Cell Metabolism and Metabolic Control

Control of Enzyme Activity

1. Control Mechanisms

Amount ("Coarse" Control)

• Synthesis and breakdown of enzymes.

• This is a long-term control mechanism, as discussed in Lecture 3.

Activity ("Fine" Control)

• Short-term control (milliseconds to seconds).

• Rapid control.

• Refer to Chapter 6 in "Lehninger Principles of Biochemistry" by Nelson and Cox.

2. Fine Control - Substrate Linked

Total Substrate Availability

• The reaction rate is dependent on substrate concentration.

• Michaelis-Menten kinetics:
  V = {V{max} [S]}{Km + [S]}

• The reaction is controlled by the preceding step in a pathway.

• This control is passive.

• If substrate concentration [S] is low (e.g., [S] < Km), then V < V{max}, and the reaction velocity V is controlled by [S].

3. Fine Control - Enzyme Linked Inhibitors

Types of Inhibitors

• Irreversible: Cannot be removed.

• Reversible: Can be removed.

• Reversible inhibitors are the basis of most drug action.

General Properties

• Chemicals that reduce the rate of enzymatic reactions.

• Usually specific and work at low concentrations.

• Block the enzyme but do not usually destroy it.

4. Irreversible Inhibition

Covalent Modification

• Usually occurs at the active site.

• Often toxic.

• The net effect is to remove the enzyme from the reaction.

• Reaction scheme:
  E + S \rightleftharpoons ES \longrightarrow E + P
  + I
  \downarrow
  EI

Example: Diisopropylphosphofluoridate

• Prototype for the nerve gas sarin.

• Modifies serine in acetylcholinesterase.

• Forms a covalent bond with the active site serine.

5. Reversible Inhibition

Noncovalent Interactions

a) Competitive Inhibition

• Inhibitors resemble the enzyme's substrate.

• Bind in the active site but do not react.

• Excess substrate can overcome this type of inhibition.

Example: Succinate Dehydrogenase

• Succinate is converted to fumarate.

• Malonate, due to its structural similarity to succinate, acts as a competitive inhibitor.

  • Succinate: COO- - CH2 - CH2 - COO-

  • Fumarate: \text{COO}^- - \text{CH} = \text{CH} - \text{COO}^-

  • Malonate: \text{COO}^- - \text{CH}_2 - \text{COO}^-

• A large excess of succinate (substrate) overcomes the competitive inhibition by malonate.

• Kinetic Effects:

  • V_{max} is unchanged.

  • K_M is increased.

b) Non-Competitive Inhibition

• The enzyme has two sites:

  • The active site.

  • An inhibitor site (allosteric site).

• Inhibitor binding does not prevent substrate binding in the active site but stops enzyme activity.

• Allostery is involved.

Example: Fructose 1,6-bisphosphatase

• Inhibited non-competitively by AMP.

  • High AMP indicates the cell needs energy and should metabolize glucose (ATP) instead of synthesizing it (consuming ATP).

• Kinetic Effects:

  • K_m remains the same.

  • V_{max} is reduced.

Kinetic Scheme

E + S \rightleftharpoons ES \longrightarrow E + P
+ I
\downarrow
EI + S \rightleftharpoons EIS

6. Inhibition in the Regulation of Pathway Flux

Feedback Regulation

Linear Pathways

• End product controls its own rate of production.

• Prevents buildup of intermediates.

• Limits overproduction.

• Typically affects the first, reversible step.
  A \longrightarrow B \longrightarrow C \longrightarrow D \longrightarrow E

• If [E] accumulates, it inhibits the conversion of A to B.

Example: Isoleucine Synthesis

• In E. coli, threonine deaminase is feedback inhibited by isoleucine.

• As isoleucine accumulates, it allosterically inhibits the enzyme for the first step of the pathway.

Branched Pathways

• Biosynthetic pathways often have branches leading to multiple end products.

• There can be differential requirements for the end products.

Sequential Feedback Inhibition

• Each end product inhibits its own branch.
  A \longrightarrow B \longrightarrow C \longrightarrow D
  \searrow
  E
  F \longrightarrow G

Nested Feedback Inhibition

• More complex regulation involving multiple inhibitors affecting multiple enzymes.
  A \longrightarrow B \longrightarrow C \longrightarrow D
  \searrow
  E
  F \longrightarrow G

Multiple Enzyme Mechanisms

Isoenzymes

• Different molecular forms of an enzyme catalyzing the same reaction.

  • Regulatory properties (e.g., K_m).

  • Cofactor requirements.

  • Localization.

• Arise due to:

  • Genetic differences.

  • Post-translational modification.

  • Multi-subunit proteins.

Isoenzymes in Complex Branched Pathways

• Can mediate feedback inhibition.

• Example: Aspartokinase in E. coli

  • Synthesis of threonine, methionine, and lysine from aspartate.

  • Three distinct forms of aspartokinase, each with its own mode of allosteric regulation.

7. Allosteric Regulation

Allostery

• Allos: other.

• steros: shape.

• Multi-subunit proteins with multiple active sites.

  • Homoallostery: Cooperative substrate binding.

  • Heteroallostery: Regulation by effector molecules.

Homoallostery

• Sigmoidal kinetic curves.

• Binding of a substrate to one active site can affect the properties of other sites in the same enzyme molecule, leading to cooperative substrate binding.

Allosteric Effectors (Modulators)

• Generally small chemicals.

• Can be positive (improve catalysis) or negative (reduce catalysis).

• Leads to Heteroallostery

Kinetics of Allosteric Regulators

• Differ from Michaelis-Menten kinetics.

• Involve T state (low activity) and R state (high activity).

Example: Aspartate Carbamoyltransferase (ATCase)

• Allosteric regulation.

• Catalyzes the "commitment" step in pyrimidine biosynthesis.
  Aspartate + Carbamoyl \ Phosphate \longrightarrow Carbamoyl \ Aspartate \longrightarrow Pyrimidines

• Inhibited by CTP and activated by ATP.

• CTP shifts the enzyme towards the inactive T state, while ATP shifts it towards the active R state.

8. Regulation by Covalent Modification

General Characteristics

• Reversible or irreversible.

• Play important roles in regulating enzyme function.

• Require expenditure of energy.

• Often used in signaling.

Examples

• Acetylation of lysine or amino terminal groups.

• Methylation of glutamate or aspartate residues.

• Nucleotidylation of tyrosine residues.

• ADP ribosylation primarily of arginine residues.

• Phosphorylation (major covalent modification).

Kinases and Phosphatases

• Two major classes:

  • Specificity for Ser and Thr residues.

  • Specificity for Tyr residues.

Effects of Phosphorylation

• Introduction of a bulky, charged group (phosphate) can:

  • Alter hydrogen bonds.

  • Introduce a negative charge.

  • Change enzyme conformation, substrate binding and catalysis.

• Kinase specificity is determined by consensus sequences around the phosphorylation site.

Example: Glycogen Phosphorylase

• Glycogen is converted to glucose-1-phosphate by glycogen phosphorylase.

• Epinephrine (adrenaline) activates glycogen phosphorylase.

• Exists as a dimer with two interconvertible forms:

  • Active: phosphorylase a.

  • Inactive: phosphorylase b.

• Allosteric activation by AMP.

Nucleotidylation

• Addition of:

  • AMP (adenylation).

  • UMP (uridylation).

• Involved in:

  • Assimilation of ammonia.

  • Biogenesis of organic nitrogen.

  • Carbamoyl Phosphate \longrightarrow Arg, Pyrimidines, Urea

  • Asp, Glu \longrightarrow Gln

  • $\longrightarrow$ Purines, Trp, His, Nucleotides, Amino sugars

9. Glutamate Dehydrogenase

Function

• Reductive amination of α-ketoglutarate (a-KG).

• Reversible; involved in:

  • Assimilation of ammonia.

  • a-KG --> citric acid cycle.

Allosteric Regulation

• Synthesis of a-KG:

  • Inhibited by ATP or GTP.

  • Stimulated by ADP or GDP.

10. Glutamine Synthetase

Reaction

• Glutamate + NH_3 + ATP \longrightarrow Glutamine + ADP + Pi

Regulation

• Central role in nitrogen metabolism.

• Involved in:

  • Biosynthesis of amino acids.

  • Purines and pyrimidines.

  • Detoxification of ammonia.

  • Supply of a-KG.

• Tightly regulated via:

  • Allosteric regulation (cumulative feedback inhibition).

  • Covalent modification (adenylation).

Feedback Inhibition

• 8 specific inhibitors.

• GS has 12 subunits, each with 8 binding sites (96 allosteric effector sites).

Nucleotidylation of Glutamine Synthetase

• Regulated by adenylation.

• Specific Tyr residue (Tyr-397, near the active site) is modified.

• AMP is transferred from ATP, inactivating GS.

• From 1 to 12 GS monomers in the holoenzyme can be modified, leading to progressive inactivation as the ratio of modified to unmodified GS subunits increases.

Adenylation and Deadenylation

• Catalyzed by the same enzyme: adenylyl transferase.

• Regulatory protein: PII.

• Uridylation of PII determines whether the complex catalyzes adenylation or deadenylation:

  • PII – UMP \longrightarrow Deadenylation.

  • PII \longrightarrow Adenylation.

• Responsive: Activated when nitrogen (glutamine) is high, stimulating adenylation and inhibiting GS activity. When nitrogen is low α-ketoglutarate accumulates stimulating GS activity.

Summary of Lectures 1 & 2

1. Coarse and fine control.

2. Substrate-linked control.

3. Enzyme-linked control (inhibitors).

4. Feedback regulation:

   • Linear pathways.

   • Branched pathways:

     • Sequential.

     • Nested (single, isoenzymes).

5. Allosteric regulation.

6. Regulation by covalent modification.