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Publication Abstract from PubMed

Enzymatic cleavage of transmembrane anchors to release proteins from the membrane controls diverse signaling pathways and is implicated in more than a dozen diseases. How catalysis works within the viscous, water-excluding, two-dimensional membrane is unknown. We developed an inducible reconstitution system to interrogate rhomboid proteolysis quantitatively within the membrane in real time. Remarkably, rhomboid proteases displayed no physiological affinity for substrates (Kd approximately 190 muM/0.1 mol%). Instead, approximately 10,000-fold differences in proteolytic efficiency with substrate mutants and diverse rhomboid proteases were reflected in kcat values alone. Analysis of gate-open mutant and solvent isotope effects revealed that substrate gating, not hydrolysis, is rate limiting. Ultimately, a single proteolytic event within the membrane normally takes minutes. Rhomboid intramembrane proteolysis is thus a slow, kinetically controlled reaction not driven by transmembrane protein-protein affinity. These properties are unlike those of other studied proteases or membrane proteins but are strikingly reminiscent of one subset of DNA-repair enzymes, raising important mechanistic and drug-design implications.

Proteolysis inside the Membrane Is a Rate-Governed Reaction Not Driven by Substrate Affinity.,Dickey SW, Baker RP, Cho S, Urban S Cell. 2013 Dec 5;155(6):1270-81. doi: 10.1016/j.cell.2013.10.053. PMID:24315097^[3]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.