Mechanism of Inhibition of Aliphatic Epoxide Carboxylation by the Coenzyme M Analog 2-Bromoethanesulfonate

Abstract

The bacterial metabolism of epoxypropane formed from propylene oxidation uses the atypical cofactor coenzyme M (CoM, 2-mercaptoethanesulfonate) as the nucleophile for epoxide ring opening and as a carrier of intermediates that undergo dehydrogenation, reductive cleavage, and carboxylation to form acetoacetate in a three-step metabolic pathway. 2-Ketopropyl-CoM carboxylase/oxidoreductase (2-KPCC), the terminal enzyme of this pathway, is the only known member of the disulfide oxidoreductase family of enzymes that is a carboxylase. In the present work, the CoM analog 2-bromoethanesulfonate (BES) is shown to be a reversible inhibitor of 2-KPCC and hydroxypropyl-CoM dehydrogenase but not of epoxyalkane:CoM transferase. Further investigations revealed that BES is a time-dependent inactivator of dithiothreitol-reduced 2-KPCC, where the redox active cysteines are in the free thiol forms. BES did not inactivate air-oxidized 2-KPCC, where the redox active cysteine pair is in the disulfide form. The inactivation of 2-KPCC exhibited saturation kinetics, and CoM slowed the rate of inactivation. Mass spectral analysis demonstrated that BES inactivation of reduced 2-KPCC occurs with covalent modification of the interchange thiol (Cys 82 ) by a group with a molecular mass identical to that of ethylsulfonate. The flavin thiol Cys 87 was not alkylated by BES under reducing conditions, and no amino acid residues were modified by BES in the oxidized enzyme. The UVvisible spectrum of BES-modifed 2-KPCC showed the characteristic charge transfer absorbance expected with alkylation at Cys 82 . These results identify BES as a reactive CoM analog that specifically alkylates the interchange thiol that facilitates thioether bond cleavage and enolacetone formation during catalysis.The bacterial metabolism of epoxides formed from shortchain aliphatic alkene epoxidation occurs by a three-step metabolic pathway that uses the atypical cofactor coenzyme M (CoM, 2-mercaptoethanesulfonate) 3 as a nucleophile for epoxide ring opening and as a carrier of intermediates that undergo dehydrogenation, reductive cleavage, and carboxylation (Fig.

Roles of the Redox-Active Disulfide and Histidine Residues Forming a Catalytic Dyad in Reactions Catalyzed by 2-Ketopropyl Coenzyme M Oxidoreductase/Carboxylase

“…Purified 2-KPCC containing the HP tag from different enzyme preparations exhibited specific activities consistently in the range of 50 to 55 nmol of acetone produced/min/mg when assayed according to equation 4. This activity is in the same range as that measured for acetone formation with preparations of native 2-KPCC under identical assay conditions (note that rates of 2-KPC carboxylation and protonation when using DTT are 60 to 65% of the corresponding rates when NADPH is used as the reductant and that the rate of 2-KPC protonation is about 25% of the rate of 2-KPC carboxylation) (8,10). Removal of the HP tag resulted in no change in 2-KPCC activity.…”

“…In comparison, the apparent K m and V max values for native 2-KPCC determined previously by the discontinuous assay were 0.63 mM and 412 nmol/min/ mg, respectively (10). The difference in V max to that reported here can be attributed to the use of DTT as the reductant in the discontinuous assay, which results in rates about 60% of the rates with the physiological reductant NADPH (8,10).…”

“…Recently, the CoM analog 2-bromoethanesulfonate was shown to be a highly specific affinity label for 2-KPCC that specifically alkylates the interchange thiol (C82) of 2-KPCC (8). As shown in Fig.…”

“…The biochemical and kinetic properties of r-2-KPCC containing the HP tag show that it is essentially identical to that of native 2-KPCC and thus suitable for construction of, and comparative studies with, sitedirected variants. The characterization of five of these sitedirected variants has allowed key tenets of the mechanism of thioether bond cleavage and substrate carboxylation proposed from mechanistic (8,10) and structural (28,30,31) studies to be tested.…”

“…Samples of native 2-KPCC (3.6 mg/ml) were incubated anoxically in the presence of 10 mM DTT and 10 mM 2-bromoethanesulfonate (BES) for 4 h as described previously (8). The samples were then desalted using prepacked columns of Sephadex G-25 (PD-10; Pharmacia) equilibrated in 50 mM Tris buffer (pH 7.4).…”