<p>Proteases are defined by their nucleophile but require additional residues to regulate their active sites, most often arranged as catalytic triads that control the generation and resolution of acyl-enzyme intermediates. Threonine N-terminal nucleophiles represent a diverse family of proteases and transferases that possess two active site nucleophiles, the side chain hydroxyl and the free amino-terminus, and require autocatalytic cleavage of their N-terminal propeptides. Here we provide evidence that the proteasome, which mediates intracellular protein degradation and contains three different threonine protease subunits, utilizes a unique catalytic pentad mechanism. In addition to the previously defined lysine/aspartate pair which regulates threonine’s side chain, a second serine/aspartate pair appears to regulate threonine’s amino-terminus. The pentad is required for substrate proteolysis and assembly-coupled autocatalytic cleavage, the latter triggered by alignment of the full pentad upon fusion of two half-proteasome precursors. A similar pentad mechanism was required by the ornithine acetyltransferase Arg7, suggesting that this may be a general property of threonine N-terminal nucleophiles. Finally, we show that two patient-derived proteasome mutations compromise function of the serine/aspartate unit in yeast, suggesting that defective pentad function may underlie some human proteasomopathies.</p>