Open AccessDirect demonstration of the isomerization component of the monoterpene cyclase reaction using a cyclopropylcarbinyl pyrophosphate substrate analog.
Author(s) -
Carl J. Wheeler,
Rodney Croteau
Publication year - 1987
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.84.14.4856
Subject(s) - pyrophosphate , monoterpene , chemistry , farnesyl pyrophosphate , isomerization , allylic rearrangement , isopentenyl pyrophosphate , substrate (aquarium) , cyclase , stereochemistry , substrate analog , catalysis , enzyme , biosynthesis , organic chemistry , active site , biology , ecology
The tightly coupled nature of the reaction sequence catalyzed by monoterpene cyclases has precluded direct observation of the topologically required isomerization step leading from geranyl pyrophosphate to the presumptive, enzyme-bound, tertiary allylic intermediate linalyl pyrophosphate, which ultimately cyclizes to the various monoterpene skeletons. By using a partially purified monoterpene cyclase preparation and 2,3-cyclopropylgeranyl pyrophosphate, a substrate analog designed to uncouple the reaction sequence, the production of the corresponding tertiary homoallylic pyrophosphate isomer was demonstrated. This provides direct evidence for the usually cryptic isomerase component of the overall catalytic cycle. A number of other related products generated by reaction of cyclase with the analog were also identified, the structures and proportions of which were consistent with the intermediacy in catalysis of a cyclopropylcarbinyl cation X pyrophosphate anion pair. Kinetic parameters for the analog were compared with those of the natural substrate geranyl pyrophosphate. The results presented confirm mechanistic similarities in the enzymatic ionization and subsequent transformation of allylic pyrophosphate and cyclopropylcarbinyl pyrophosphate intermediates of isoprenoid metabolism.