Intramembrane proteases, in contrast to these well-studied soluble proteases, are a more recently-discovered class of extraordinary enzymes that evolved independently to catalyze hydrolysis immersed within the membrane ( De Strooper and Annaert, 2010 Fluhrer et al., 2009 Makinoshima and Glickman, 2006 Urban and Dickey, 2011 Wolfe, 2009). The catalytic efficiency of a protease is the quotient of these two parameters, and typically ranges from 10 4–10 7 M −1s −1 (10 8 reflects enzymes whose activity is limited by diffusion). Coupled with structural analyses, these studies have established that both cytosolic and extracellular proteases are designed to bind their substrates specifically at discrete sites, with affinity reflected in the Michaelis constant (K M), and endowed with catalytic residues that function in rate enhancement, reflected in the turnover number (k cat). Kinetic dissection of protease catalysis has been key in revealing these properties ( Huntington, 2012 Perona and Craik, 1997 Timmer et al., 2009). Ultimately deciphering how a protease shapes the signaling characteristics of healthy cells, or targeting it for therapeutic intervention in disease, requires a sophisticated understanding of its enzymatic properties. Aside from controlling essential processes in all forms of life, protease inhibition has proven to be a particularly effective therapeutic strategy, especially in hypertension and antiviral treatment ( Drag and Salvesen, 2010). The purpose of these enzymatic events ranges from shredding damaged proteins that might otherwise harm the cell, to sculpting signal precursors to initiate cell communication ( Lopez-Otin and Bond, 2008). These properties are unlike those of other studied proteases or membrane proteins but strikingly reminiscent of one subset of DNA-repair enzymes, raising important mechanistic and drug-design implications.Įach protein in a living cell will be cleaved by a protease ( Doucet et al., 2008 Lopez-Otin and Bond, 2008). Rhomboid intramembrane proteolysis is thus a slow, kinetically controlled reaction not driven by transmembrane protein-protein affinity. Ultimately a single proteolytic event within the membrane normally takes minutes. Analysis of gate-open mutant and solvent isotope effects revealed that substrate gating, not hydrolysis, is rate limiting. Instead, ~10,000-fold differences in proteolytic efficiency with substrate mutants and diverse rhomboid proteases were reflected in k cat values alone. ![]() ![]() ![]() Remarkably, rhomboid proteases displayed no physiological affinity for substrates (K d ~190 μM, or 0.1 mol%). We developed an inducible reconstitution system to interrogate rhomboid proteolysis quantitatively within the membrane in real time. How catalysis works within the viscous, water-excluding, two-dimensional membrane is unknown. Enzymatic cleavage of transmembrane anchors to release proteins from the membrane controls diverse signaling pathways and is implicated in over a dozen diseases.
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