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


Ubiquitin C-terminal hydrolases (UCHs) are cysteine proteases featuring a classical Cys-His-Asp catalytic triad, and also a highly conserved Gln that is thought to be a part of the oxyanion hole. However, the contribution of this side chain to catalysis by UCHs is not known. Herein, we demonstrate that the Gln side chain contributes to rate enhancement in UCHL1, UCHL3, and UCHL5. Mutation of the Gln to Ala in these enzymes impairs the catalytic efficiency, mainly because of a 16-fold to 30-fold reduction in k(cat) , which is consistent with a loss of approximately 2 kcal·mol(-1) in transition state stabilization. However, the contribution to transition state stabilization observed here is rather modest for the side chain's role in oxyanion stabilization. Interestingly, we discovered that the carbonyl oxygen of this side chain is engaged in a C-H···O hydrogen-bonding contact with the CεH group of the catalytic His. Upon further analysis, we found that this interaction is a common active site structural feature in most cysteine proteases, including papain, belonging to families with the QCH(N/D) type of active site configuration. It is possible that removal of the Gln side chain might have abolished the C-H···O interaction, which typically accounts for 2 kcal·mol(-1) of stabilization, leading to the effect on catalysis observed here. Additional studies performed on UCHL3 by mutating the Gln to Glu (strong C-H···O acceptor but oxyanion destabilizer) and to Lys (strong oxyanion stabilizer but lacking C-H···O hydrogen-bonding capability) suggest that the C-H···O hydrogen bond could contribute to catalysis.

DOI: 10.1111/j.1742-4658.2012.08507.x
Citations per Year

695 Citations

Semantic Scholar estimates that this publication has 695 citations based on the available data.

See our FAQ for additional information.

Cite this paper

@article{Boudreaux2012ContributionOA, title={Contribution of active site glutamine to rate enhancement in ubiquitin C-terminal hydrolases.}, author={David Boudreaux and Joseph Rashon Chaney and Tushar Kanti Maiti and Chittaranjan Das}, journal={The FEBS journal}, year={2012}, volume={279 6}, pages={1106-18} }