Studies of the cryptic allylic pyrophosphate isomerase activity of trichodiene synthase using the anomalous substrate 6,7-dihydrofarnesyl pyrophosphate.
Two enantiomeric analogues of farnesyl pyrophosphate (1) were tested as inhibitors and anomalous substrates of trichodiene synthase, which catalyzes the cyclization of trans,trans-farnesyl pyrophosphate (1) to the sesquiterpene hydrocarbon trichodiene (2). The reaction has been shown to involve preliminary isomerization of 1 to the tertiary allylic isomer nerolidyl pyrophosphate (3) which is cyclized without detectable release of the intermediate from the active site of the cyclase. Both (7S)-trans-6,7-dihydrofarnesyl pyrophosphate (7a) and (7R)-trans-6,7-dihydrofarnesyl pyrophosphate (7b), prepared from (3R)- and (3S)- citronellol (9a and 9b), respectively, proved to be modest competitive inhibitors of trichodiene synthase. The values of Ki(7a), 395 nM, and Ki(7b), 220 nM, were 10-15 times the observed Km for 1 and half the Ki of inorganic pyrophosphate alone. Incubation of either 7a or 7b with trichodiene synthase resulted in formation of a mixture of products which by radio/gas-liquid chromatographic and GC/selected ion mass spectrometric analysis was shown to be composed of 80-85% isomeric trienes 19-21 and 15-20% allylic alcohols 12 and 18. Examination of the water-soluble products resulting from incubation of 7a also revealed the generation of 24% of the isomeric cis-6,7-dihydrofarnesyl pyrophosphate (26). The combined rate of formation of anomalous alcoholic and olefinic products was 10% the Vmax determined for the conversion of 1 to 2. The results can be explained by initial enzyme-catalyzed isomerization of dihydrofarnesyl pyrophosphate (7) to the corresponding tertiary allylic isomer dihydronerolidyl pyrophosphate (8). Since the latter intermediate is unable to cyclize due to the absence of the 6,7-double bond, ionization of 8 and quenching of the resulting ion pair by deprotonation, capture of water, or collapse to the isomeric primary pyrophosphate esters will generate the observed spectrum of anomalous products.
Cane
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DE
Department of Chemistry, Brown University, Providence, Rhode Island 02912.
Pawlak
J L
JL
Horak
R M
RM
D001665:Binding Sites; D019755:Carbon-Carbon Lyases; D005670:Fusarium / Q000201:enzymology; D007535:Isomerases / Q000378:metabolism*; D007536:Isomerism; D007700:Kinetics; D008190:Lyases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D015394:Molecular Structure; D011106:Polyisoprenyl Phosphates / Q000138:chemical synthesis / Q000494:pharmacology; D013379:Substrate Specificity
D011106:Polyisoprenyl Phosphates; C065312:6,7-dihydrofarnesyl pyrophosphate; D008190:Lyases; D019755:Carbon-Carbon Lyases; C051130:trichodiene synthetase; D007535:Isomerases