Contribution of active site glutamine to rate enhancement in ubiquitin C-terminal hydrolases.

摘要: Ubiquitin carboxy terminal hydrolases (UCHs) are cysteine proteases featuring a classical cysteine-histidine-aspartate catalytic triad, also highly conserved glutamine thought to be part of the oxyanion hole. However, contribution this side chain catalysis by UCH enzymes is not known. Herein, we demonstrate that contributes rate enhancement in UCHL1, UCHL3 and UCHL5. Mutation alanine these impairs efficiency mainly due 16 30-fold reduction kcat, which consistent with loss approximately 2 kcal/mol transition-state stabilization. stabilization observed here rather modest for chain’s role Interestingly, discovered carbonyl oxygen engaged C—H•••O hydrogen-bonding contact CeH group histidine. Upon further analysis, found interaction common active-site structural feature most proteases, including papain, belonging families QCH(N/D) type configuration. It possible removal might have abolished interaction, typically accounts stabilization, leading effect on here. Additional studies performed mutating glutamate (strong acceptor but destabilizer) lysine stabilizer lacking property) suggest hydrogen bond could contribute catalysis.