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The crystal structure of a mu class glutathione S-transferase (EC 2.5.1.18) from
rat liver (isoenzyme 3-3) in complex with the physiological substrate
glutathione (GSH) has been solved at 2.2-A resolution by multiple isomorphous
replacement methods. The enzyme crystallized in the monoclinic space group C2
with unit cell dimensions of a = 87.98 A, b = 69.41 A, c = 81.34 A, and beta =
106.07 degrees. Oligonucleotide-directed site-specific mutagenesis played an
important role in the solution of the structure in that the cysteine mutants
C86S, C114S, and C173S were used to help locate the positions of mercuric ion
sites in nonisomorphous derivatives with ethylmercuric phosphate and to align
the sequence with the model derived from MIR phases. A complete model for the
protein was not obtained until part of the solvent structure was interpreted.
The dimer in the asymmetric unit refined to a crystallographic R = 0.171 for
19,298 data and I > or = 1.5 sigma (I). The final model consists of 4150
atoms, including all non-hydrogen atoms of 434 amino acid residues, two GSH
molecules, and oxygen atoms of 474 water molecules. The dimeric enzyme is
globular in shape with dimensions of 53 x 62 x 56 A. Crystal contacts are
primarily responsible for conformational differences between the two subunits
which are related by a noncrystallographic 2-fold axis. The structure of the
type 3 subunit can be divided into two domains separated by a short linker, a
smaller alpha/beta domain (domain I, residues 1-82), and a larger alpha domain
(domain II, residues 90-217). Domain I contains four beta-strands which form a
central mixed beta-sheet and three alpha-helices which are arranged in a beta
alpha beta alpha beta beta alpha motif. Domain II is composed of five
alpha-helices. Domain I can be considered the glutathione binding domain, while
domain II seems to be primarily responsible for xenobiotic substrate binding.
The active site is located in a deep (19-A) cavity which is composed of three
relatively mobile structural elements: the long loop (residues 33-42) of domain
I, the alpha 4/alpha 5 helix-turn-helix segment, and the C-terminal tail. GSH is
bound at the active site in an extended conformation at one end of the
beta-sheet of domain I with its backbone facing the cavity and the sulfur
pointing toward the subunit to which it is bound.(ABSTRACT TRUNCATED AT 400
WORDS)
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