Enzyme Regulation: Allosteric Control, Covalent Modification, and Zymogen Activation

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Enzyme Regulation

Key Concepts in Regulation

Enzyme regulation is essential for controlling metabolic pathways and cellular processes. The main mechanisms include allosteric control, reversible covalent modification, and proteolytic activation.

Allosteric Control: The activity of some proteins is modulated by small signaling molecules. Binding of these molecules induces conformational changes that alter enzyme activity.
Reversible Covalent Modification: Many proteins are regulated by the attachment of chemical groups, most commonly phosphorylation (addition of a phosphate group).
Proteolytic Activation: Some enzymes are synthesized as inactive precursors (zymogens) and activated by specific cleavage events.

Learning Objectives

Understand that allosteric enzymes do not follow Michaelis-Menten kinetics and exhibit sigmoidal velocity-substrate profiles.
Describe allosteric regulation in the ATCase system, including activation by substrate binding, feedback inhibition by CTP, and positive regulation by ATP.
Predict the qualitative effects of allosteric regulators on ATCase kinetic curves.
Recognize major forms of covalent modification, especially phosphorylation, and how it is modulated by kinases and phosphatases.
Explain proteolytic activation of zymogens, with examples such as chymotrypsin.

Allosteric Regulation

Mechanism and Properties

Allosteric enzymes typically consist of multiple subunits that influence each other's activity. Substrate binding at one site can affect the affinity at other sites, leading to cooperative conformational changes between less active (T state) and more active (R state) forms.

Small molecule regulators can bind to allosteric sites, shifting the equilibrium between T and R states to adjust enzyme activity.
Allosteric enzymes do not follow Michaelis-Menten kinetics; instead, they display sigmoidal (S-shaped) velocity vs. substrate concentration curves.

Example: Hemoglobin is a classic allosteric protein, but in metabolism, ATCase is a key example.

Aspartate Transcarbamoylase (ATCase)

Role in Pyrimidine Biosynthesis

Aspartate transcarbamoylase (ATCase) catalyzes the first committed step in the biosynthesis of pyrimidine nucleotides, such as CTP, which are essential for nucleic acids, energy storage, and enzyme cofactors.

Substrates: Carbamoyl phosphate and aspartate
Product: N-Carbamoylaspartate
End-product: Cytidine triphosphate (CTP)

Reaction:

Carbamoyl phosphate + Aspartate \xrightarrow{ATCase} N-Carbamoylaspartate + Pi

Quaternary Structure of ATCase

Subunit Composition

ATCase is a multimeric enzyme composed of two types of subunits:

c subunit (catalytic; 34 kD): Forms trimers when isolated
r subunit (regulatory; 17 kD): Forms dimers when isolated

The active complex is a dodecamer: C6R6 (six catalytic and six regulatory subunits).

Identification of Active Sites Using an Inhibitor

PALA as a Structural Mimic

The compound PALA (N-(Phosphonacetyl)-L-aspartate) is a structural analog of a reaction intermediate and acts as a potent inhibitor of ATCase. PALA binds between two catalytic subunits, identifying the location of the active site.

Bound substrates: Carbamoyl phosphate and aspartate
Reaction intermediate: N-Carbamoylaspartate
PALA: Mimics the transition state, binds tightly, and induces conformational changes

Example: X-ray crystallography shows PALA binding causes a shift from the less active T state to the more active R state, though PALA itself is inhibitory due to competitive binding.

Summary Table: Mechanisms of Enzyme Regulation

Mechanism	Description	Example
Allosteric Control	Regulation by small molecules binding at sites other than the active site, causing conformational changes	ATCase regulation by CTP (inhibition) and ATP (activation)
Reversible Covalent Modification	Attachment/removal of chemical groups (e.g., phosphorylation) to modulate activity	Phosphorylation of glycogen phosphorylase
Proteolytic Activation	Activation of enzymes by cleavage of inactive precursors (zymogens)	Activation of chymotrypsinogen to chymotrypsin

Key Equations

Allosteric Equilibrium Constant:
Free Energy Difference:

Additional info:

Allosteric enzymes are often regulated by both homotropic (substrate itself) and heterotropic (other molecules) effectors.
Feedback inhibition is a common regulatory strategy, as seen with CTP inhibiting ATCase.
Phosphorylation is catalyzed by kinases and reversed by phosphatases, allowing rapid and reversible control of enzyme activity.
Zymogen activation is irreversible and is crucial for processes such as digestion and blood clotting.