FIG 5.
Glutathionylation inhibits wild-type (WT) SARS-Cov-2 Mpro activity but not C300S Mpro activity. (A) Activity of WT and C300S Mpro (1 μM enzyme) following 30-min preincubation of 1.2 μM Mpro with 10 mM oxidized glutathione. (B) Mpro activity for a WT monoglutathionylated Mpro preparation that had excess GSSG removed (containing approximately 30% monoglutathionylated Mpro and 4% diglutathionylated) and a C300S monoglutathionylated Mpro preparation (containing approximately 18% monoglutathionylated Mpro) preincubated for 10 min without DTT (-DTT) or with 20 mM DTT (+DTT). The amount of monoglutathionylated Mpro was estimated using the relative abundances of native Mpro and glutathionylated Mpro following deconvolution of the eluting Mpro species from SEC/MALDI-TOF MS analysis. Values represent the averages ± standard deviations of three separate experiments (n = 3). Statistical significance: *, P value of <0.05; ns, not significant (P > 0.05).