Figure 3: Architecture and proposed reaction mechanism of the proteasomal active site.

(a) Hydrogen-bonding network at the mature WT β5 proteasomal active site (dotted lines). Thr1OH is hydrogen-bonded to Lys33NH₂ (2.7 Å), which in turn interacts with Asp17O^δ. The Thr1 N terminus is engaged in hydrogen bonds with Ser129O^γ, the carbonyl oxygen of residue 168, Ser169O^γ and Asp166O^δ. (b) The orientations of the active-site residues involved in hydrogen bonding are strictly conserved in each proteolytic centre, as shown by superposition of the β subunits. (c) Structural superposition of the WT β5 and the β5-K33A pp trans mutant active site. In the latter, a water molecule (red sphere) is found at the position where in the WT structure the side chain amine group of Lys33 is located. Similarly to Lys33, the water molecule hydrogen bonds to Arg19O, Asp17O^δ and Thr1OH. Note, the strong interaction with the water molecule causes a minor shift of Thr1, while all other active-site residues remain in place. (d) Proposed chemical reaction mechanism for autocatalytic precursor processing and proteolysis in the proteasome. The active-site Thr1 is depicted in blue, the propeptide segment and the peptide substrate are coloured in green, whereas the scissile peptide bond is highlighted in red. Autolysis (left set of structures) is initiated by deprotonation of Thr1OH via Lys33NH₂ and the formation of a tetrahedral transition state. The strictly conserved oxyanion hole Gly47NH stabilizing the negatively charged intermediate is illustrated as a semicircle. Collapse of the transition state frees the Thr1 N terminus (by completing an N-to-O acyl shift of the propeptide), which is subsequently protonated by Asp166OH via Ser129OH. Next, Thr1NH₂ polarizes a water molecule for the nucleophilic attack of the acyl-enzyme intermediate. On hydrolysis of the latter, the active-site Thr1 is ready for catalysis (right set of structures). Substrate processing starts with nucleophilic attack of the carbonyl carbon atom of the scissile peptide bond. The charged Thr1 N terminus may engage in the orientation of the amide moiety and donate a proton to the emerging N terminus of the C-terminal cleavage product. The resulting deprotonated Thr1NH₂ finally activates a water molecule for hydrolysis of the acyl-enzyme.