Stereochemical course of the reaction catalyzed by the pyridoxal phosphate-dependent enzyme 1-aminocyclopropane-1-carboxylate synthase

Abstract

(+/-)-S-adenosyl-DL-(3R*,4S*)-[3,4-2H2]-methionine [a 1:1 mixture of (3R,4S) and (3S,4R)] and (+/-)-S-adenosyl-DL-(3R*,4R*)-[3,4-2H2]methionine [a 1:1 mixture of (3R,4R) and (3S,4S)] were synthesized from (Z)- and (E)-[1,2-2H2]ethene, respectively. Key steps in the synthesis were the antiperiplanar addition of methanesulfenyl chloride to (Z)-[1,2-2H2]ethene, to give a 1:1 mixture of (R,R)- and (S,S)-1-chloro-2-(methylthio)[1,2-2H2)ethane, followed by alkylation with sodium acetamidomalonate and hydrolysis to give an equal mixture of four stereoisomers of [3,4-2H2]methionine [(2R,3R,4S), (2R,3S,4R), (2S,3R,4S), and (2S,3S,4R)]. The other four stereoisomers of [3,4-2H2]methionine were prepared from (E)-(1,2-2H2]ethene. The two sets of stereoisomers of [3,4-2H2]methionine were chemically converted to S-adenosylhomocysteine, methylated to give the corresponding (+/-)-S-adenosyl-DL-methionines, and then incubated with 1-aminocyclopropane-1-carboxylate synthase partially purified from tomato (Lycopersicon esculentum, L.) pericarp tissue. The stereochemistry of the resulting samples of 1-aminocyclopropane-1-carboxylic acid was determined by comparison with the 1H NMR of the chemically synthesized and regio- and stereo-specifically deuterated compound. The results indicate that the hydrogens at the beta carbon of the methionine portion of S-adenosylmethionine do not participate in the reaction and that the ring closure occurs with inversion of configuration at the gamma carbon of the methionine portion of S-adenosyl-methionine, probably through a direct SN2-type displacement of the 5'-methylthio-5'-deoxyadenosine moiety by a carbanion equivalent formed at the alpha carbon of the methionine portion of S-adenosylmethionine.