Proteolysis inside the membrane is a rate-governed reaction not driven by substrate affinity

Area of Science:

• Membrane biology
• Enzymology
• Biochemistry

Background:

• Enzymatic cleavage of transmembrane anchors releases proteins, controlling signaling pathways and implicated in numerous diseases.
• The mechanism of intramembrane proteolysis within the lipid bilayer remains poorly understood.

Purpose of the Study:

• To quantitatively interrogate rhomboid proteolysis within the membrane in real time.
• To elucidate the kinetic and mechanistic features of intramembrane proteolysis.

Main Methods:

• Developed an inducible reconstitution system for real-time analysis of rhomboid proteolysis.
• Utilized substrate mutants and solvent isotope effects to analyze reaction kinetics.
• Investigated enzyme-substrate affinity and catalytic efficiency (kcat).

Main Results:

• Rhomboid proteases exhibit low physiological affinity for substrates (Kd ~190 microM).
• Proteolytic efficiency differences are primarily reflected in kcat values, not affinity.
• Substrate gating, not hydrolysis, was identified as the rate-limiting step.
• Individual proteolytic events within the membrane occur over minutes.

Conclusions:

• Rhomboid intramembrane proteolysis is a slow, kinetically controlled process, distinct from affinity-driven reactions.
• The mechanism shares similarities with certain DNA-repair enzymes, suggesting broader biological relevance.
• Findings offer insights for mechanistic understanding and potential drug design targeting intramembrane proteases.