(Received for publication, July 3, 1995; and in revised form, September 6, 1995)
From the
Rat glutathione transferase (GST) 8-8 displays high
catalytic activity with ,
-unsaturated carbonyl compounds,
including lipid peroxidation products such as 4-hydroxyalkenals. The
catalytic efficiency of the related class Alpha GST 1-1 is
substantially lower with the same substrates. Chimeric enzymes were
prepared by replacing N-terminal subunit 8 segments of different
lengths (6, 25, or 100 residues) with corresponding sequences from
subunit 1 using recombinant DNA techniques. The chimeric subunit
r1(25)r8, containing 25 amino acid residues from subunit 1, had the
same low activity with alkenal substrates as that displayed by subunit
1. Mutation of Ala-12 into Gly in r1(25)r8 gave rise to the high
alkenal activity characteristic of subunit 8, showing the importance of
amino acid residue 12 for the activity. However, other structural
determinants are also essential, as demonstrated by the corresponding
Ala-12
Gly mutation in subunit 1, which did not afford high
alkenal activity. The results show that a single point mutation in a
GST subunit may give rise to a 100-fold increase in catalytic
efficiency with certain substrates. Introduction of such mutations may
have contributed to the biological evolution of GST isoenzymes with
altered substrate specificities and may also find use in the
engineering of GSTs for novel functions.
The cytosolic glutathione transferases (GSTs) ()are
homologous proteins that have been divided into four distinct
structural classes (Mannervik et al., 1985; Meyer et
al., 1991). Members of a given class usually have amino acid
sequence identities >70%, whereas isoenzymes of different classes
have sequence identities <35% (Mannervik and Danielson, 1988).
Crystal structures of members of classes Alpha (Sinning et
al., 1993), Mu (Ji et al., 1992; Raghunathan et
al., 1994), and Pi (Reinemer et al., 1991, 1992; Dirr et al., 1994;
García-Sáez et
al., 1994) have been determined, and the similarities in protein
fold among members from the different classes as well as the
differences in the detailed topography of the secondary structures are
fully consistent with the view that the GST classes are branches on a
common evolutionary tree (Mannervik, 1985).
Attempts have been made to classify glutathione transferases on the basis of activities with different substrates (Boyland and Chasseaud, 1969), but in general substrate specificities are largely overlapping, and members within the same structural class can diverge very significantly in their substrate preferences. For example, the human class Alpha enzymes GST A1-1 and GST A2-2 differ only in 11 out of the 222 amino acids encoded per subunit (Lai et al., 1984). Only four of the variant amino acids are located at the active site (Sinning et al., 1993). These limited structural differences appear to govern the significant differences in substrate specificities reported (Chow et al., 1988; Burgess et al., 1989).
In the rat, a class Alpha
isoenzyme, GST 8-8, has been identified, which is characterized
by particularly high catalytic activity with 4-hydroxyalkenals (Jensson et al., 1986) and other activated alkenes (Stenberg et
al., 1992). The substrates include many highly cytotoxic
,
-unsaturated carbonyl compounds produced by lipid
peroxidation, radical reactions, and other processes elicited by
oxidative stress (Berhane et al., 1994). In terms of protein
design as well as from the evolutionary viewpoint, it is of interest to
elucidate the structural basis for the high catalytic activity of GST
8-8 with these pathophysiologically important substrates. We have
previously demonstrated that fully functional chimeric GSTs can be
constructed by replacing the C-terminal one-third of a human GST
structure with the corresponding segment of a rat GST (Mannervik et
al., 1990; Björnestedt et al., 1992).
In the present investigation it was therefore decided to construct
chimeric GSTs from segments of rat GST 8-8 and rat GST 1-1
in an attempt to locate amino acid residues essential for the high
catalytic activity with alkene substrates. GST 1-1 is a class
Alpha enzyme that is 59% sequence identical with GST 8-8
(Stenberg et al., 1992). Its catalytic activity with long
chain 4-hydroxyalkenals is 1 order of magnitude lower than that of GST
8-8, but the catalytic efficiency of GST 1-1 is still 1
order of magnitude higher than the other rat GSTs investigated
(Danielson et al., 1987). The results of the present
investigation show that the characteristic high catalytic efficiency of
GST 8-8 with activated alkenes is critically dependent on Gly-12
in the active-site region.
Figure 1: Amino acid sequences of GST subunits 1 and 8. Positions of identity between the sequences are indicated by dashes. The initiator methionine is missing in the recombinant subunit 1, but it is retained in subunit 8. Fusion points for chimeric structures involving an N-terminal segment of subunit 1 and the remainder to the C terminus from subunit 8 are indicated by vertical arrows. The glutathione-binding (G-site) residues are indicated by g, and the second-substrate binding (H-site) residues are indicated by h below the sequences, based on comparison with the human class Alpha subunit A1 structure (Sinning et al., 1993). The exons of the corresponding genomic DNA, determined for subunit 1 (Telakowski-Hopkins et al., 1986), are shown below the sequences.
All of the chimeric and mutant enzymes displayed affinity for the glutathione affinity matrix used in the purification, indicating that the glutathione-binding site was functional. GST r1(6)r8, r1(25)r8, and all other variant forms were catalytically active, expressed in moderate to high yields, and appeared to be stable in the pure state. However, GST r1(100)r8 was unstable and obtained in low yield. It is worth noting that the base composition in the 5` coding end strongly affected the expression levels of the proteins. For example, GST r1(25)r8 N11Q was obtained in 10-fold higher yield than GST r1(25)r8, as a result of substituting two bases in codon 11. This dependence on optimal codons at the beginning of the mRNA was utilized to increase the expression levels of GST 8-8 in a rational way. Five codons in the 5` coding region of the subunit 8 cDNA, which are rarely present in highly biased genes in E. coli (Andersson and Kurland, 1990), were selected for silent random mutagenesis. Thus, a library of expression vectors was created with the goal of improving the protein expression level. This successful approach led to the isolation of a clone capable of expressing GST 8-8 at 25-fold increased level compared with the previously published pKGTRA8 (Stenberg et al., 1992).
N-terminal amino acid sequence analysis of GST r1(25)r8 A12G confirmed the expected primary structure SGKPVLHYFNGRGRMECI. Likewise, in the heterologously expressed GST 1-1, the initiator Met is removed by the E. coli Met aminopeptidase, leaving a Ser as the N-terminal residue of the recombinant protein (Wang et al., 1989). In contrast, the N-terminal Met is retained in GST 8-8 (Stenberg et al., 1992), as expected for a sequence with Glu as the penultimate amino acid residue (Dalbøge et al., 1990).
Earlier investigations have shown that rat class Alpha GST 8-8 is much more efficient than GST 1-1 in inactivating 4-hydroxyalkenals by catalyzing their conjugation with glutathione (Danielson et al., 1987). Although the sequence identity between GST 1-1 and GST 8-8 is only 59% (Fig. 1), indirect evidence for heterodimer formation has been reported (Johnson et al., 1990). Also, the overall fold is assumed to be similar for isoenzymes belonging to the same structural class. The inherent substrate diversity (GST 8-8 is more than 100-fold more active with 4-hydroxypentenal than is GST 1-1) in combination with structural similarities make GST 1-1 and GST 8-8 an ideal pair for the construction of functional chimeras with the goal of elucidating the structural basis for function.
The chimeric GST
r1(25)r8, in which the N-terminal segment of 25 amino acid residues of
subunit 8 have been replaced, is altered in nine positions as compared
with GST 8-8 (Fig. 1). The four substitutions closest to
the N terminus of these nine alterations, caused no or little effect on
the catalytic behavior as demonstrated by the characteristics of the
chimeric GST r1(6)r8. When three of the remaining five variant residues
were subjected to site-directed mutagenesis, the H8Y and N11Q
alterations caused only modest effects on the catalytic efficiency
toward ,
-unsaturated carbonyl compounds. As judged by
comparison with the three-dimensional structure of the homologous human
class Alpha GST A1-1 (Sinning et al., 1993), residues 18
and 25 are far removed from the active site and were therefore not
mutated. However, the A12G substitution in GST r1(25)r8 essentially
restored the catalytic properties of GST 8-8. The effect of the
mutation was reflected in a
G value of 10 kJ/mol of
incremental transition state stabilization (calculated from the k
/K
values, cf.
Danielson et al., 1987) when monitored with 4-hydroxynonenal
for GST r1(25)r8 A12G as compared with GST 1-1. In contrast, only
0.3 and 0.8 kJ/mol were calculated for GST 1-1 A12G and GST
r1(25)r8, respectively, as compared with GST 1-1, showing that
the effect of the A12G mutation is dependent on the structural context.
Based on the three-dimensional structure of human GST A1-1 (Sinning et al., 1993), which is the human enzyme most similar to rat GST 1-1, Ala in position 12 is one of the residues forming the active site. However, it is situated approximately 9 Å from the sulfur of enzyme-bound glutathione and 9 Å from the electrophilic group of the second substrate, which reacts with the sulfur in the catalyzed reaction. Assuming that the overall architecture of the active sites of GST 1-1 and GST 8-8 is similar to that of human GST A1-1, residue 12 is probably not in direct contact with either GSH or the electrophilic substrate, but mediates its effect via interactions with other structural elements that contribute to the active site. Gly allows greater variation in the conformation of the polypeptide chain than do other amino acid residues (Creighton, 1993) and may induce changes in the topology of the active site when it replaces Ala.
The second-substrate binding site, or H-site, in human GST A1-1 is highly hydrophobic, a property that appears to be preserved in both GST 1-1 and GST 8-8. However, 9 of the approximately 15 amino acids that contribute to the H-site differ between GST 1-1 and GST 8-8 (Table 2). A larger substrate-binding cavity in GST 8-8, corresponding to approximately five methyl groups, can be calculated from the volume enclosed by the van der Waals radii of the 9 residues differing in the H-site between subunit 1 and 8 (Creighton, 1993). However, this approximate calculation does not consider possible structural readjustments induced by the amino acid substitutions in the active site.
Comparison of class Alpha primary structures reveals that mouse GST A4-4 (Zimniak et al., 1992), human GST 5.8 (Singhal et al., 1994a), chicken GST CL3 (Chang et al., 1992), and rat GST 8-8 form a distinct subgroup within the Alpha class (Singhal et al., 1994b). For example, the amino acid sequence of rat GST 8-8 is 91% identical to that of mouse GST A4-4. All GSTs from this subgroup are highly active with 4-hydroxynonenal with some uncertainty for GST CL3, which has not been assayed with this substrate.
It is clear from the properties of
the GST 1-1 Ala-12 Gly mutant that the Ala-12
Gly
substitution alone in the GST subunit 1 framework cannot confer the
high alkenal activity exhibited by GST 8-8 onto GST 1-1.
Contributions by other residues, yet to be defined, are also needed. A
closer examination of postulated H-site residues within the subgroup of
sequence-related isoenzymes with high 4-hydroxyalkenal activity reveals
that Gly-12, Pro-110, Phe-111, Val-213, Val-216, and Leu-220 are
conserved, whereas other residues are variable (Table 2). Further
experiments are needed to clarify whether or not it would be possible
to redesign the active site of GST 1-1 by substitution of a
limited number of residues in the H-site to mimic the substrate profile
of GST 8-8.
It is also noteworthy that the residue in position
12 is 1 out of the 11 amino acids that differ between the human
isoenzymes GST A1-1 and GST A2-2 (Table 2), which
show divergent substrate specificities (Chow et al., 1988;
Burgess et al., 1989). Furthermore, the amino acid at the
topologically equivalent position in two class Mu isoenzymes has been
suggested as a determinant for stereoselectivity toward
,
-unsaturated ketones (Zhang et al., 1992; Shan and
Armstrong, 1994). Van Ness et al. (1994) have constructed
chimeric GSTs in an attempt to identify the contribution of three
different regions of a mouse enzyme to the high activity with the
hepatocarcinogen aflatoxin B
8,9-epoxide. However, the
specific residues of importance for the substrate selectivity were not
identified.
This investigation complements previous studies showing that construction of chimeric GSTs is a feasible approach to modular design of new functional entities (Zhang and Armstrong, 1990; Mannervik et al., 1990; Zhang et al., 1992; Björnestedt et al., 1992; Van Ness et al., 1994). The positive outcome of these experiments lends support to the proposal that recombination of segments of genomic DNA may have been an evolutionary mechanism for generation of novel GST isoenzymes (Mannervik, 1985). The chimera r1(25)r8 corresponds to the product that would result from a switching from the gene for subunit 1 to the gene for subunit 8 in a position between exons 2 and 3 (residues 26-29 are identical between the subunits, cf. Fig. 1).
GSTs are capable of interacting with a very broad
range of molecular structures (Mannervik and Danielson, 1988) and have
attractive features for protein engineering, including high yield
expression in E. coli and lack of post-translational
modifications. It should be possible to redesign GSTs to obtain
catalysts for GSH conjugation of a very broad range of electrophilic
compounds by optimizing the active site structure of the enzyme for the
particular reaction to be catalyzed. This has recently been attempted
via selection of GST mutants by use of phage display (Widersten and
Mannervik, 1995; Mannervik et al., 1995). Conventional wisdom
in the field of detoxication enzymes maintains that such enzymes have
low catalytic efficiencies in order to be able to react with a broad
range of substrates (Jakoby, 1980). According to this paradigm, it
would appear difficult to obtain mutant enzymes with high catalytic
turnover. However, the present work shows that high catalytic
efficiency can be obtained by a single point mutation to give a variant
GST, i.e. r1(25)r8 Ala-12 Gly, with high catalytic
efficiency not only in relative terms, but also on an absolute scale
(k
/K
> 10
M
s
).