Evidence supporting a catalytic pentad mechanism for the proteasome and other N-terminal nucleophile enzymes

Darlene Fung, Aida Razi, Michael Pandos, Benjamin Velez, Erignacio Fermin Perez, Lea Adams, Shaun Rawson, Richard M. Walsh and John Hanna ()
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Darlene Fung: Harvard Medical School and Brigham and Women’s Hospital
Aida Razi: Harvard Medical School and Brigham and Women’s Hospital
Michael Pandos: Harvard Medical School and Brigham and Women’s Hospital
Benjamin Velez: Harvard Medical School and Brigham and Women’s Hospital
Erignacio Fermin Perez: Harvard Medical School and Brigham and Women’s Hospital
Lea Adams: Harvard Medical School and Brigham and Women’s Hospital
Shaun Rawson: Harvard Medical School
Richard M. Walsh: Harvard Medical School
John Hanna: Harvard Medical School and Brigham and Women’s Hospital

Nature Communications, 2025, vol. 16, issue 1, 1-12

Abstract: Abstract 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.

Date: 2025
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DOI: 10.1038/s41467-025-58077-x

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