FIGURE 1.

Schematic illustration of enzymes, substrates, and assay system. a, different composition of β subunits in eubacterial and eukaryotic proteasomes. The Mtb proteasome has seven identical β subunits, whereas eukaryotic proteasomes have seven different β subunits. Only three β subunits of eukaryotic proteasomes are active once their Thr¹ active sites are exposed by autocatalytic removal of propeptide: β1, caspase-like; β2, trypsin-like; and β5, chymotrypsin-like. Eubacterial proteasomes are generally considered to be chymotrypsin-like when assayed with small peptide substrates. b, structure of the acetyl-P3-P2-P1-AMC substrate library. R₁, R₂, and R₃ refers to the side chains of P₁, P₂, P₃ amino acids, respectively; and S₁, S₂, and S₃ refer to the binding pockets of the proteasome for P₁, P₂, and P₃ amino acid side chains, respectively. c, schematic illustration of microfluidic TF460 assay system. Stream splitting and simultaneous fluorescence detection facilitated subtraction of the background fluorescence of each unhydrolyzed substrate; after substrate in buffer was pulled into the channel through vacuum, the stream was split 50:50 into parallel channels and mixed with enzyme and buffer, respectively. The final annotated data were obtained by subtracting background fluorescence of the unhydrolyzed substrate from that of the enzymatic reaction.