Mechanism-Based Inactivation of Ammonia Monooxygenase in Nitrosomonas europaea by Allylsulfide

Abstract

Allylsulfide caused an irreversible inactivation of ammonia monooxygenase (AMO) activity (ammoniadependent 02 uptake) in Nitrosomonas europaea. The hydroxylamine oxidoreductase activity (hydrazinedependent 02 uptake) of cells was unaffected by allylsulfide. Anaerobic conditions or the presence of allylthiourea, a reversible noncompetitive AMO inhibitor, protected AMO from inactivation by allylsulfide. Ammonia did not protect AMO from inactivation by allylsulfide but instead increased the rate of inactivation. The inactivation of AMO followed pseudo-first-order kinetics, but the observed rates did not saturate with increasing allylsulfide concentrations. The time course of recovery of AMO-dependent nitrite production after complete inactivation by allylsulfide required de novo protein synthesis. Incubation of cells with allylsulfide prevented the 14C label from 14C2H2 (a suicide mechanism-based inactivator of AMO) from being incorporated into the 27-kDa polypeptide of AMO. Some compounds structurally related to allylsulfide were unable to inactivate AMO. We conclude that allylsulfide is a specific, mechanism-based inactivator of AMO in N. europaea.

Scope and Strategies for Regulation of Nitrification in Agricultural Systems—Challenges and Opportunities

“…Competitive inhibitors are substrates that utilize the same binding site as NH 3 . Keener and Arp (1993) proposed NH 3 - Juliette et al, 1993 binding sites to which competitive inhibitors bind and also an alternative binding site (such as for O 2 and another for electron donation) to which noncompetitive inhibitors bind. It has been hypothesized that the noncompetitive binding site of AMO is likely to be a hydrophobic region, which is not well-defined as indicated by the wide structural diversity of noncompetitive inhibitors affecting ammonia oxidation (Keener and Arp, 1993).…”

“…With mechanism-based inhibition causing irreversible damage to AMO activity (i.e., loss of enzyme for Nitrosomonas), de novo synthesis of this enzyme is required for Nitrosomonas to resume metabolic activity (Hyman and Wood, 1985). Allylsulfide, also a suicidal inhibitor, whose oxidation product by AMO is highly unstable and rapidly binds to AMO, damages irreversibly the enzyme function (Juliette et al, 1993). Several S-containing amino acids have an inhibitory effect on AMO, through the formation of volatile S compounds such as carbon disulfide (CS 2 ).…”

Oxidation of methyl fluoride and dimethyl ether by ammonia monooxygenase in Nitrosomonas europaea

“…The alternative approach to specific substrate inhibitors of AMO such as MF or DME is the use of mechanism-based inactivators of AMO. These include n-alkynes (6, 8) and allylsulfide (9). Many of these compounds are gaseous and are not transformed significantly by AMO.…”

Effect of Application Solvents on Heterotrophic and Nitrifying Populations in Soil Microcosms

“…16, 1997 451 includes the halogenated methanes and ethanes. Other recent work has suggested that AMO inhibition can be both specific and nonspecific [17]. Specific AMO inhibition occurs when the AMO enzyme is irreversibly bound by a chlorinated organic, while nonspecific inhibition is the result of AMO oxidation of a chlorinated organic that leads to the formation of intermediates that are toxic to cell constituents.…”