Fig. (8) Inactivation of IleRS by bromomethyl ketones derived from amino acids.The enzyme (2 µM) was incubated at 37°C in 50 mM Hepes/Na (pH 7.8), with IBMK (■), NleBMK (+), VBMK ○ or FBMK (◆), at a final concentration of 2 mM. Control experiments (□) were carried out in the absence of reagent. Aliquots of the incubation mixtures were withdrawn, quenched by dilution with a buffer that contained 50 mM 2-mercaptoethanol, and assayed for the isoleucine-dependent isotopic [³²P]PPi-ATP exchange activity, as described under Methods. For the protection experiments, the enzyme was pre-incubated with 8 mM MgATP (▴) or 8 mM Lisoleucine (●), prior to IBMK addition. The combination of 8 mM MgATP and 8 mM L-isoleucine was as efficient as 8 mM Lisoleucine alone in the protection of IleRS against inactivation by IBMK. Pre-incubation of IleRS with 8 mM L-valine prior to IBMK addition did not affect the kinetics of inactivation of the synthetase by this cognate amino acid analog (■).Data are plotted as the percent of activity remaining versus incubation time.