PRE-STEADY-STATE STUDY OF RECOMBINANT SESQUITERPENE CYCLASES

Citation
Jr. Mathis et al., PRE-STEADY-STATE STUDY OF RECOMBINANT SESQUITERPENE CYCLASES, Biochemistry, 36(27), 1997, pp. 8340-8348
Citations number
38
Categorie Soggetti
Biology
Journal title
ISSN journal
00062960
Volume
36
Issue
27
Year of publication
1997
Pages
8340 - 8348
Database
ISI
SICI code
0006-2960(1997)36:27<8340:PSORSC>2.0.ZU;2-6
Abstract
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.