ASPARTATE 458 of Human Glutathione Synthetase is Important for Cooperativity and Active Site Structure Page: 2
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Cooperativity for allosteric enzymes is typically quantified by the Hill coefficient (H): Hill
coefficients of> 1 indicate positive cooperativity; H < 1 indicates negative cooperativity,
and H= 1 defines a non-cooperative enzyme. Though there are many forms of regulation,
negative cooperativity is rare and poorly understood [4,5].
Mammalian glutathione synthetase (rat and human GS) displays negative cooperativity for
y-glutamyl substrate: y-glutamyl-a-aminobutyrate [6-8] or y-glutamylcysteine [6]. Also, the
Arabidopsis thaliana GS enzyme displays negative cooperativity to y-glutamylcysteine [9].
The human GS (hGS) enzyme catalyzes formation of glutathione via ligation of glycine to
y-glutamylcysteine using ATP [10,11]. The compound y-glutamyl-a-aminobutyrate is also a
natural GS substrate, and in fact the product of its GS reaction, ophthalmic acid is found in
the lens and may be a hepatic oxidative stress biomarker [12]. Homodimeric hGS is a
member of the ATP-grasp superfamily of carboxylate-amine ATP-dependent ligases, which
catalyze formation of peptide linkages via an acyl-phosphate intermediate [13,14]. Each
hGS monomer has three conserved loops - G-loop, S-loop and A-loop - in the vicinity of
the active sites of each subunit; notably, the A-loop is located near the glycine substrate and
ATP (Fig. la).
Our previous hGS research suggests that the G-, A- and S-loops are essential for hGS
activity [15]. A strong ionic bond between the Gly371 (G-loop) backbone and the Asp458
(A-loop) carboxyl is present in both reactant binding and product release, and facilitates
active site closure [15]. In the present research, A-loop residue Asp458 was examined for its
hydrogen bonding potential in inter-, intra- and ligand-loop interactions and to delineate its
role in hGS catalysis.
Methods and Materials
Materials
Oligonucleotide primers for mutagenesis and sequencing were synthesized by Integrated
DNA Technologies. The QuikChangen site-directed mutagenesis kit was from Stratagene.
y-Glutamyl-a-aminobutyrate (GAB) was synthesized[ 16]. Isopropyl-1-thio-f3-D-
galactopyranoside (IPTG) was from American Bioanalytical. Other reagents were from
Sigma (St. Louis, MO).
Construction of hGS Mutant Enzymes
The cDNA that encodes the wild-type human glutathione synthetase was subcloned into
pET-15b expression vector (Novagen), which provides a His tag at the N-terminus [17].
Site-directed mutagenesis was performed on double-stranded plasmid (dsDNA) by PCR
using the QuickChanger Site-Directed Mutagenesis Kit (Stratagene). The internal primers
used for hGS mutant enzymes are shown in Table 1. All mutations were confirmed by
sequencing the hGS DNA (Genewiz, Inc.).
Glutathione Synthetase Growth and Purification
The overexpression and protein purification protocol used for the recombinant wild-type and
mutant hGS enzymes, as previously described [17], except cell lysis occurred with pressure,
(15 kPSI, One Shot Model, Constant Systems, Inc.). All purification steps were carried out
at 4 C [17]. The purity of all hGS enzymes was confirmed by SDS-PAGE.
Enzyme Assays and Kinetic Analysis
Analyses were carried out in duplicate using purified recombinant hGS as described [17,18].
In brief, the enzyme activity was measured using the pymuvate kinase (PK)/Iactate
dehydrogenase (LDH) coupled assay. y-Glutamyl-o-aminobutyrate (GAB; a -Biochem Biophys Res Commun. Author manuscript; available in PiMC 2012 August 5.
Brown et al.
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Brown, Teresa R.; Drummond, Michael L.; Barelier, Sarah; Crutchfield, Amanda S.; Dinescu, Adriana; Slavens, Kerri D. et al. ASPARTATE 458 of Human Glutathione Synthetase is Important for Cooperativity and Active Site Structure, article, August 5, 2011; [Amsterdam, Netherlands]. (https://digital.library.unt.edu/ark:/67531/metadc181678/m1/2/: accessed March 29, 2024), University of North Texas Libraries, UNT Digital Library, https://digital.library.unt.edu; crediting UNT College of Arts and Sciences.