HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hearne, J. L. Articles by Colman, R. F. Search for Related Content PubMed PubMed Citation Articles by Hearne, J. L. Articles by Colman, R. F. Social Bookmarking                   What's this?

Delineation of xenobiotic substrate sites in rat glutathione S-transferase M1-1

Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, USA

(RECEIVED June 14, 2005; FINAL REVISION July 15, 2005; ACCEPTED July 17, 2005)

Glutathione S-transferases catalyze the conjugation of glutathione with endogenous and exogenous xenobiotics. Hu and Colman (1995) proposed that there are two distinct substrate sites in rat GST M1-1, a 1-chloro-2,4-dintrobenzene (CDNB) substrate site located in the vicinity of tyrosine-115, and a monobromobimane (mBBr) substrate site. To determine whether the mBBr substrate site is distinguishable from the CDNB substrate site, we tested S-(hydroxyethyl)bimane, a nonreactive derivative of mBBr, for its ability to compete kinetically with the substrates. We find that S-(hydroxyethyl)bimane is a competitive inhibitor (K_I = 0.36 µM) when mBBr is used as substrate, but not when CDNB is used as substrate, demonstrating that these two sites are distinct. Using site-directed mutagenesis, we have localized the mBBr substrate site to an area midway through -helix 4 (residues 90–114) and have identified residues that are important in the enzymatic reaction. Substitution of alanine at positions along -helix 4 reveals that mutations at positions 103, 104, and 109 exhibit a greater perturbation of the enzymatic reaction with mBBr than with CDNB as substrate. Various other substitutions at positions 103 and 104 reveal that a hydrophobic residue is necessary at each of these positions to maintain optimal affinity of the enzyme for mBBr and preserve the secondary structure of the enzyme. Substitutions at position 109 indicate that this residue is important in the enzyme’s affinity for mBBr but has a minimal effect on V_max. These results demonstrate that the promiscuity of rat GST M1-1 is in part due to at least two distinct substrate sites.

Keywords: glutathione S-transferase M1-1; monobromobimane; substrate site; site-directed mutagenesis

Abbreviations: GST, glutathione S-transferase • GSH, glutathione • CDNB, 1-chloro-2,4-dinitrobenzene • mBBr, monobromobimane, 4-bromo-methyl-3,6,7-trimethyl-1,5-diazabicyclo-[3.3.0]octa-3,6-diene-2,8-dione;Tris, Tris(hydroxymethyl)aminomethane • LB, Luria-Bertani • EDTA, disodium ethylenediamine tetraacetate • DMF, N,N'-dimethylformamide • IPTG, isopropyl--D-thiogalactoside • CD, circular dichroism • TFA, trifluoro-acetic acid • BTP, 1,3-Bis [tris (hydroxymethyl) methylamino] propane • ESI-MS, electrospray ionization mass spectrometry • rpm, revolutions per minute

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051651905.

Reprint request to: Roberta F. Colman, Department of Chemistry and Biochemistry, University of Delaware, Newark, DE 19716, USA; e-mail: rfcolman{at}chem.udel.edu; fax: (302) 831-6335.

CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				J. L. Hearne and R. F. Colman Contribution of the mu loop to the structure and function of rat glutathione transferase M1-1 Protein Sci., June 1, 2006; 15(6): 1277 - 1289. [Abstract] [Full Text] [PDF]