Steric Course of the Reactions Catalyzed by 5-Enolpyruvylshikimate-3-phosphate Synthase, Chorismate Mutase, and Anthranilate Synthase

Article abstract of DOI:10.1021/ja00300a040

To probe the steric course of several reactions in the shikimate pathway, (1R,2R)-<1-(2)H,(3)H>glycerol was synthesized and fed to cultures of Klebsiella pneumoniae mutant 62-1.The accumulated chorismic acid, labeled from endogenously formed (E)-<3-(2)H,(3)H>phosphoenolpyruvate, was isolated and degraded by hydrogenation of the side-chain double bond and cleavage of the ether linkage to D,L-lactic-acid.Resolution of the latter and determination of the methyl group configuration in the two enantiomers demonstrated predominant E configuration of the <3'-(2)H,(3)H>chorismic acid.It follows that the addition and the elimination step in the 5-enolpyruvylshikimate-3-phosphate (ESP) synthase reaction proceed with opposite stereochemistry, i.e., anti/syn or syn/anti.Incubation of the above chorismic acid with anthranilate synthase produced <3'-(2)H,(3)H>pyruvate of predominantly S configuration, indicating stereospecific protonation on the re face of the side chain.Conversion of the (E)-<3'-(2)H,(3)H>chorismic acid into phenylalanine and tyrosine with whole cells of E. coli and configurational analysis of these amino acids by conversion into pyruvate carrying a chiral methyl group with phenylalanine hydroxylase and/or tyrosine phenol-lyase indicated that the chorismate mutase reaction proceeds via a chair transition state.