RT Journal Article SR Electronic T1 Variety in the USP deubiquitinase catalytic mechanism JF Life Science Alliance JO Life Sci. Alliance FD Life Science Alliance LLC SP e202302533 DO 10.26508/lsa.202302533 VO 7 IS 4 A1 Keijzer, Niels A1 Priyanka, Anu A1 Stijf-Bultsma, Yvette A1 Fish, Alexander A1 Gersch, Malte A1 Sixma, Titia K YR 2024 UL https://www.life-science-alliance.org/content/7/4/e202302533.abstract AB The ubiquitin-specific protease (USP) family of deubiquitinases (DUBs) controls cellular ubiquitin-dependent signaling events. This generates therapeutic potential, with active-site inhibitors in preclinical and clinical studies. Understanding of the USP active site is primarily guided by USP7 data, where the catalytic triad consists of cysteine, histidine, and a third residue (third critical residue), which polarizes the histidine through a hydrogen bond. A conserved aspartate (fourth critical residue) is directly adjacent to this third critical residue. Although both critical residues accommodate catalysis in USP2, these residues have not been comprehensively investigated in other USPs. Here, we quantitatively investigate their roles in five USPs. Although USP7 relies on the third critical residue for catalysis, this residue is dispensable in USP1, USP15, USP40, and USP48, where the fourth critical residue is vital instead. Furthermore, these residues vary in importance for nucleophilic attack. The diverging catalytic mechanisms of USP1 and USP7 are independent of substrate and retained in cells for USP1. This unexpected variety of catalytic mechanisms in this well-conserved protein family may generate opportunities for selective targeting of individual USPs.