Abstract
Enzymes from Salvia officinalis, capable of catalyzing the electrophilic isomerization and subsequent cyclization of geranyl pyrophosphate (3,8-dimethylocta-2E,6-dienyl pyrophosphate) to the monoterpenes (+)-alpha-pinene and (+)-bornyl pyrophosphate, were examined with a series of substrate analogs modified in carbon chain length and in the geometric and electronic character of the C2-C3 and C6-C7 olefinic domains. Inhibition studies with these monoterpene cyclases indicated that the pyrophosphate ester function was the principal determinant of substrate recognition and that the C2-C3 olefin was recognized largely on the basis of geometry, whereas the primary basis of interaction with the C6-C7 olefin was electronic. A related group of allylic pyrophosphates was tested for the ability to undergo enzyme-catalyzed ionization to afford olefinic and/or alcoholic products. From the relative reaction rates it was deduced that the alignment of the allylic pi-system with the C1-OP bond was essential for ionization of the substrate and that specific interaction with the distal C6-C7 isopropylidene function served not only to optimize orbital alignment but also to exclude water from the active site, and thus determine the partitioning of cationic intermediates into olefins or alcohols. From the combination of results, the interrelationships of substrate functional groups within the active site could be approximated and the topology of geranyl pyrophosphate binding to the cyclase thereby formulated.
Highlights
Enzymes from Salvia officinalisc,apable of catalyz- petent to cyclize
All monoterpene cyclases investigated to ing theelectrophilic isomerization and subsequent cy- date are capabloef catalyzing both threequired isomerization clization of geranyl pyrophosphate
Inhibition studies with thesme onoterpene cyclases in- allylic intermediate, linalyl pyrophosphate (2a) (2-4). In this dicated that thepyrophosphate ester function was the “ionization-isomerization’’ step, which removes the topologiprincipal determinanotf substrate recognition and that cal barrierto cyclization, thefirstformalchiralcenteris the C2-C3 olefin was recognized largely on the basis of geometry, whereas theprimary basis of interaction with theC6-C7 olefin was electronic
Summary
Inhibition studies with thesme onoterpene cyclases in- allylic intermediate, linalyl pyrophosphate (2a) (2-4) In this dicated that thepyrophosphate ester function was the “ionization-isomerization’’ step, which removes the topologiprincipal determinanotf substrate recognition and that cal barrierto cyclization, thefirstformalchiralcenteris the C2-C3 olefin was recognized largely on the basis of geometry, whereas theprimary basis of interaction with theC6-C7 olefin was electronic. From the relative reaction sponding monocyclic ( R ) - or (5’)-a-terpinyl cationzpyrates it was deduced that the alignment of the allylic rophosphate anion pair (3).These mechanistic steps appear r-system with theC1-OP bond was essential forionization of the substrate and that specific interaction with the distal C6-C7 isopropylidene function served to optimize orbital alignment and to exclude water from the active site, and determine the partitioning of cationic intermediates into olefins to be common to all monoterpene cyclase transformations, withsubsequent steps involving eithertermination of the reaction by deprotonation or nucleophilic capture or further electrophilic cyclization, hydride shifts, or Wagner-Meerwein rearrangements before termination. By examining the inhibitory propertiesandcatalytic competence ( i e . enzyme-catalyzed ionization) of a series of modified substrate analogs, it has beenpossible to deduce the structural parameters of principal importance in both binding andionization
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