An Escherichia coli expression system was used to generate hexahistidy
l-tagged plant sesquiterpene cyclases, which were readily purified by
a single affinity chromatographic step. Genes for Hyoscyamus muticus v
etispiradiene synthase (HVS), a chimeric 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 accumul
ating to 50% of the total protein and 35% of the soluble protein. From
initial velocity experiments, the Michaelis constant for HVS was 3.5
mu M, while CH3 and CH4 exhibited smaller values of 0.7 and 0.4 mu M,
respectively. Steady-stale catalytic constants were from 0.02 to 0.04
s(-1). A combination of pre-steady-state rapid quench experiments, iso
tope trapping experiments, and experiments to measure the burst rate c
onstant as a function of substrate concentration revealed that turnove
r in all three cyclases is limited by a step after the initial chemica
l step involving rupture of the carbon-oxygen bond in farnesyl diphosp
hate (FPP). Rate constants for the limiting step were 10-70-fold small
er than for the initial chemical step, Dissociation constants for the
enzyme-substrate complex (20-70 mu M) were determined from the pre-ste
ady-state experiments and were significantly larger than the observed
Michaelis constants. A mechanism that involves an initial, rapid equil
ibration 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 enzyme
s, Interestingly, the multifunctional chimeric enzyme CH4 exhibited bo
th a righter binding of FPP and a faster conversion of FPP to products
than either of its wild-type parents.