Enzyme Regulation - Detailed Notes

Enzyme Regulation

Enzyme Regulation Overview

• Enzyme activity is regulated to ensure enzymes function at the correct time and place, coordinating biochemical processes within an organism.
• This lecture will examine the major physiological mechanisms for regulation of enzymatic activity and thus cellular metabolism.

Metabolic Pathways and Regulation

• Metabolic processes in living cells involve sequential steps, each catalyzed by a different enzyme.
• A metabolic pathway converts an initial product (molecule A) to a final product (molecule F) through a series of enzyme-catalyzed reactions.
• The pathway includes intermediates, where the product of one reaction serves as the substrate for the next.
• Regulation typically occurs at a specific step within the multi-step process.

Feedback Inhibition

• Feedback inhibition is a common strategy for regulating cellular metabolism.
• The final product (e.g., molecule Z) inhibits the initial, unique step of its synthesis pathway.
• Molecule Z is neither a substrate nor a product of the enzyme converting B to X.
• This doesn't inhibit other uses for molecule B, such as synthesis of molecule C.
• Feedback inhibition is a practical physiological control mechanism to ensure reactions are initiated only when needed and to prevent waste of resources.
• It stops the sequence at the beginning when sufficient product is available, preventing accumulation of unneeded intermediates.

Mechanisms of Enzyme Regulation

• Drugs can act as substrate analogs, binding at the active site and acting as competitive enzyme inhibitors.
• Physiological mechanisms for enzyme regulation involve various molecular interactions.
• Allosteric control and reversible covalent enzyme modification regulate major metabolic pathways like glycolysis and fatty acid synthesis.
• Proteolytic activation of enzymes is usually an extracellular mechanism, keeping enzymes inactive until the right place and time.

Allosteric Regulation

• Allosteric regulation involves a regulatory molecule binding to a site on the enzyme distinct from the active site.
• This binding changes the enzyme's tertiary and quaternary structures, altering the active site conformation.
• Allosteric proteins are often complex with multiple functional (active) sites and distinct regulatory sites.
• The kinetics of allosteric enzymes show cooperativity, where substrate binding to one active site affects binding at other active sites in the same molecule.
• Examples include bacterial aspartate transcarbamylase (ATCase) and hemoglobin.
• Hemoglobin, a transport protein, illustrates cooperativity: oxygen binding at one site affects affinity at other sites, and a regulatory molecule (2,3-BPG) affects oxygen binding.

Aspartate Transcarbamylase (ATCase)

• ATCase synthesizes N-carbamoylaspartate, an intermediate in pyrimidine synthesis (uracil and cytosine).
• The enzyme is inhibited by CTP, an end product of the pathway.
• CTP binds to an allosteric regulatory site on ATCase.
• Catalytic and regulatory sites are on different polypeptides in ATCase.
• Catalytic pieces are polypeptide trimers, and regulatory pieces are dimers.
• The complete molecule has two catalytic trimers and three regulatory dimers.

ATCase Kinetics and Cooperativity

• Increased substrate (aspartate) concentration generates a sigmoidal curve for ATCase activity, unlike the Michaelis-Menten curve.
• At low substrate concentrations, the relationship between substrate concentration and product formation is not linear; cooperativity is observed.
• Aspartate binding to ATCase enhances the ability of other aspartate molecules to bind to other active sites.
• Cooperativity requires the presence of the regulatory subunits.
• Purified catalytic subunits do not generate sigmoidal kinetics.

Allosteric Effects and Conformations

• Allosteric effects involve shape changes; ATCase has two conformations: tense (T) and relaxed (R).
• The T state is relatively inactive, and the R state is active.
• Ligand (substrate) binding converts a subunit from T to R form, modifying adjacent subunits to more readily bind ligand.
• Aspartate enhances the stability of the R state.
• CTP binding to a regulatory dimer enhances the stability of the T state, inhibiting enzyme activity.

Hemoglobin and Cooperativity

• Oxygen binding to hemoglobin is a classic example of cooperativity.
• Each hemoglobin molecule is a tetramer with an oxygen-binding site (heme) on each subunit.
• The binding of oxygen (fractional saturation) is a function of oxygen concentration (partial pressure).
• Without cooperativity, the curve would be simple; with cooperativity, it's sigmoidal.

Hemoglobin Function and Oxygen Delivery

• Hemoglobin must pick up oxygen at high concentrations (lungs) and release it at low concentrations (tissues).
• The change in fractional saturation between oxygen tensions is greater with cooperativity.
• This allows the same number of hemoglobin molecules to deliver more oxygen to the tissues.

Allosteric Modulators of Hemoglobin

• Hemoglobin is affected by the allosteric modulator 2,3-BPG.
• 2,3-BPG is produced in red blood cells and fits in a binding pocket of deoxyhemoglobin (without bound oxygen).
• This reduces hemoglobin's ability to bind oxygen, facilitating oxygen release at the tissues.
• Hydrogen ions also act as an allosteric modulator (Bohr effect), releasing oxygen from oxyhemoglobin when carbon dioxide concentrations increase.
• Carbon dioxide produces carbonic acid (H2CO3), which dissociates into hydrogen ion and bicarbonate.
• This enhances oxygen release to metabolically active tissues.

Fetal Hemoglobin

• Fetal hemoglobin differs from adult hemoglobin: (\alpha2 \gamma2) vs. adult (\alpha2 \beta2).
• The presence of 𝛾 subunits results in lower 2,3-BPG binding affinity.
• Fetal hemoglobin holds onto oxygen more tightly than adult hemoglobin at low oxygen tensions.
• This permits efficient oxygen transfer from maternal circulation across the placenta to the fetus.

Reversible Covalent Modification

• Activities of many proteins are regulated by reversible covalent modification.
• Enzymes and membrane channels are reversibly phosphorylated and dephosphorylated.
• Depending on the protein, phosphorylation can enhance or inhibit activity.
• Histone activities are regulated by reversible acetylation and deacetylation.
• Phosphorylation and acetylation add polar functional groups.
• Attachment of hydrophobic or nonpolar lipid chains anchors the protein to a membrane.

Protein Kinases

• Protein kinases catalyze phosphorylation of proteins.
• Two major classes: those that phosphorylate serine/threonine residues and those that phosphorylate tyrosine residues.
• Some protein kinases are specific for one protein; others are multifunctional.
• Both the phosphate group and energy for the reaction come from ATP.

Protein Phosphatases

• Protein phosphatases remove covalently attached phosphate groups from proteins, reversing the effects of protein kinases.
• The reaction is hydrolysis, using water to release inorganic phosphate (not the reverse of protein kinase reaction, so ATP is not generated).

Effects of Phosphorylation

• Phosphorylation adds a large, negatively charged functional group, altering electrostatic interactions.
• Each phosphate group can form as many as three different hydrogen bonds.
• The net effect can markedly alter substrate binding and catalytic activity.

Regulation by Phosphorylation/Dephosphorylation

• Reversible phosphorylation and dephosphorylation is a major mechanism for regulating enzyme activity in response to hormonal signals.
• Some enzymes, like glycogen synthase, are active without phosphate and inactivated by phosphorylation.
• Other enzymes, like glycogen phosphorylase, are inactive when dephosphorylated and activated by protein kinase.

Protein Kinase and Phosphatase Cycle

• The paired activities of a protein kinase and a protein phosphatase on the same protein form a cycle.
• This cycle alternates an enzyme between active and inactive forms.

Regulation of Protein Kinases

• Protein kinase activities are also regulated.
• Protein kinase A (PKA) is activated in response to hormones like epinephrine and glucagon.
• The hormones initiate synthesis of cyclic AMP (cAMP), a second messenger, from ATP.
• cAMP activates PKA, which phosphorylates many cellular proteins and contributes to coordinated regulation of metabolic pathways.

cAMP and Protein Kinase A

• cAMP is an allosteric regulator of protein kinase A (PKA).
• PKA is a multimeric protein with catalytic (C) and regulatory (R) subunits.
• When separate, catalytic subunits are active; when bound to regulatory subunits, catalytic subunits are inactive.
• Regulatory subunits have a pseudosubstrate sequence that binds tightly to the active site of catalytic subunits; but is not itself phosphorylated.
• Binding of cAMP to the regulatory subunit changes its conformation and releases the pseudosubstrate from the active site, activating the catalytic subunits.

Proteolysis

• Proteolysis is another mechanism for enzyme activation and is irreversible.
• The enzyme is synthesized as an inactive proenzyme or zymogen.
• A protease cleaves one or more peptide bonds to activate the enzyme.
• This mechanism keeps certain enzymes, such as digestive proteases, from hydrolyzing the proteins of the cells in which they are made.

Chymotrypsin Activation

• Chymotrypsin is synthesized as a zymogen called chymotrypsinogen.
• Trypsin hydrolyzes a specific peptide bond of chymotrypsinogen in the small intestine.
• The initial 245 amino acid polypeptide is split into two pieces (15 and 230 amino acids), which remain attached by disulfide bonds to form active enzyme
• Chymotrypsin can then hydrolyze one of its own bonds, creating a second active form with three covalently bound shorter polypeptides.

Structural Changes in Chymotrypsin Activation

• Chymotrypsinogen and chymotrypsin appear structurally similar, but one is inactive and the other active.
• Trypsin's action converts one peptide bond into a free carboxyl group and a free amino group, causing structural changes.
• The newly free amino group (isoleucine 16) forms an electrostatic bond with aspartate 194, initiating conformational changes.
• These changes include formation of the substrate-binding cavity, which accepts aromatic and bulky nonpolar groups.
• A second conformational change brings the three amino acid side-chains of the catalytic triad into the appropriate physical relationship for activity.

Zymogen Activation Cascade in Digestion

• A cascade of zymogen activation occurs in digestion.
• The pancreas secretes protease zymogens: trypsin, elastase, carboxypeptidase, and chymotrypsin.
• Carboxypeptidase releases the carboxy-terminal amino acid, while others cleave within the polypeptide chain.
• The pancreas also secretes phospholipase A2 (hydrolyzes phospholipids) in zymogen form.
• Enteropeptidase, synthesized by cells lining the small intestine, hydrolyzes one bond in trypsinogen, producing active trypsin.
• Trypsin hydrolyzes bonds in other zymogens (and in trypsinogen), producing the full spectrum of active pancreatic digestive enzymes.