Fig. 4: A catalytic pentad is required for autocatalytic activation and proteolysis by β1. | Nature Communications

Fig. 4: A catalytic pentad is required for autocatalytic activation and proteolysis by β1.

From: Evidence supporting a catalytic pentad mechanism for the proteasome and other N-terminal nucleophile enzymes

Fig. 4: A catalytic pentad is required for autocatalytic activation and proteolysis by β1.The alternative text for this image may have been generated using AI.

a Structural arrangement of the proposed pentad residues as seen in mature wild-type CP (PDB: 5CZ4). Lys52, Asp36, and Thr20 represent the canonical catalytic triad, while Ser148 and Asp185 represent additional residues proposed to complete the catalytic pentad. Dashed lines, hydrogen bonds. b Phenotypic analysis of the indicated mutants, expressed from low-copy centromeric plasmids harboring the endogenous promoter/terminator elements in a strain where endogenous β1 is under the control of the pGAL1 promoter. In glucose-containing media, expression of the endogenous β1 locus is repressed and plasmid-derived β1 is the sole source of this protein. Plates were cultured at 30 °C for 2 (no drug) and 7 days (canavanine, 1.5 μg/mL). c Analysis of wild-type and mutant CP by native gel electrophoresis followed by immunoblotting with antibodies against α5, immature (propeptide-bearing) β5, and Pba1/2. d Proteasome activity assays using active site-specific fluorescent substrate probes. Background fluorescence has been subtracted and relative activity has been normalized to untreated controls. Individual points represent biologic duplicates. e Structures of the helix-loop-helix motif responsible for the spatial proximity of the Ser/Asp pair within the catalytic pentad (PDB: 5CZ4). Similar results were obtained in 2 independent experiments (b, c).

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