(a) Mass spectra of tryptic N-terminal heptapeptides from Mtb proteasomes that were untreated (i), HT1171-treated (ii), treated with glutaraldehyde/Na(CN)BH3 after trypsin digestion (iii), or treated with glutaraldehyde/Na(CN)BH3 after HT1171 treatment and trypsin digestion (iv). All ions were confirmed by MS/MS analysis (Fig. S7a–d). Reaction equations illustrate proposed modification of active site Thr1 by oxathiazol-2-one (i
ii), and modification of primary amino groups at Thr1 and Lys7 with glutaraldehyde and Na(CN)BH3 (i
iii and ii
iv). (b) Proposed mechanism of proteasome inactivation by oxathiazol-2-one. Paths marked by a, b, d lead to irreversible inhibition. In paths marked by c, hydrolysis of the inhibitor-enzyme intermediate allows the proteasome to degrade the oxathiazol-2-one without losing activity.