Figure 5. Human TRMT1 peptides are cleaved with similar catalytic efficiencies to known M^pro substrates.

A) Kinetics of nsp4/5, nsp8/9, and TRMT1 peptide cleavage by M^pro. To initiate the reaction, 50nM enzyme was added to 100–0.097 μM peptide. Each fluorogenic peptide was conjugated with a quenching moiety, and upon peptide cleavage, the fluorescence of the cleavage product was measured to determine initial rates of the reaction. Nsp4/5 cleavage rates were faster than those observed for the nsp8/9 or TRMT1 peptides, but nsp8/9 and TRMT1 sequences exhibit similar M^pro-mediated cleavage rates. B) The catalytic efficiency (k_cat/K_M) of TRMT1 peptide cleavage by M^pro is similar to that for nsp8/9 peptide cleavage; both of these substrates are cleaved significantly slower than the nsp4/5 sequence. This suggests that TRMT1 is a feasible substrate for M^pro. C) Illustration of changes in M^pro Met49, Asn142, and Gln189 residue positioning in TRMT1-bound (white) versus nsp4/5-bound (orange) structures. The TRMT1 peptide is shown in blue; nsp4/5 peptide is not shown. D) No major changes in catalytic efficiency are observed for nsp4/5 and TRMT1 peptide cleavage upon mutagenesis of key M^pro residues involved in TRMT1 binding and recognition. Primary fluorogenic kinetic data used to construct the plots and determine the kinetic parameters shown in A – D are listed in Dataset S1; numerical k_cat, K_M, and k_cat/K_M values are listed in Table S3.