Mechanistic studies of the protonolytic carbon-mercury bond cleavage by organomercurial lyase from Escherichia coli (R831) suggest that the reaction proceeds via an SE2 pathway. Studies with stereochemically defined substrates cis-2-butenyl-2-mercuric chloride (1) and endo-norbornyl-2-mercuric bromide (2) reveal that a high degree of configurational retention occurs during the bond cleavage, while studies with exo-3-acetoxynortricyclyl-5-mercuric bromide (3) and cis-exo-2-acetoxy-bicyclo[2.2.1]hept-5-enyl-3-mercuric bromide (4) show that the protonolysis proceeds without accompanying skeletal rearrangement. Kinetic data for the enzymatic reactions of cis-2-butenyl-2-mercuric chloride (1) and trans-1-propenyl-1-mercuric chloride (6) indicate that these substrates show enhanced reaction rates of ca. 10-200-fold over alkylvinylmercurials and unsubstituted vinylmercurials, suggesting that the olefinic methyl substituent may stabilize an intermediate bearing some positive charge. Enzymatic reaction of 2-butenyl-1-mercuric bromide (5) yields a 72/23/5 mixture of 1-butene/trans-2-butene/cis-2-butene, indicative of intervening SE2' cleavage. The observation of significant solvent deuterium isotope effects at pH 7.4 of Vmax (H2O)/Vmax(D2O) = 2.1 for cis-2-butenyl-2-mercuric chloride (1) turnover and Vmax(H2O)/Vmax(D2O) = 4.9 for ethylmercuric chloride turnover provides additional support for a kinetically important proton delivery. Finally, the stoichiometric formation of butene and Hg(II) from 1 and methane and Hg(II) from methylmercuric chloride eliminates the possibility of an SN1 solvolytic mechanism. As the first well-characterized enzymatic reaction of an organometallic substrate and the first example of an enzyme-mediated SE2 reaction the organomercurial lyase catalyzed carbon-mercury bond cleavage provides an arena for investigating novel enzyme structure-function relationships.