Inactivation of toluene 2-monooxygenase in Burkholderia cepacia G4 by alkynes

High concentrations of acetylene (10 to 50% [vol/vol] gas phase) were requi red to inhibit the growth of Burkholderia cepacia G4 on toluene, while 1% ( vol/vol) (gas phase) propyne or 1-butyne completely inhibited growth. Low c oncentrations of longer-chain alkynes (C-5 to C-10) were also effective inh ibitors of toluene-dependent growth, and 2- and 3-alkynes were more potent inhibitors than their 1-alkyne counterparts. Exposure of toluene-grown B. c epacia G4 to alkynes resulted in the irreversible loss of toluene- and o-cr esol-dependent O-2 uptake activities, while acetate- and 3-methylcatechol-d ependent O-2 uptake activities were unaffected. Toluene-dependent O-2 uptak e decreased upon the addition of 1-butyne in a concentration- and time-depe ndent manner. The loss of activity followed first-order kinetics, with appa rent rate constants ranging from 0.25 min(-1) to 2.45 min(-1). Increasing c oncentrations of toluene afforded protection from the inhibitory effects of 1-butyne. Furthermore, oxygen, supplied as H2O2, was required for inhibiti on by 1-butyne. These results suggest that alkynes are specific, mechanism- based inactivators of toluene 2-monooxygenase in B. cepacia G4, although th e simplest alkyne, acetylene, was relatively ineffective compared to longer alkynes. Alkene analogs of acetylene and propyne-ethylene and propylene-we re not inactivators of toluene 2-monooxygenase activity in B. cepacia G4 bu t were oxidized to their respective epoxides, with apparent K-s and V-max v alues of 39.7 mu M and 112.3 nmol min(-1) mg of protein(-1) for ethylene an d 32.3 mu M and 89.2 nmol min(-1) mg of protein(-1) for propylene.