Pre-steady-state study of recombinant sesquiterpene cyclases

Jeffery R. Mathis, Kyoungwhan Back, Courtney Starks, Joe Noel, C. Dale Poulter, Joseph Chappell

Research output: Contribution to journalArticlepeer-review

68 Scopus citations


An Escherichia coli expression system was used to generate hexahistidyl- tagged plant sesquiterpene cyclases, which were readily purified by a single affinity chromatographic step. Genes for Hyoscyamus muticus vetispiradiene synthase (HVS), a chimetic 5-epi-aristolochene synthase (CH3), and a chimeric sesquiterpene cyclase possessing multifunctional epi-aristolochene and vetispiradiene activity (CH4) were expressed in bacterial cells, which resulted in the sesquiterpene cyclases accumulating to 50% of the total protein and 35% of the soluble protein. From initial velocity experiments, the Michaelis constant for HVS was 3.5 μM, while CH3 and CH4 exhibited smaller values of 0.7 and 0.4 μM, respectively. Steady-state catalytic constants were from 0.02 to 0.04 s-1. A combination of pre-steady-state rapid quench experiments, isotope trapping experiments, and experiments to measure the burst rate constant as a function of substrate concentration revealed that turnover in all three cyclases is limited by a step after the initial chemical step involving rupture of the carbon-oxygen bond in famesyl diphosphate (FPP). Rate constants for the limiting step were 10-70-fold smaller than for the initial chemical step. Dissociation constants for the enzyme-substrate complex (20-70 μM) were determined from the pre-steady- state experiments and were significantly larger than the observed Michaelis constants. A mechanism that involves an initial, rapid equilibration of enzyme with substrate to form an enzyme-substrate complex, followed by a slower conversion of FPP to an enzyme-bound hydrocarbon and a subsequent rate-limiting step, is proposed for the three enzymes. Interestingly, the multifunctional chimeric enzyme CH4 exhibited both a tighter binding of FPP and a faster conversion of FPP tn products than either of its wild-type parents.

Original languageEnglish
Pages (from-to)8340-8348
Number of pages9
Issue number27
StatePublished - Jul 8 1997

ASJC Scopus subject areas

  • Biochemistry


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