Kinetic mechanism and enantioselectivity of halohydrin dehalogenase from Agrobacterium radiobacter.
Halohydrin dehalogenase (HheC) from Agrobacterium radiobacter AD1 catalyzes the reversible intramolecular nucleophilic displacement of a halogen by a hydroxyl group in vicinal haloalcohols, producing the corresponding epoxides. The enzyme displays high enantioselectivity toward some aromatic halohydrins. To understand the kinetic mechanism and enantioselectivity of the enzyme, steady-state and pre-steady-state kinetic analysis was performed with p-nitro-2-bromo-1-phenylethanol (PNSHH) as a model substrate. Steady-state kinetic analyses indicated that the k(cat) of the enzyme with the (R)-enantiomer (22 s(-1)) is 3-fold higher than with the (S)-enantiomer and that the K(m) for the (R)-enantiomer (0.009 mM) is about 45-fold lower than that for the (S)-enantiomer, resulting in a high enantiopreference for the (R)-enantiomer. Product inhibition studies revealed that HheC follows an ordered Uni Bi mechanism for both enantiomers, with halide as the first product to be released. To identify the rate-limiting step in the catalytic cycle, pre-steady-state experiments were performed using stopped-flow and rapid-quench methods. The results revealed the existence of a pre-steady-state burst phase during conversion of (R)-PNSHH, whereas no such burst was observed with the (S)-enantiomer. This indicates that a product release step is rate-limiting for the (R)-enantiomer but not for the (S)-enantiomer. This was further examined by doing single-turnover experiments, which revealed that during conversion of the (R)-enantiomer the rate of bromide release is 21 s(-1). Furthermore, multiple turnover analyses showed that the binding of (R)-PNSHH is a rapid equilibrium step and that the rate of formation of product ternary complex is 380 s(-1). Taken together, these findings enabled the formulation of an ordered Uni Bi kinetic mechanism for the conversion of (R)-PNSHH by HheC in which all of the rate constants are obtained. The high enantiopreference for the (R)-enantiomer can be explained by weak substrate binding of the (S)-enantiomer and a lower rate of reaction at the active site.
Tang
Lixia
L
Laboratory of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, NL-9747 AG, Groningen, The Netherlands.
Lutje Spelberg
Jeffrey H
JH
Fraaije
Marco W
MW
Janssen
Dick B
DB
D001665:Binding Sites; D001965:Bromides / Q000032:analysis; D004852:Epoxy Compounds / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D006867:Hydrolases / Q000737:chemistry* / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D010626:Phenylethyl Alcohol / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism; D012231:Rhizobium / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D013237:Stereoisomerism; D013379:Substrate Specificity
D001965:Bromides; D004852:Epoxy Compounds; C033332:4-nitrostyrene oxide; D006867:Hydrolases; C430848:halohydrin dehalogenase; D010626:Phenylethyl Alcohol