Active site generation of a protonically unstable suicide substrate from astable precursor: Glucose oxidase and dibromonitromethane

Bromonitromethane is an inefficient suicide substrate for glucose oxidase ( in contrast to the case of CH3CCl=NO2- and D-amino acid oxidase) because, i n the enzyme-substrate encounter step, the required ionization states of en zyme (EH0+, pK(a) similar to 3.5) and substrate (CHBr=NO2-, pK(a) similar t o 8.3) cannot be highly populated simultaneously. Because reprotonation of CHBr-NO2- is rapid at the pH value used for the assay of glucose oxidase, p resentation of the enzyme with the preformed anion could not be exploited i n this case. We circumvent this difficulty by allowing the enzyme to reduct ively dehalogenate CHBr2-NO2, thereby generating the desired protonically u nstable suicide substrate in situ (E-r + CHBr2NO2 --> E-o + CHBr=NO2- + HBr + Hf). Irreversible inactivation of the enzyme, because of the formation o f a dead-end N-5 formylflavin adduct, is more than 100-fold faster when CHB r=NO2- is generated in situ than when it is externally applied. The remaini ng competitive fates of CHBr=NO2- at the active site are protonation and re lease or oxidation to HCOBr (or HCONO2). Strong support for these conclusio ns comes from (1) the brisk evolution of CH3CBr=NO2- (which is too bulky to act further as an efficient suicide substrate) from the enzyme-catalyzed r eductive debromination of CH3CBr2NO2, (2) the 1:1 stoichiometry of enzyme i nactivation, and (3) the identification of the modified flavin as 5-formyl- 1,5-dihydro-FAD.