From the Department of Chemistry and Biochemistry, Miami University, Oxford, Ohio 45056
Received for publication, June 13, 2000, and in revised form, November 16, 2000
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ABSTRACT |
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Glyoxalase II participates in the cellular
detoxification of cytotoxic and mutagenic 2-oxoaldehydes. Because of
its role in chemical detoxification, glyoxalase II has been studied as
a potential anti-cancer and/or anti-protozoal target; however, very
little is known about the active site and reaction mechanism of this important enzyme. To characterize the active site and kinetic mechanism
of the enzyme, a detailed mutational study of Arabidopsis glyoxalase II was conducted. Data presented here demonstrate for the
first time that the cytoplasmic form of Arabidopsis
glyoxalase II contains an iron-zinc binuclear metal center that is
essential for activity. Both metals participate in substrate binding,
transition state stabilization, and the hydrolysis reaction. Subtle
alterations in the geometry and/or electrostatics of the binuclear
center have profound effects on the activity of the enzyme. Additional residues important in substrate binding have also been identified. An
overall reaction mechanism for glyoxalase II is proposed based on the
mutational and kinetic data from this study and crystallographic data
on human glyoxalase II. Information presented here provides new
insights into the active site and reaction mechanism of glyoxalase II
that can be used for the rational design of glyoxalase II inhibitors.
The glyoxalase system consists of two enzymes that convert
cytotoxic 2-oxoaldehydes into hydroxy acids utilizing the cofactor glutathione. Under physiological conditions, glyoxalase I
(lactoylglutathione lyase) catalyzes the formation of
S-D-lactoylglutathione
(SLG)1 in the presence of
methylglyoxal and glutathione (1). Methylglyoxal, a cytotoxic compound
that can inactivate proteins, modify guanylate residues in DNA, and
create interstrand cross-links (2-4), is formed as a by-product of
several biochemical reactions including that of triose-phosphate
isomerase (2). Glyoxalase II (hydroxyacylglutathione hydrolase)
hydrolyzes SLG to form D-lactic acid and regenerate glutathione (1). SLG is also known to exhibit cytotoxic effects through
the inhibition of DNA synthesis (5). Therefore, the glyoxalase system
is thought to play a major role in chemical detoxification (5,
6).
The glyoxalase system has been the subject of intense study because of
its potential implications in anti-protozoal and anti-tumor drug design
(6-8). Increased cellular levels of methylglyoxal and glyoxalase
activity are associated with tumor cells, the malaria parasite, and
several complications associated with diabetes (6). Inhibitors of
glyoxalase I and glyoxalase II have been designed as potential
anti-tumor agents; however, all inhibitors tested to date exhibited
problems that limited their therapeutic usefulness (6-9). It should be
noted that these inhibition studies were all conducted with little or
no information on the active site structure or the kinetic mechanism of
the enzyme. Therefore, it is clear that a more detailed understanding
of the active site of glyoxalase II and its reaction mechanism is
essential to the rational design and implementation of clinically
useful inhibitors.
Glyoxalase II has been purified and cloned from a number of sources
(10-15), including Arabidopsis thaliana (16, 17). Five glyoxalase II isozymes have been identified in Arabidopsis
including three, GLX2-1, 2-4, and 2-5,
which appear to be mitochondrial forms; GLX2-2, a
cytoplasmic form; and GLX2-3, which has not yet been
localized (16). The GLX2-2 gene has been successfully cloned and overexpressed in Escherichia coli yielding protein
concentrations as high as 100 µM (18). Early biochemical
studies suggested that glyoxalase II, unlike glyoxalase I, does not
require bound metal ions for activity (13, 15, 17, 19). However,
through the analysis of recombinant GLX2-2, we demonstrated that
glyoxalase II is a Zn(II)-requiring enzyme (18). All species of
glyoxalase II, including human, yeast, and Arabidopsis,
contain a highly conserved metal binding domain
(THXHXDH) that is also present in the family of
metallo- Early chemical modification, inhibition, and pH dependence studies on
glyoxalase II suggested that an arginine, an amine, and a critical
histidine are involved in the active site (13, 25). Given that Zn(II)
is critical for activity (18), the critical histidine is most likely
one of the metal ligands. In addition to the highly conserved
metal-binding ligands, several other highly conserved residues,
including arginine and lysine residues, have been identified (18). To
probe the functionality of some of these residues, we have generated
and analyzed a series of site-directed mutants of glyoxalase II.
Conserved residues predicted to participate in substrate and metal
binding were mutated, and the altered forms of the enzyme were analyzed
for conformation, metal content, and kinetic properties. Most of the
mutations did not lead to a significant drop in substrate affinity;
however, one mutation, R248W, did cause a 10-fold increase in
Km. All of the mutations affecting metal ligands
resulted in a large drop in activity, suggesting that the metals must
be bound in an optimal geometry for catalysis. Evidence was also
obtained that the binuclear metal site of glyoxalase II is able to bind both zinc and iron. These results provide significant new insight into
the metal binding and active sites of glyoxalase II and its mechanism
of catalysis.
Materials
Deep Vent polymerase and other overlap PCR reagents were
purchased from New England Biolabs (Beverly, MA); the oligonucleotides were synthesized by Integrated DNA Technologies, Inc (Coralville, IA).
All of the chromatographic reagents, columns, and fast protein liquid
chromatography system were obtained from Amersham Pharmacia Biotech.
The SLG was purchased from Sigma. All other molecular biology reagents
were purchased commercially and were of highest quality.
Methods
Site-directed Mutagenesis--
Site-specific mutations were
introduced into the glyoxalase II gene (GLX2-2) through
overlap extension PCR (26). The wild type template, pT7-7/GLX2-2, was
previously described by Maiti et al. (16). Numbering of
amino acids corresponds to the Arabidopsis GLX2-2 sequence
(GenBankTM accession number O24496). One mutation,
R248W, found in the wild type cDNA template, was corrected through
overlap PCR (TGG to CGC). The forward and reverse primers were between
26 and 32 bases long with the mutation in the center. The mutations generated were as follows: H54N, CAT to AAC; D58C, GAT to TGT; H59S,
CAT to AGT; H59C, CAT to TGC; K74A, AAA to GCA; C140A, TGT to GCG;
K142A, AAG to GCA; H172R, CAT to CGC; N178A, AAC to GCA; and R225A, CGT
to GCA. All mutated PCR products were cloned into pT7-7 (27) and sequenced.
Protein Production and Purification--
The expression plasmids
were transformed into BL21(DE3)pLysS E. coli cells and
overexpressed as described previously (18). The induction time was
extended to 30 h, and the induction temperature was lowered to
15 °C to increase the yield of soluble protein. The wild type and
mutant proteins, except for H59C and H172R, were purified using a
Q-Sepharose column as described previously (18). The H59C and H172R
enzymes could not be purified; therefore, these proteins were assessed
in crude soluble extracts. Kinetic data and metal content were obtained
and averaged from three preparations of each enzyme.
Steady State Kinetics and Crude Assays--
The steady state
kinetic parameters of purified wild type and mutant enzymes were
determined by measuring the rate of hydrolysis of SLG at 240 nm using a
Cary IE UV-visible spectrophotometer as previously described (18).
To prepare crude soluble protein extracts of the wild type, H59C, and
H172R enzymes, 50-ml cultures of each strain were grown at 30 °C in
ZY media (2% (w/v) tryptone, 1.5% yeast extract, 0.5% NaCl, 1%
(v/v) glycerol) supplemented with 500 µM
ZnSO4 and 150 µg/ml ampicillin. The cultures were induced
with 0.1 mM
isopropyl-1-thio- End Product Inhibition Studies of SLG Hydrolysis--
Inhibition
studies were performed on glyoxalase II using glutathione and
D-lactic acid, the products of SLG hydrolysis. Steady state
kinetic studies of purified wild type glyoxalase II in the presence of
varying concentrations of glutathione (0.8, 1.7, 5, and 6.7 mM) or lithium D-lactate (103, 205, and 410 mM) were conducted by measuring the rate of hydrolysis of
SLG. Glutathione and lithium D- lactate were prepared in 10 mM MOPS, pH 7.2; the pH of the lithium
D-lactate solution was adjusted to 7.2 after dissolution of
the salt. The enzyme and inhibitor were added first to allow the
inhibitor time to interact with the enzyme. The substrate was added at
time 0. For all reactions Lineweaver-Burk plots were generated to
determine the mode of inhibition (28). Greater experimental error is
associated with the results obtained from assays conducted at high
inhibitor concentrations (low activity) than those at lower inhibitor
concentrations, resulting in some lines that do not cross at a common
point. Slope replots were generated to confirm the mode of inhibition
and the KI for each product (28).
Solvent Isotope Effects--
Solvent isotope effect studies were
performed on the wild type and D58C mutant enzymes to assess the
presence of a rate-limiting proton transfer. Steady state kinetic
studies of purified wild type or D58C mutant enzymes were conducted by
measuring the rate of SLG hydrolysis at 240 nm in 0, 25, 50, 75, and
100% D2O. The kH/kD
and
(k/Km)H/(k/Km)D
ratios were then calculated from the steady state kinetic constants obtained in 10 mM MOPS, pH 7.2, made with 0 and 100%
D2O.
Metal Analysis--
The metal content of purified wild type and
mutant proteins was measured with a Varian inductively coupled plasma
spectrometer (ICP) with atomic emission spectroscopy detection as
previously described (18). Purified protein was diluted in 10 mM MOPS, pH 7.2, to a concentration of 10 µM
and analyzed for zinc, cobalt, and iron.
Whole Cell Metal Analysis--
Whole cell metal analysis was
performed on soluble extracts from wild type and mutant cultures
through a modification of the method of D'souza and Holz (29).
Fifty-milliliter cultures of BL21 (DE3) pLysS E. coli cells
containing pT7-7, GLX2-2/pT7-7, and the
GLX2-2/pT7-7 plasmids containing the H59C and H172R
mutations were grown at 30 °C in ZY media minus ZnSO4
with 150 µg/ml ampicillin. The cultures were induced with 0.1 mM isopropyl-1-thio- Fluorescence Emission--
The intrinsic fluorescence of the
wild type and mutant proteins was measured as described by Dragani
et al. (30). Protein solutions of 0.2 mg/ml were prepared in
10 mM MOPS buffer and analyzed with a PerkinElmer Life
Sciences Luminescence Spectrometer. The blank-corrected emission
spectra were measured at 2 nm/s with excitation wavelengths of 280 and
295 nm from three enzyme preparations and were averaged.
Overexpression and Purification of Wild Type and Mutant Glyoxalase
II--
Several highly conserved amino acids were mutated, and the
effect of these mutations on enzyme activity was analyzed to elucidate the roles of metal ligand and substrate-binding residues in the hydrolysis of SLG by glyoxalase II. Four metal-binding ligands of
glyoxalase II (His-54, Asp-58, His-59, and His-172) were mutated to
assess the sensitivity of metal binding and activity to changes in
polarity, electrostatics, and geometry of the metal ions. In addition,
several other highly conserved residues (Lys-74, Asn-178, Cys-140, Lys-142, and Arg-225) were replaced to evaluate their role in
substrate binding.
Wild type and mutant proteins were overexpressed in E. coli
and purified through fast protein liquid chromatography. All but three
of the enzymes were produced at high levels and readily purified.
Typically 40-80 mg of protein with 95-99% purity was obtained from a
1-liter culture. Two of the enzymes, metal ligand mutants H59C and
H172R, could not be purified. Although the proteins accumulate to
relatively high levels in the cell, neither enzyme bound efficiently to
Q-Sepharose, preventing purification at levels appropriate for
Michaelis-Menten kinetics and metal analysis. The H59C and H172R
mutations appear to destabilize the structure of the proteins, which
may result in denaturation during purification. The loss of column
affinity as a result of mutations in metal ligands of other proteins
has previously been reported (31). The K74A mutation results in a
highly insoluble enzyme. It could not be purified, and therefore was
not analyzed. Lysine 74 is located on the outside of the protein in the
middle of a Analysis of Crude Protein Extracts of Wild Type and Mutant
Glyoxalase II--
Because the H59C and H172R proteins could not be
purified, crude protein extracts were analyzed for glyoxalase II
activity. Both enzymes could be produced at levels relatively
comparable to wild type glyoxalase II and were found at least in part
in the soluble protein fraction (see Fig. 1 of Supplemental Material). Levels of soluble enzyme with the H172R mutation were, however, consistently lower than that observed for the wild type enzyme. Therefore, the relative amount of soluble glyoxalase II was determined in all experiments. When the relative amounts of glyoxalase II present
in the extracts were taken into consideration, the H59C and H172R
proteins were found to exhibit ~1 and 0.05% of the crude protein
activity seen in wild type GLX2-2, respectively (Table I). The reduced activity seen in the H59C
and H172R enzymes is similar to that seen in the other metal ligand
mutants (see below).
The in vivo metal content of crude protein extracts of cells
overexpressing the H59C and H172R enzymes were also compared with cells
expressing the wild type enzyme using ICP with atomic emission
spectroscopy detection to determine if the mutant enzymes are still
able to bind metal. A variation of this method has been used to
determine the physiologically relevant metal ions present in methionyl
aminopeptidase (29). Zinc and iron uptake was calculated by measuring
the difference between the metal content of cells containing the empty
expression vector (pT7-7) and those overexpressing the
GLX2-2 enzymes. Since glyoxalase II is expressed at high
levels after induction, comparison of the whole cell metal
concentrations in the presence and absence of glyoxalase II expression
should reflect the relative ability of the enzymes to bind metal. This method is not, however, able to give the stoichiometry of the metal in
the enzymes. Cells overexpressing wild type GLX2-2 contain approximately twice as much zinc (3.79 ± 0.66 µM/mg/ml protein) and iron (3.75 ± 0.37 µM/mg/ml protein) as control cells with no GLX2-2
expression (1.75 ± 0.23 µM Zn/mg/ml protein and
1.84 ± 0.57 µM Fe/mg/ml protein). Cells expressing
the H59C enzymes contain iron levels (3.96 ± 0.42 µM/mg/ml protein) similar to those observed in cells
overexpressing the wild type enzyme, whereas cells expressing the H172R
enzyme did not accumulate iron above control levels (1.77 ± 0.45 µM/mg/ml protein). Cells expressing both the H59C and
H172R enzymes failed to accumulate zinc and contained essentially the
same levels of zinc as the control cells (1.71 ± 0.37 and
1.67 ± 0.47 µM/mg/ml protein, respectively).
These results suggest that the H59C mutation alters the binding site
for metal 2, which is bound by Asp-58, His-59, Asp-133, His-172, and a
bridging water molecule (23), such that it prevents metal binding.
Although cysteine should be capable of binding either zinc or iron, the
change in charge caused by this substitution may significantly alter
the electrostatic environment of the active site thus preventing metal
ion binding. Overexpression of the H172R enzyme did not result in a
detectable uptake of either metal. Therefore, the H172R mutation seems
to cause significant changes in the protein, which affect the binding
of both metal 2 and metal 1, which is bound by His-54, His-56, His-110,
Asp-133, and a bridging water molecule (23).
Metal Analysis of Purified Wild Type and Mutant Glyoxalase
II--
The total metal content of purified wild type and mutant
proteins was measured using ICP with atomic emission spectroscopy detection to determine whether any of the mutations affect the ability
of glyoxalase II to bind metal ions. All of the proteins, with the
exception of the C140A and R225A enzymes, contained approximately two
metal ions as shown in Table II. Previous
data suggested that glyoxalase II binds two zinc ions; however, the
GLX2-2 enzyme analyzed in this earlier study contained the mutation
R248W (18). As shown in Table II, enzyme containing the R248W mutation
binds approximately 2 mol of zinc and 0.4 mol of iron. In contrast, the
wild type protein binds ~0.8 mol of zinc and 1.5 mol of iron. Therefore, it appears that both zinc and iron can occupy the metal sites of fully active glyoxalase II.
Although the total amount of metal bound by different forms of the
enzyme was relatively constant at 2 mol of metal/mol of enzyme, the
ratio of zinc to iron in glyoxalase II appears to be influenced by
growth conditions and the environment around the metal-binding ligands.
The metal content of wild type enzyme preparations ranged from 1:1 to
1:2 (Zinc:iron) with the average Zinc:iron ratio being 1:2. The
D58C, K142A, and N178A enzymes bind iron and zinc at levels and in
ratios nearly identical to wild type (1:1.7, 1:1.7, and 1:2.5,
respectively). The R225A enzyme binds approximately half of the total
metal seen in wild type GLX2-2 and seems to have a higher affinity for
iron than does wild type (1:4.3). The C140A and R248W enzymes have
higher affinities for zinc than wild type, with ratios of 1.6:1 and
5.1:1, respectively. The increased Zinc:iron ratio seen in the C140A
enzyme is the result of increased zinc binding without a corresponding
decrease in the amount of iron bound. Therefore, this enzyme binds
twice as much total metal as does wild type glyoxalase II. Data
presented in Tables I and II suggest that metal site 1 has a preference for iron, whereas metal site 2 binds zinc. The H59C mutation in metal
site 1 results in an enzyme that binds normal levels of iron and no
zinc, whereas the H54N mutation in metal site 2 results in an enzyme
that binds zinc but little iron. Whereas further experiments are
required to confirm these observations, these results provide
preliminary insights into the nature of the metal-binding sites of
glyoxalase II.
Steady State Kinetic Analysis of Purified Wild Type and Mutant
Glyoxalase II--
Steady state kinetic studies on the wild type and
mutant proteins were performed to determine the effects of the
mutations on substrate binding and catalysis. The steady state
constants, kcat and Km,
extracted from kinetic data fitted to the Michaelis-Menten equation,
for the wild type and mutant enzymes are shown in Table II. The
Km value obtained for wild type enzyme was similar
to data previously obtained for Arabidopsis glyoxalase II
(17). Very few of the mutations affected the Km values when using SLG as substrate. Amino acid residues Asn-178, Cys-140, Lys-142, and Arg-248 were previously proposed to interact with
the glutathione portion of SLG in human glyoxalase II (23). The
Km values for the N178A and C140A enzymes are
similar to that of wild type, suggesting that these residues do not
play an important role in substrate binding. In contrast, the K142A and
R248W substitutions did result in 3-10-fold higher
Km values, respectively. Removing the positive side
chains of these two amino acid residues most likely impaired their
affinity for the carbonyl groups on glutathione. Replacement of Arg-248
with a tryptophan residue results in an enzyme with a
Km value that is 10-fold higher than wild type. This
suggests that Arg-248 plays an important role in substrate binding. The
H54N enzyme also displayed a reduced affinity for SLG. Histidine 54 was
not previously implicated with substrate binding. It does, however, bind metal 1, which is located within binding distance of the lactoyl
carbonyl. Therefore, changes in this residue can be expected to affect
substrate binding.
Most of the mutations examined had an effect on the activity of the
enzyme. All of the metal ligand substitutions, H54N, D58C, H59C, and
H172R, caused a significant reduction in the
kcat values (~99% reduction) of the enzymes.
The K142A mutation, which is predicted to alter the position of a
second shell metal ligand (23), caused a less severe reduction in
activity (~55% loss). Given that the H54N, D58C, and K142A enzymes
bind normal amounts of metal, these data suggest that enzyme activity
depends not only on the correct metal stoichiometry but also on the
geometry of the metals bound within the active site. The N178A mutation caused a 70% reduction in activity. The change in polarity generated by the asparagine to alanine substitution may alter the local electrostatics of the active site thus reducing activity.
Purified enzyme containing the R225A mutation displayed a significant
reduction in activity and metal content. However, crude protein
extracts from cells expressing the R225A enzyme displayed activity
comparable to wild type (Table I). This suggests that the enzyme is
initially correctly folded and active. The low enzymatic activity and
metal content of the purified enzyme suggest that the mutation may
cause structural alterations that result in denaturation during
purification. Consistent with this hypothesis is the observation that
preparations of the R225A enzyme displayed highly variable fluorescence
patterns, suggesting that the preparations contained protein at varying
stages of denaturation (see below).
The C140A mutation yielded an enzyme that is more active than wild
type. In contrast to all of the other mutations, which reduced enzyme
activity, the kcat of the C140A enzyme is
~170% of the wild type enzyme, whereas the Km
values of the two enzymes are identical. The possibility that the
increased metal content and activity was a result of an error in enzyme concentration caused by a change in the molar absorptivity was considered. To evaluate this possibility the concentration of the
purified enzyme was analyzed visually through SDS-polyacrylamide gel
electrophoresis with other glyoxalase II enzymes, spectroscopically through UV absorption at 280 nm, and chemically through the BCA assay.
All methods yielded similar protein concentrations indicating that the
concentration value used in kcat and metal
content determinations is correct. Therefore, the C140A mutation
results in an increase in metal binding and enzyme activity.
Fluorescence Emission Spectra of Purified Wild Type and Mutant
Glyoxalase II--
Fluorescence emission spectra were obtained for
purified wild type and mutant proteins using excitation wavelengths of
280 and 295 nm to determine the effect of the mutations on the general conformation of the enzyme. GLX2-2 contains two tryptophan residues at
amino acid residues 57 and 198. It has previously been demonstrated that mutations that alter the general structure of glyoxalase II change
the environment around the two tryptophan residues and result in an
increased fluorescence emission (30). Most of the mutant enzymes,
including the D58C enzyme, exhibited a fluorescence emission spectrum
similar to that of wild type GLX2-2, suggesting that no significant
change in the general structure of the proteins occurred as a result of
the mutations (Fig. 1C). As
seen in Fig. 1, A and B, two of the enzymes, H54N
and C140A, exhibited a much greater intrinsic fluorescence intensity
than the wild type enzyme. This suggests that these mutations, which
are both at the active site of the enzyme, cause a significant change
in the structure of the proteins. In contrast to the other enzymes, the
fluorescence emission pattern for the purified R225A enzyme varied
significantly from preparation to preparation, suggesting that the
preparations may contain varying amounts of misfolded and/or denatured
protein.
Solvent Isotope Effects--
The reaction mechanism of glyoxalase
II, specifically if a proton transfer occurs during the rate-limiting
step of SLG hydrolysis, was investigated by conducting solvent isotope
effect kinetics on the wild type and D58C mutant enzymes. The
hydrolysis of SLG by wild type glyoxalase II shows very little solvent
isotope effect, kH/kD = 1.4 ± 0.2;
(k/Km)H/(k/Km)D = 1.0 ± 0.5. These data are similar to that previously determined for human liver glyoxalase II (13). If there is a rate-limiting proton
transfer occurring between the bridging water and aspartic acid 58, its
replacement with a cysteine should prevent this proton transfer from
occurring, which would lower the solvent isotope effect. The D58C
enzyme exhibited solvent isotope effects similar to that determined for
the wild type enzyme, kH/kD = 1.5 ± 0.1;
(k/Km)H/(k/Km)D = 1.3 ± 0.2. Although we cannot completely rule out the
possibility, our data suggest that the rate-limiting step of
Arabidopsis GLX2-2 does not involve a proton in flight.
Typically a kH/kD = 1.11 is
indicative of a carbonium ion in the transition state, as seen with
lysozyme (32). In contrast the metallo- End Product Inhibition Studies--
To determine the order of
product release after the hydrolysis of SLG, end product inhibition
studies were performed. Steady state kinetic studies, using the
substrate SLG, were conducted on wild type GLX2-2 in the presence of
varying concentrations of glutathione (0.8-6.7 mM) or
lithium D-lactic acid (100-410 mM). Inhibition
by glutathione was seen at concentrations as low as 0.8 mM,
whereas inhibition by D-lactic acid was seen at
concentrations as low as 20 mM. Steady state kinetics was
conducted at 10, 30, 50, and 70% inhibition by glutathione and at 18, 33 and 50% inhibition by D-lactic acid. Lineweaver-Burk
plots for glutathione were typical of a mixed mode of inhibition,
whereas D-lactic acid exhibited weak competitive inhibition
(Supplemental Figs. 2 and 3, respectively). The slope replots resulted
in KI values of 0.4 and 122 mM for
glutathione and D-lactic acid, respectively. These data differ from earlier biochemical studies on human liver glyoxalase II
that suggest glutathione is a weak competitive inhibitor with a
KI = 4.0 mM, and D-lactic
acid is not an inhibitor (19). The significant differences in the
KI values of glutathione and D-lactic
acid suggest that product release after the hydrolysis of SLG is
ordered (28). The low KI for glutathione suggests
that it is bound more tightly into the active site and therefore is
released after D-lactic acid.
In this report we present results of mutational and kinetic
studies that were conducted on glyoxalase II to gain insight into the
role of highly conserved amino acids in metal ion and substrate binding. Early studies suggested that glyoxalase II is not a
metalloenzyme (13, 15, 17, 19). However, in 1997 we showed that
glyoxalase II in fact requires 2 mol of Zn(II) per mol of enzyme for
catalytic function (18). The protein used in this study was
subsequently found to contain a mutation, R248W. Evidence presented
here demonstrates that wild type Arabidopsis glyoxalase II
contains a binuclear metal-binding site that is able to bind both zinc
and iron. As shown in Table II, enzyme preparations containing the
R248W mutation bind 2 mol of zinc and 0.4 mol of iron, whereas wild
type Arabidopsis glyoxalase II binds an average of 0.8 mol
of zinc and 1.5 mol of iron. Furthermore, data on several of the
mutations presented here indicate that the relative stoichiometry of
the metals bound by glyoxalase II is influenced by the environment
around the metal ligands. The R248W and C140A mutant enzymes bind more
zinc than iron, whereas the wild type enzyme has a preference
for iron. The observation that Arabidopsis glyoxalase II can
bind both zinc and iron is consistent with recent results on human
glyoxalase II, which was found to contain ~1.5 mol of zinc and 0.7 mol of iron (23). In this study the authors concluded that the enzyme is a binuclear zinc protein. However, the resolution of the crystal structure was not sufficient to distinguish between zinc and iron in
the protein.
Binuclear iron-zinc proteins have been found throughout nature,
including the kidney bean purple acid phosphatases and protein phosphatases 1, 2A, and 2B (35, 36). Purple acid phosphatases can have
Fe(III)-Fe(II), Fe(III)-Mn(II), or Fe(III)-Zn(II) binuclear metal centers. In the mammalian dinuclear iron purple acid phosphatases zinc can readily replace iron to form functional Fe-Zn enzymes (37),
whereas iron can replace zinc in the Zn-Fe purple acid phosphatases
from plants (36). Our current results indicate that glyoxalase II can
utilize both zinc and iron. Further experiments are required to
determine the exact nature of the binuclear metal center in the enzyme
and to determine whether it is able to utilize other metals, such as
manganese. The ability to utilize both zinc and iron is not seen
in the metallo- Early chemical modification, inhibition, and pH dependence studies
suggested that an arginine, an amine, and a critical histidine are
involved in the active site of glyoxalase II (13, 25). The critical
arginine may be Arg-240, which is essential for substrate binding, as
shown by the 10-fold reduction in SLG affinity when Arg-248 is
substituted by tryptophan. The critical amine is not Lys-142; changing
this residue to an alanine did not result in a large loss of activity.
There are no other conserved amines in the active site with the
exception of the imidazole rings of the metal ligand histidines
(16).
The essential histidine residue previously identified is most likely
one or more of the metal ligands. Our finding that any alteration in
the metal-binding ligands of glyoxalase II results in a significant
reduction in activity clearly demonstrates the essential role of the
binuclear metal site in the catalysis of this enzyme. Substitutions of
metal-binding ligands that are expected to alter the geometry of the
metal ions but still allow metal binding (H54N, D58C, and H59C) all
resulted in a dramatic decrease in enzyme activity. In addition, the
structure of glyoxalase II cannot accommodate the loss of one metal, as
knocking out one metal ligand through the H172R mutation yielded a near
inactive enzyme with virtually no bound metal.
It has been proposed that the active site of human glyoxalase II is
also dependent on a GCG hairpin; the cysteine of this hairpin was
proposed to be involved with substrate binding and stabilizing several
highly conserved active site residues (23). Replacing this cysteine
(140) with alanine in GLX2-2 results in dramatic structural changes, as
shown by the significant increase in the intrinsic fluorescence of the
enzyme. Interestingly, this alteration does not reduce enzymatic
activity. Instead the activity increases almost 2-fold. It was also
noted that the metal content of the C140A enzyme is twice that of wild
type glyoxalase II. Quantification of the C140A protein using several
methods indicated that the 2-fold increases in activity and metal
content are not due to errors in enzyme concentration. The increased
fluorescence of the C140A mutant enzyme indicates that the environment
around the active site tryptophan residues (57 and 198) has been
changed by this mutation. It has been suggested previously that
substrate binding is the rate-limiting step for glyoxalase II (39). It is possible that the C140A mutation alters the structure of the enzyme
to facilitate access of substrate to the active site. Further experiments, including crystallographic studies, are necessary to
confirm this proposal.
Many binuclear metalloenzymes, such as the metallo- By using data from this study, the crystallographic data on human
glyoxalase II, and results from earlier biochemical studies, an overall
reaction mechanism can be offered (Fig.
2). Arginine 248 apparently participates
in the initial binding of S-lactoylglutathione; Lys-142 and
Asn-178 also participate in substrate binding but to a lesser extent.
In contrast to the proposal of Cameron and coworkers (23), Cys-140
seems to have little to no initial binding interaction with SLG; the
Km of the C140A enzyme is identical to wild type.
The binuclear metal center also plays a role in substrate binding, with
both metal ions potentially helping to orient SLG. Metal 2 is proposed
to be in close proximity to the sulfur of the glutathione portion of
SLG, whereas metal 1 is proposed to be in close proximity to the
lactoyl carbonyl of SLG (23). The metals may help orient SLG and
stabilize the excess negative charge formed in a tetrahedral transition
state. The participation of metal ions in orienting substrate for
nucleophilic attack has been proposed in several other binuclear
metalloenzymes, including the metallo-
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-lactamases, which are known to require Zn(II) (16, 18, 20,
21). Based on its similarity to the metallo-
-lactamases, we
predicted that glyoxalase II binds two Zn(II) ions, utilizing five
histidines, two aspartic acids, and a bridging water molecule (18).
These predictions have been restated by Melino et al. (22)
and supported through the recent determination of the human glyoxalase
II crystal structure (23). The crystallographic data indicates that the
metal binding and active sites of glyoxalase II resemble those in the
metallo-
-lactamase L1 from Stenotrophomonas maltophilia
(24).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside at
A600 of 0.7 and transferred to a shaker
at 15 °C. After 24 h, the cultures were harvested and washed
three times with Nanopure water. The cell pellets were resuspended in
10 ml of 10 mM MOPS, pH 7.2, and lysed by two passes
through a French press at 16,000 pounds/square inch. A soluble extract
was obtained after centrifugation at 12,500 rpm for 45 min. The amount
of crude protein in the soluble extract was quantitated through
SDS-polyacrylamide gel electrophoresis and the BCA protein assay. The
total activity of crude soluble protein was determined from the rate of
hydrolysis of 100 nmol of SLG in a 1-ml reaction volume over a 2-min
period at 25 °C and standardized relative to the amount of
glyoxalase II in the extract.
-D-galactopyranoside at
A600 of 0.7 and transferred to a shaker at
15 °C. After 24 h, the cells were harvested, washed three times
with Nanopure water and three times with 10 mM MOPS, pH
7.2, and a crude protein extract was obtained as described above. The
extracts were diluted with Nanopure water to a concentration of 2 mg/ml
and analyzed for zinc, cobalt, and iron content using ICP.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
strand (23). The drastic change in protein solubility
likely results from dramatic structural changes. All other mutant
proteins were successfully expressed in soluble, active forms.
Activity of crude extracts of wild type and mutant glyoxalase II
Kinetic constants and metal content of purified wild type and mutant
glyoxalase II
View larger version (10K):
[in a new window]
Fig. 1.
The fluorescence emission spectra of wild
type and mutant glyoxalase II obtained with an excitation wavelength of
280 nm. A, H54N (---) and wild type (···);
B, C140A (---) and wild type (···); C, D58C
(---) and wild type (···). The error bar represents
the error at the maximum point of emission observed for three
trials.
-lactamases, which utilize a
rate-limiting proton transfer exhibit a
kH/kD = 2.5-3.5 (33, 34). The
fact that the D58C mutation did not significantly lower the solvent
isotope effect indicates that Asp-58 does not participate in a
rate-limiting proton transfer in the catalytic mechanism of glyoxalase II.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-lactamases, which only bind zinc (38), even though
the metal-binding ligands of glyoxalase II most closely resemble those
of the metallo-
-lactamase L1 from S. maltophilia (24). It
is interesting that two enzymes with essentially the same metal-binding
ligands can exhibit such different metal-binding preferences.
-lactamases and
leucine aminopeptidase, appear to utilize a hydroxide, arising from a
bridging water molecule in the nucleophilic attack of substrate (33,
40). Residues that hydrogen-bond to the nucleophilic hydroxide usually
play a critical role in orienting the hydroxide and holding it in a
fixed position that reduces the entropic barrier for nucleophilic
attack on the substrate (41). We predict that Asp-58 is the critical
residue for hydroxide positioning in Arabidopsis glyoxalase
II. The aspartic acid at position 58, in human glyoxalase II, has also
been predicted to hydrogen-bond to the bridging hydroxide through its
carbonyl oxygen (23). There are several other metalloenzymes, including
the metallo-
-lactamases, that have a homologous aspartic acid in their binuclear metal-binding sites (33). In addition to removing the
possibility of a proton transfer, changing an aspartic acid to cysteine
removes the hydrogen bond partner of the nucleophilic hydroxide. The
D58C substitution caused a reduction in activity greater than 99%,
while still maintaining wild type levels of metal. This suggests an
important role for Asp-58 in orienting the nucleophilic hydroxide for
attack. The fact that the enzyme binds normal levels of metal and
exhibits wild type fluorescence emission patterns suggests that the
change in activity is not due to major structural changes. The solvent
isotope effect data do not support a role for this residue in a
rate-limiting proton transfer event but do not rule out an orientation
role for Asp-58. The homologous aspartic acid (residue 103) in the
metallo-
-lactamase from Bacteroides fragilis has been
proposed to play an identical role in orienting the nucleophilic
hydroxide for attack of the substrate nitrocefin, rather than
participating in the reaction (33, 34).
-lactamases and leucine
aminopeptidase (33, 38, 40). Once SLG has been bound and oriented for
nucleophilic attack, the hydroxide, now bound only to metal 2 and held
in position by Asp-58, can attack the lactoyl carbonyl. After initial
nucleophilic attack, a tetrahedral transition state is formed.
View larger version (18K):
[in a new window]
Fig. 2.
The proposed mechanism for SLG
hydrolysis by glyoxalase II. Metal 1 (M1),
metal 2 (M2), Asn-178, Lys-142, Cys-140, and
Arg-248 participate in the initial binding of SLG. The nucleophilic
hydroxide, held in position by Asp-58, attacks the lactoyl carbonyl,
yielding a tetrahedral transition state. Ligand exchange occurs at
metal 1, where D-lactic acid is replaced by
water, and a water on metal 1 protonates the glutathione leaving group.
Numbering of amino acids corresponds to the Arabidopsis
GLX2-2 sequence (GenBankTM accession number O24496).
GS, glutathione.
The tetrahedral transition state is predicted to be stabilized by
interactions with metal 2, at the sulfur of glutathione and metal 1 at
the former lactoyl carbonyl. In leucine aminopeptidase, a lysine
residue homologous to Lys-142 of glyoxalase II has been shown to
participate in transition state stabilization (40). Replacing this
lysine with alanine in leucine aminopeptidase resulted in a 99%
reduction in activity (40). Substituting Lys-142 with alanine in
glyoxalase II did not lead to as drastic a reduction in activity
(55%), suggesting that Lys-142 is not stabilizing the transition
state. However, it is possible that the binuclear metal center in
GLX2-2 might accommodate for the loss of Lys-142. Glyoxalase II may
complete the hydrolysis reaction in a manner similar to the B. fragilis metallo--lactamase. In this enzyme, it was predicted
that Zn2-OH2, serving as an acid, protonates the negatively charged nitrogen of the leaving group, inducing product
release. Ligand exchange occurs at Zn1 as the acyl group of
the product is replaced with a solvent water molecule, regenerating the
active site of the metallo-
-lactamase (33). In glyoxalase II, metal
2, serving as an acid, may protonate the negatively charged sulfur of
the leaving group, inducing glutathione release. Ligand exchange may
occur at metal 1 as lactic acid is replaced with the hydroxide on metal
2, regenerating the active site.
In conclusion, the characterization of site-directed mutations of metal
ligand and substrate-binding residues has provided important new
information about the nature of the binuclear metal center and the
mechanism of SLG hydrolysis of glyoxalase II. They have identified
critical active site residues of glyoxalase II and helped differentiate
the roles of several highly conserved residues in metal binding,
substrate binding, and catalysis. Specifically, we have shown for the
first time that the binuclear metal center of glyoxalase II can readily
accommodate different metal ions, including both zinc and iron and that
metal preference is readily influenced by both first and second shell
metal ligands. The two metal ions are absolutely essential for full
enzymatic activity. Both metal ions appear to participate in substrate
binding, transition state stabilization, and the hydrolysis reaction.
We provide strong evidence to support the hypothesis that Asp-58 plays
a critical role in orienting the hydroxide for attack of SLG. In
addition we have confirmed the role of residues Arg-248, Lys-142, and
Asn-178 in substrate binding, and we demonstrated that contrary to
predictions made based on the crystal structure that Cys-140 does not
have a major role in substrate binding.
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ACKNOWLEDGEMENT |
---|
We thank Daniel N. Sobieski for helping with the solvent isotope effect studies on wild type glyoxalase II.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Science Foundation Grants MCB-9817083 and CHE-9619696.The costs of publication of this article were defrayed in part by the payment of page charges. The 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 Figs. 1\N3).
To whom correspondence should be addressed. Tel.: 513-529-1659;
Fax: 513-529-5715; E-mail: makaroca@muohio.edu.
Published, JBC Papers in Press, November 20, 2000, DOI 10.1074/jbc.M005090200
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ABBREVIATIONS |
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The abbreviations used are: SLG, S-D-lactoylglutathione; PCR, polymerase chain reaction; MOPS, 4-morpholinepropanesulfonic acid; ICP, inductively coupled plasma.
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REFERENCES |
---|
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---|
1. | Thornalley, P. (1990) Biochem. J. 269, 1-11[Medline] [Order article via Infotrieve] |
2. | Rahman, A., Shahabuddin, A., and Hadi, S. (1990) J. Biochem. Toxicol. 5, 161-166[Medline] [Order article via Infotrieve] |
3. |
Lo, T.,
Westwood, M.,
McLellan, A.,
Selwood, T.,
and Thornalley, P.
(1994)
J. Biol. Chem.
269,
32299-32305 |
4. | Papoulis, A., Al-Abed, Y., and Bucala, R. (1995) Biochemistry 34, 648-655[Medline] [Order article via Infotrieve] |
5. | Thornalley, P. (1993) Mol. Aspects Med. 14, 287-371[CrossRef][Medline] [Order article via Infotrieve] |
6. | Thornalley, P. J. (1996) Gen. Pharmacol. 27, 565-573[CrossRef][Medline] [Order article via Infotrieve] |
7. | Allen, R., Lo, T., and Thornalley, P. J. (1993) Biochem. Soc. Trans. 21, 535-540[Medline] [Order article via Infotrieve] |
8. | Elia, A. C., Chyna, M. K., Principato, G. B., Giovannini, E., Rosi, G., and Norton, S. J. (1995) Biochem. Mol. Biol. Int. 35, 763-771[Medline] [Order article via Infotrieve] |
9. | Norton, S. J., Elia, A. C., Chyan, M. K., Gillis, G., Frenzel, C., and Principata, G. B. (1993) Biochem. Soc. Trans. 21, 545-549[Medline] [Order article via Infotrieve] |
10. | Allen, R., Lo, T., and Thornalley, P. (1993) Eur. J. Biochem. 213, 1261-1267[Abstract] |
11. | Talesa, V., Uotila, L., Koivusalo, M., Principato, G. B., Giovannini, E., and Rosi, G. (1989) Biochim. Biophys. Acta 993, 7-11[Medline] [Order article via Infotrieve] |
12. | Murata, K., Inoue, Y., Watanabe, K., Fukuda, Y., Saikusa, T., Shimosaka, M., and Kimura, A. (1986) Agric. Biol. Chem. 50, 135-142 |
13. | Vander Jagt, D. L. (1993) Biochem. Soc. Trans. 21, 522-527[Medline] [Order article via Infotrieve] |
14. | Talesa, V., Rosi, G., Contenti, S., Mangiabene, C., Lupattelli, M., Norton, S. J., Giovannini, E., and Principato, G. B. (1990) Biochem. Int. 22, 1115-1120[Medline] [Order article via Infotrieve] |
15. |
Ridderstrom, M.,
Saccucci, F.,
Hellman, U.,
Bergman, T.,
Principato, G.,
and Mannervik, B.
(1996)
J. Biol. Chem.
271,
319-323 |
16. | Maiti, M. K., Krishnasamy, S., Owen, H. A., and Makaroff, C. A. (1997) Plant Mol. Biol. 35, 471-481[CrossRef][Medline] [Order article via Infotrieve] |
17. | Ridderstrom, M., and Mannervik, B. (1997) Biochem. J. 322, 449-454[Medline] [Order article via Infotrieve] |
18. | Crowder, M. W., Maiti, M. K., Banovic, L., and Makaroff, C. A. (1997) FEBS Lett. 418, 351-354[CrossRef][Medline] [Order article via Infotrieve] |
19. | Uotila, L. (1973) Biochemistry 12, 3944-3951[Medline] [Order article via Infotrieve] |
20. | Crowder, M. W., Wang, Z., Franklin, S. L., Zovinka, E. P., and Benkovic, S. J. (1996) Biochemistry 35, 12126-12132[CrossRef][Medline] [Order article via Infotrieve] |
21. | Concha, N. O., Rasmussen, B. A., Bush, K., and Herzberg, O. (1996) Structure 4, 823-836[Medline] [Order article via Infotrieve] |
22. | Melino, S., Capo, C., Dragani, B., Aceto, A., and Petruzzelli, R. (1998) Trends Biochem. Sci. 23, 381-382[CrossRef][Medline] [Order article via Infotrieve] |
23. | Cameron, A. D., Ridderstrom, M., Olin, B., and Mannervik, B. (1999) Structure 7, 1067-1078[CrossRef][Medline] [Order article via Infotrieve] |
24. | Ullah, J. H., Walsh, T. R., Taylor, I. A., Emery, D. C., Verma, C. S., Gamblin, S. J., and Spencer, J. (1998) J. Mol. Biol. 284, 125-136[CrossRef][Medline] [Order article via Infotrieve] |
25. | Ball, J., and Vander Jagt, D. (1981) Biochemistry 20, 899-905[Medline] [Order article via Infotrieve] |
26. | Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K., and Pease, L. R. (1989) Gene (Amst.) 77, 51-59[CrossRef][Medline] [Order article via Infotrieve] |
27. | Tabor, S., and Richardson, S. C. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 1074-1078[Abstract] |
28. | Segel, I. H. (1993) Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady State Enzyme Systems , pp. 544-555, John Wiley & Sons, Inc., New York |
29. | D'souza, V. M., and Holz, R. C. (1999) Biochemistry 38, 11079-11085[CrossRef][Medline] [Order article via Infotrieve] |
30. | Dragani, B., Cocco, R., Ridderstrom, M., Stenberg, G., Mannervik, B., and Aceto, A. (1999) J. Mol. Biol. 29, 481-490 |
31. | Geeganage, S., and Frey, P. A. (1999) Biochemistry 38, 13398-13406[CrossRef][Medline] [Order article via Infotrieve] |
32. | Schowen, K., and Schowen, R. (1982) Methods Enzymol. 87, 551-606[Medline] [Order article via Infotrieve] |
33. | Wang, Z., Fast, W., and Benkovic, S. J. (1999) Biochemistry 38, 10013-10023[CrossRef][Medline] [Order article via Infotrieve] |
34. | Yanchak, M., Taylor, R., and Crowder, M. (2000) Biochemistry 39, 11330-11339[CrossRef][Medline] [Order article via Infotrieve] |
35. | Mondragon, A., Griffith, E. C., Sun, L., Xiong, F., Armstrong, C., and Liu, J. O. (1997) Biochemistry 36, 4934-4942[CrossRef][Medline] [Order article via Infotrieve] |
36. | Twitchett, M. B., and Sykes, A. G. (1999) Eur. J. Inorg. Chem. 12, 2105-2115[CrossRef] |
37. | Wang, X., Randall, C. R., True, A. E., and Que, J., L. (1996) Biochemistry 35, 13946-13954[CrossRef][Medline] [Order article via Infotrieve] |
38. | Crowder, M. W., and Walsh, T. R. (1999) in Antimicrobial Agents and Chemotherapy, Vol. 3, Part 1 (Pandalai, S. G., ed) , pp. 105-132, Research Signpost, Trivandrum, India |
39. | Creighton, D. J., Migliorni, M., Pourmotabbed, T., and Guha, M. K. (1988) Biochemistry 27, 7376-7384[Medline] [Order article via Infotrieve] |
40. |
Strater, N.,
Sun, L.,
Kantrowitz, E. R.,
and Lipscomb, W. N.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
11151-11155 |
41. | Christianson, D. W., and Cox, J. D. (1999) Annu. Rev. Biochem. 68, 33-57[CrossRef][Medline] [Order article via Infotrieve] |