Figure 5.

Inhibition of S. pneumoniae MTAN by cHexyl-DADMe-ImmA [23], an example of data analysis for a slow-onset, tight-binding DADMe-Immucillin derivative. The reaction rate and slow-onset inhibition was monitored by the conversion of MTA [17] to 2,8-dihydroxyadenine at 293 nm in a coupled reaction with xanthine oxidase (Upper panel). The coupled assay permits use of high substrate concentration (2.0 mM, 87 × K_m for MTA) to compete against these powerful inhibitors. Control experiments demonstrate that Immucillins do not inhibit xanthine oxidase. Values of K_i (middle panel) and K_i* (lower panel) were obtained from the initial (0 to 5 min) and the final rates (25 to 30 min). Similar experiments were used to measure inhibition by slow-onset tight-binding inhibitors shown in Figure 3 and 4.