Document Detail


Kinetic mechanism and enantioselectivity of halohydrin dehalogenase from Agrobacterium radiobacter.
MedLine Citation:
PMID:  12731879     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Lixia Tang; Jeffrey H Lutje Spelberg; Marco W Fraaije; Dick B Janssen
Related Documents :
22387469 - Catalytic versatility and backupsin enzyme active sites: the case of serum paraoxonase 1.
25488109 - Optimization of poly(n-isopropylacrylamide) as an artificial amidase.
9305839 - The interplay between binding energy and catalysis in the evolution of a catalytic anti...
15686389 - How does the camp-dependent protein kinase catalyze the phosphorylation reaction: an ab...
12590569 - Structural and kinetic analysis of catalysis by a thiamin diphosphate-dependent enzyme,...
21819109 - Exploring organosilane amines as potent inhibitors and structural probes of influenza a...
3828289 - Atp-induced dissociation of rabbit skeletal actomyosin subfragment 1. characterization ...
8329439 - Kinetic characterization of the reconstituted dicarboxylate carrier from mitochondria: ...
8756689 - Isolation and characterization of subunits of acetohydroxy acid synthase isozyme iii an...
Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Biochemistry     Volume:  42     ISSN:  0006-2960     ISO Abbreviation:  Biochemistry     Publication Date:  2003 May 
Date Detail:
Created Date:  2003-05-06     Completed Date:  2003-06-24     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  0370623     Medline TA:  Biochemistry     Country:  United States    
Other Details:
Languages:  eng     Pagination:  5378-86     Citation Subset:  IM    
Affiliation:
Laboratory of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, NL-9747 AG, Groningen, The Netherlands.
Export Citation:
APA/MLA Format     Download EndNote     Download BibTex
MeSH Terms
Descriptor/Qualifier:
Binding Sites
Bromides / analysis
Epoxy Compounds / chemistry*,  metabolism
Escherichia coli / enzymology*
Hydrolases / chemistry*,  metabolism*
Hydrolysis
Kinetics
Phenylethyl Alcohol / analogs & derivatives*,  chemistry*,  metabolism
Rhizobium / enzymology*
Spectrometry, Fluorescence
Stereoisomerism
Substrate Specificity
Chemical
Reg. No./Substance:
0/Bromides; 0/Epoxy Compounds; 60-12-8/Phenylethyl Alcohol; 6388-74-5/4-nitrostyrene oxide; EC 3.-/Hydrolases; EC 3.8.1.-/halohydrin dehalogenase

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


Previous Document:  Channel formation by the binding component of Clostridium botulinum C2 toxin: glutamate 307 of C2II ...
Next Document:  The CXC motif: a functional mimic of protein disulfide isomerase.