title: A noncanonical cysteine protease USP1 is activated through active site modulation by USP1-associated factor 1.
abstract: Ubiquitin-specific proteases (USPs) constitute the largest family of the human deubiquitinating enzymes. USP1 belongs to the cysteine protease family and contains a catalytic triad comprised of C90, H593, and D751. Notably, the catalytic activity of USP1 is stimulated through the formation of a tight complex with a WD40 repeat protein UAF1 (USP1-associated factor 1). Our kinetic analyses revealed a general base catalysis in USP1/UAF1, in contrast to an ion-pair mechanism as demonstrated for papain and cathepsin. The pK(a) value of the catalytic cysteine was determined to be 8.67 ± 0.07 in a pH-dependent inactivation study of USP1/UAF1 by iodoacetamide. A normal solvent kinetic isotope effect of 2.8 for k(cat) and 3.0 for k(cat)/K(m) was observed in the USP1/UAF1-catalyzed hydrolysis of ubiquitin-AMC substrate. Moreover, proton inventory analysis supported the transfer of a single solvent-derived proton in the transition state. Our study also revealed the molecular basis for the activation of USP1 by UAF1. Although the pK(a) of the catalytic cysteine in USP1 and USP1/UAF1 was almost identical, the pK(a) of the catalytic histidine in USP1/UAF1 was 0.43 pH unit lower than that in USP1, which facilitates general base catalysis at a neutral pH and contributes to the elevated catalytic efficiency. We ruled out that the higher catalytic efficiency is due to a tighter binding of ubiquitin. Our results support a regulatory mechanism in which UAF1 activates USP1 by modulating its active site conformation. This finding has a general implication for the regulation of USPs that form complex with partner proteins.
authors.affiliation: Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, United States.
authors.forename: Mark A
authors.initials: MA
authors.lastname: Villamil
authors.forename: Junjun
authors.initials: J
authors.lastname: Chen
authors.forename: Qin
authors.initials: Q
authors.lastname: Liang
authors.forename: Zhihao
authors.initials: Z
authors.lastname: Zhuang
mesh_terms: D055162:Biocatalysis; D020134:Catalytic Domain; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012965:Sodium Chloride / Q000737:chemistry; D025801:Ubiquitin / Q000494:pharmacology
chemical_list: D009687:Nuclear Proteins; D012965:Sodium Chloride; D025801:Ubiquitin; C526165:WDR48 protein, human