title: Pre-steady-state study of recombinant sesquiterpene cyclases.
abstract: 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 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 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 microM, while CH3 and CH4 exhibited smaller values of 0.7 and 0.4 microM, 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 farnesyl 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 microM) 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 to products than either of its wild-type parents.
authors.affiliation: Department of Chemistry, University of Utah, Salt Lake City, Utah 84112, USA.
authors.forename: J R
authors.initials: JR
authors.lastname: Mathis
authors.forename: K
authors.initials: K
authors.lastname: Back
authors.forename: C
authors.initials: C
authors.lastname: Starks
authors.forename: J
authors.initials: J
authors.lastname: Noel
authors.forename: C D
authors.initials: CD
authors.lastname: Poulter
authors.forename: J
authors.initials: J
authors.lastname: Chappell
mesh_terms: D019883:Alkyl and Aryl Transferases; D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D007700:Kinetics; D010944:Plants / Q000201:enzymology; D011106:Polyisoprenyl Phosphates / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012717:Sesquiterpenes; D014166:Transferases / Q000235:genetics / Q000378:metabolism*
chemical_list: D011106:Polyisoprenyl Phosphates; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D012717:Sesquiterpenes; C004808:farnesyl pyrophosphate; D014166:Transferases; D019883:Alkyl and Aryl Transferases; C078217:5-epi-aristolochene synthase; C092952:vetispiradiene synthase