Function of Conserved Residues of Human Glutathione Synthetase: Implications for the ATP-grasp Enzymes Page: 22,412
10 p.View a full description of this article.
Extracted Text
The following text was automatically extracted from the image on this page using optical character recognition software:
Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2004/03/1 0/M401334200.DC1.htmlTHE JOURNAL OF BIOLOGICAL CHEMISTRY
2004 by The American Society for Biochemistry and Molecular Biology, Inc.Vol. 279, No. 21, Issue of May 21, pp. 22412-22421, 2004
Printed in U.S.A.Function of Conserved Residues of Human Glutathione Synthetase
IMPLICATIONS FOR THE ATP-grasp ENZYMES*[
Received for publication, February 6, 2004
Published, JBC Papers in Press, February 27, 2004, DOI 10.1074/jbc.M401334200
Adriana Dinescut, Thomas R. Cundarit, Vikas S. Bhansalill, Jia-Li Luoll,
and Mary E. Anderson**$$
From the Departments of *Chemistry and Biology, University of North Texas, Denton, Texas 76203, the **Department of
Chemistry and Physics, Texas Woman's University, Denton, Texas 76204, and the Hisun Pharmaceutical Co. Ltd.,
46 Waisha Rd., Taizhaou, Zhejiang 318000, ChinaGlutathione synthetase is an enzyme that belongs to
the glutathione synthetase ATP-binding domain-like su-
perfamily. It catalyzes the second step in the biosynthe-
sis of glutathione from y-glutamylcysteine and glycine
in an ATP-dependent manner. Glutathione synthetase
has been purified and sequenced from a variety of bio-
logical sources; still, its exact mechanism is not fully
understood. A variety of structural alignment methods
were applied and four highly conserved residues of hu-
man glutathione synthetase (Glu-144, Asn-146, Lys-305,
and Lys-364) were identified in the binding site. The
function of these was studied by experimental and com-
putational site-directed mutagenesis. The three-dimen-
sional coordinates for several human glutathione syn-
thetase mutant enzymes were obtained using molecular
mechanics and molecular dynamics simulation tech-
niques, starting from the reported crystal structure of
human glutathione synthetase. Consistent with circular
dichroism spectroscopy, our results showed no major
changes to overall enzyme structure upon residue mu-
tation. However, semiempirical calculations revealed
that ligand binding is affected by these mutations. The
key interactions between conserved residues and li-
gands were detected and found to be essential for enzy-
matic activity. Particularly, the negatively charged Glu-
144 residue plays a major role in catalysis.
Glutathione synthetase (1-4) catalyzes the second and final
step in the biosynthesis of glutathione (GSH)1 from y-glutamyl-
cysteine and glycine in an ATP-dependent manner. This proc-
ess involves formation of an enzyme-bound acyl phosphate
* The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
[ ] The on-line version of this article (available at http://www.jbc.org)
contains Supplemental Table S1 and Appendix S1.
These authors acknowledge the support of this research by the
University of North Texas through a Faculty Research Grant and
Chemical Computing for providing the Molecular Operating Environ-
ment program.
$$ Supported by a New Faculty Research Enhancement Program
Award from the Texas Woman's University and by a Chemistry Depart-
ment Welch Foundation Grant. To whom correspondence should be
addressed: Dept. of Chemistry and Physics, Texas Woman's University,
P. O. Box 425859, Denton, TX 76204-5859. Tel.: 940-898-2569; Fax:
940-898-2548; E-mail: Manderson3@mail.twu.edu.
1 The abbreviations used are: GSH, glutathione; gluABA, y-glutamyl-
a-aminobutyrate; SCOP, Structural Classification of Proteins; FSSP,
Fold classification based on Structure-Structure alignment of Proteins;
LPC, ligand-protein contacts; PM3, Parametric Method 3; MD, molec-
ular dynamics technique; r.m.s.d., root mean square deviation.(y-glutamylcysteinyl phosphate), followed by attack of the gly-
cine and formation of an enzyme-product complex, which fi-
nally dissociates with the release of GSH, ADP, and phosphate
(Pi), as shown in Reaction 1.
Glutathione is present in the majority of living cells and is
also the most abundant intracellular thiol. It has a number of
vital functions: it protects cells against oxidative damage, fa-
cilitates the formation of deoxyribonucleotides, reacts with
toxic compounds, participates as a coenzyme for enzymes such
as glyoxalase (5) and glutathione-dependent formaldehyde de-
hydrogenase (6). Glutathione is also involved in amino acid
transport, in metabolism of therapeutic drugs, mutagens, and
carcinogens, and in the maintenance of protein thiol groups
and ascorbic acid in its reduced form (7). Lowered levels of
glutathione have been associated with some diseases, for ex-
ample, human immunodeficiency, hepatitis C, type II diabetes,
ulcerative colitis, idiopathic pulmonary fibrosis, adult respira-
tory distress syndrome, and cataracts (7).
Substantial attention has been given to human glutathione
synthetase because of the biological implications for human
patients with hereditary glutathione synthetase deficiency (8).
In generalized glutathione synthetase deficiency, lowered lev-
els of GSH induce an overproduction of y-glutamylcysteine due
to the lack of feedback inhibition of y-glutamylcysteine synthe-
tase by GSH. Even though y-glutamylcysteine can compensate
for GSH in many aspects of cellular defense against oxidative
stress (9), the increased amounts of y-glutamylcysteine lead to
accumulation of 5-oxoproline (8, 10, 11). On the basis of clinical
symptoms, patients with glutathione synthetase deficiency can
be classified into three phenotypes: mild, moderate, and severe
(or generalized). Patients with mild glutathione synthetase
deficiency have hemolytic anemia as their only clinical symp-
tom. Those with moderate glutathione synthetase deficiency
usually display symptoms starting from the neonatal period,
i.e. metabolic acidosis, 5-oxoprolinuria, and hemolytic anemia.
Those with severe glutathione synthetase deficiency also de-
velop progressive neurological symptoms such as seizures and
psychomotor retardation (12). The severe form of glutathione
synthetase deficiency is caused by mutations in the coding
sequence of human glutathione synthetase that lead to a re-
duction of enzyme activity (13). However, studies on patients
affected by this genetic disorder indicate a residual activity of
glutathione synthetase, suggesting that a complete loss of its
function is probably lethal (14, 15).
Glutathione synthetase has been purified and sequenced
from a variety of sources (2, 16-23). The first highly purified
mammalian glutathione synthetase was isolated from rat kid-
ney in 1979 (24) and then was cloned and sequenced in 1995
(25). In the same year 1995, Gali et al. (26) reported the amino
acid sequence for human glutathione synthetase. Currently,This paper is available on line at http://www.jbc.org
o
0
o
5-
0)
Q
CD
0
3
0
o0
C
z
m
0
m
"11
O
0
--I
I
--I
m
x
022412
Upcoming Pages
Here’s what’s next.
Search Inside
This article can be searched. Note: Results may vary based on the legibility of text within the document.
Tools / Downloads
Get a copy of this page or view the extracted text.
Citing and Sharing
Basic information for referencing this web page. We also provide extended guidance on usage rights, references, copying or embedding.
Reference the current page of this Article.
Dinescu, Adriana; Cundari, Thomas R., 1964-; Bhansali, Vikas S.; Luo, Jia-Li & Anderson, Mary E. Function of Conserved Residues of Human Glutathione Synthetase: Implications for the ATP-grasp Enzymes, article, February 27, 2004; [Rockville, Maryland]. (https://digital.library.unt.edu/ark:/67531/metadc75414/m1/1/: accessed April 25, 2024), University of North Texas Libraries, UNT Digital Library, https://digital.library.unt.edu; crediting UNT College of Arts and Sciences.