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.