2 levels.

2,3-BPG Modulation of Hemoglobin

• 2,3-BPG is present in red blood cells.
• It stabilizes the T state of deoxyhemoglobin, reducing oxygen affinity.
• This facilitates oxygen release in target tissues.

Fetal Hemoglobin

• Fetal hemoglobin: α2γ2 instead of adult α2β2.
• Lower affinity for 2,3-BPG due to the γ_2 subunits.
• Ensures efficient oxygen transfer from maternal to fetal red blood cells.

Regulation via Covalent Modification

• Reversible covalent modification regulates enzyme activity.
• Phosphorylation/dephosphorylation is the most common.
• Other modifications include acetylation of histones and lipid additions for membrane anchoring.

Protein Kinases

• Protein kinases phosphorylate proteins by transferring a phosphate group from ATP to serine, threonine, or tyrosine residues.
• There are >500 human protein kinases.

Protein Phosphatases

• Protein phosphatases remove phosphate groups from phosphorylated proteins, releasing inorganic phosphate (P_i).
• Phosphorylation status depends on the relative activities of kinases and phosphatases.

Phosphorylation and Protein Structure

• Phosphorylation alters substrate binding and catalytic activity.
• The phosphoryl group ( -OPO_3^{2-} ) introduces negative charges and promotes hydrogen bonds.
• The energy from the phosphate bond can shift equilibrium between protein structures.

Phosphorylation and Enzyme Activity

• Some enzymes are inactivated by phosphorylation (e.g., glycogen synthase).
• Others are activated (e.g., glycogen phosphorylase).

Kinase-Phosphatase Cycle

• Phosphorylation and dephosphorylation are reversible, occurring rapidly or over hours.

Regulation of Protein Kinase Activity

• Protein kinase A (PKA) is a key enzyme in hormone-regulated enzyme activation.
• Activated by epinephrine and glucagon via cyclic AMP (cAMP).
• cAMP, a second messenger formed from ATP cyclization, activates PKA, which then phosphorylates intracellular enzymes.

Activation of Protein Kinase A

• PKA has regulatory (R) and catalytic (C) subunits.
• The pseudosubstrate portion of R blocks catalytic sites.
• cAMP binding to R subunits releases the catalytic subunits, allowing them to phosphorylate substrate proteins.

Proteolytic Activation of Enzymes

• Some enzymes are synthesized as inactive zymogens or proenzymes, activated by cleavage of specific peptide bonds.
• Activation is irreversible.
• Used for digestive enzymes and peptide hormones like insulin.

Chymotrypsinogen to Chymotrypsin

• Chymotrypsin is initially synthesized as chymotrypsinogen in the pancreas.
• Trypsin cleaves a peptide bond in the small intestine.
• The resulting π-enzyme cleaves itself to form the α form. Both are active.
• The separated chains stay linked by disulfide bonds.

Inactivity of Chymotrypsinogen

• Chymotrypsinogen is an inactive zymogen.
• Cleavage forms π-chymotrypsin, causing conformational changes.
• The new amino terminal isoleucine 16 forms an ionic bond with aspartate, stabilizing the active site.
• Without these changes, the enzyme is inactive.

Zymogen Activation of Digestion

• Enteropeptidase, produced in the duodenum, activates trypsinogen to trypsin.
• Trypsin then activates other pancreatic zymogens.
• Secretion as zymogens prevents autodigestion of the pancreas.