From the
The conformational mobility of glyoxalase I (Glx I) during
catalysis has been probed using stable analogs of the enediol
intermediate that forms along the reaction pathway: GSC(O)N(OH)R, where
GS = glutathionyl and R = CH
The isomerase glyoxalase I (Glx I,(
Unfortunately, the recently
available primary structures of human Glx I (6, 7) and Pseudomonas
putida Glx I
(8) do not show high levels of sequence
homology with other enzymes of known structure and function.
Nevertheless, whether by chance or by heredity, Glx I catalyzes a
reaction that is stereochemically and mechanistically analogous to that
catalyzed by triosephosphate isomerase (TIM), which interconverts
dihydroxyacetone phosphate and D-glyceraldehyde 3-phosphate
via a cis-enediol(ate) intermediate
(9) . Interestingly,
a significant fraction of the intracellular methylglyoxal that is
detoxified by Glx I has been calculated to arise from the incorrect
processing of the enzyme-bound enediol(ate) intermediate formed along
the reaction pathway of TIM
(10) . Conceivably, the two enzymes
might be distantly related through evolution.
During the course of
studies aimed at identifying catalytically important conformational
changes in Glx I, we have uncovered evidence for a flexible peptide
loop near the active site. Proteolytic susceptibility measurements and
mutagenesis experiments suggest that the loop is functionally analogous
to the ``catalytic loop'' of TIM. This is the first evidence
that Glx I and TIM are related at the level of enzyme structure and
function.
Circular dichroism (CD) spectra
were obtained using a Jasco J-710 spectropolarimeter. The CD spectra
were corrected for contributions due to buffer and at least two scans
were averaged for each spectrum.
In order to
determine the sites of proteolytic cleavage, the peptide fragments were
transferred from SDS-PAGE gels to polyvinylidene difluoride membranes
by electroblotting, stained with Coomassie Brilliant Blue R-250 and
subjected to N-terminal analysis. Identification of the sites of
proteolytic cleavage was based on analyzing at least 10 amino acids at
the N terminus of the peptides.
Our interest in the conformational mobility of Glx I
originated with the problem of designing tight-binding inhibitors of
Glx I that also serve as substrates for the hydrolase Glx
II
(3, 4) . Recently, we suggested that inhibitors of
this type might function as potential tumor-selective anticancer agents
because of the critical role that glyoxalase plays in removing toxic
methylglyoxal from cells, and because of the abnormally low levels of
Glx II activity in many types of cancer cells. Thus, tumor selectivity
could arise from the reduced ability of cancer cells to hydrolyze the
inhibitors.
In support of the feasibility of this anticancer
strategy, we also reported that S-N-hydroxycarbamoyl esters of
GSH (e.g.
1-4) serve as slow substrates
for bovine liver Glx II and strong competitive inhibitors of human
erythrocyte Glx I. Tight binding to Glx I appears to result from the
fact that these compounds are stable analogs of the tightly bound
enediol(ate) intermediate that forms along the reaction
pathway
(3, 4) , shown in Fig. D1. Moreover, the
high affinity of Glx I for the enediol analogs, versus
substrate/product, appears to result from differential binding of
functional groups that are both near to and far from the
reaction center
(4) . This implies that different conformational
forms of Glx I are involved in binding substrate, intermediate and
product species. Indeed, Mannervik and co-workers
(17) previously proposed that catalysis might be coupled to a
significant change in enzyme conformation. Their hypothesis was based
on the observation that binding of the competitive inhibitor
S-(p-bromobenzyl)glutathione to the active site of
human erythrocyte Glx I induces a 10% decrease in protein fluorescence,
while binding of the product S-D-lactoylglutathione
does not affect protein fluorescence.
Consistent with the presence of
a flexible peptide loop near the active site of human Glx I, the
presence of either
2 or
3 diminishes
the rate of proteolytic cleavage by Pronase E (e.g.Fig. 2
). In the absence of enediol analog, Pronase E cleaves
Glx I into two major peptide fragments that migrate on SDS-PAGE gels at
In
order to test this hypothesis, site-directed mutagenesis was used to
construct a mutant form of P. putida Glx I in which Glu-93 is
replaced by Asp, giving E93D Glx I. We reasoned that if Glu-93
contributes the active site base the mutant enzyme would show little
catalytic activity, based on the fact that the specific activity of
E165D TIM is about 10
We thank Kevin Lawton of the University of Maryland
Blood Bank for supplying the outdated human erythrocytes used in the
preparation of Glx I, and Ray Sowder of the Frederick Cancer Center for
N-terminal analyses.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(1),
C
H
(2), C
H
Cl
(3), or C
H
Br (4). For human
erythrocyte Glx I, catalysis is unlikely to be coupled to major changes
in protein secondary structure, as the circular dichroism spectrum of
the enzyme (190-260 nm) is insensitive to saturating
concentrations of either enediol analog or
S-D-lactoylglutathione, the product of the Glx I
reaction. However, a small conformational change is indicated by the
fact that binding of enediol analog to the active site decreases
intrinsic protein fluorescence by 11%, and protects the enzyme from
proteolytic cleavage by Pronase E at the C-side of Ala-92 and Leu-93.
In contrast, binding of S-D-lactoylglutathione does
not affect protein fluorescence, and increases the rate of proteolytic
cleavage by 1.5-fold. These observations are consistent with a model of
catalysis in which a flexible peptide loop folds over and stabilizes
the enediol intermediate bound to the active site. Indeed, a highly
conserved sequence of amino acid residues is found near the proteolytic
cleavage sites, for human Glx I(100-111) and Pseudomonas
putida Glx I(93-105), that shows significant sequence
homology to the ``catalytic loop'' of chicken muscle
triosephosphate isomerase (TIM)(165-176). The active site base
(Glu-165) of TIM, which catalyzes the proton transfer reaction during
isomerization, corresponds in position to Glu-93 of P. putida Glx I. Consistent with a functional role for Glu-93, a mutant
enzyme in which Glu-93 is replaced by Asp shows no detectable catalytic
activity.
)
EC
4.4.1.5) is recognized to be a highly efficient catalyst for
interconverting glutathione (GSH)-methylglyoxal thiohemiacetal and
S-D-lactoylgluta-thione via a cis-enediol
proton-transfer mechanism (for reviews, see Refs. 1 and 2)
(Fig. R1).
Figure R1:
Reaction 1.
This enzyme, together with the thioester hydrolase
glyoxalase II (Glx II), plays a vital role in chemically removing
cytotoxic methylglyoxal from cells as D-lactate. From a
practical perspective, Glx I has been the subject of renewed interest
as a possible anticancer target because of speculation that the subtle
differences in the activities of the glyoxalase enzymes between normal
cells and cancer cells might serve as the basis for the development of
Glx I inhibitors that are tumor-selective
(3, 4) , and
because of the recent demonstration that GSH-based inhibitors of Glx I
can be efficiently delivered into leukemia cells as the
[glycyl,-glutamyl]diethyl
esters
(5) . Thus, obtaining detailed structural and mechanistic
information on Glx I is of considerable importance, in order to provide
a solid basis for inhibitor design.
Materials
Pronase E (Type XIV) from
Streptomyces griseus,
S-D-lactoylglutathione, and phenylmethylsulfonyl
fluoride were purchased from Sigma and used without further
purification. The enediol analogs were synthesized as described
previously
(3, 4) . Glx I was purified to apparent
homogeneity from human erythrocytes, following the procedure of
Aronsset al.(11) . The purified enzyme was at
least 95% pure, as judged by SDS-PAGE. All other reagents were of the
highest purity commercially available.
Spectrophotometric Methods
Ligand-induced
quenching of Glx I fluorescence was measured using an SLM 48000S
spectrofluorimeter (25 °C). The fluorescence intensity of solutions
containing Glx I and ligand was corrected for contributions due to
buffer, and to inner filter effects arising from absorption of exciting
light by ligand. The inner filter correction was obtained from the
decrease in the fluorescence intensity of a standard solution of
L-tryptophan in the presence of known concentrations of
ligand. The dissociation constant (K ) of
the ligand with the enzyme was obtained from nonlinear regression
analysis of the fluorescence titration data using the following
equation: F
= F
- R(F
-
F
), where F
is the
fluorescence intensity at a given concentration of the ligand and
F
and F
are the fluorescence
intensities at zero and at saturating concentrations of ligand,
respectively. The term R is the fraction of enzyme bound to
ligand, defined as (0.5 (([E
] +
[L
] +
K
) -
(([E
] +
[L
] +
K
)
-
4[E
][L
])
))/[E
],
where [L
] is the total concentration of
ligand and [E
] is the total
concentration of Glx I active sites.
Proteolytic Susceptibility
The cleavage of human
Glx I by Pronase E was followed by SDS-PAGE. Aliquots were withdrawn
from digestion mixtures as a function of time and quenched by the
addition of phenylmethylsulfonyl fluoride (in 100% ethanol) to a final
concentration of 9 mM. These fractions were then
heat-denatured in the presence of 2-mercaptoethanol and resolved on
16.5% SDS-polyacrylamide minigels following the method of Schagger and
von Jagow
(12) . The molecular weight markers (Promega) spanned
the range 2.5-31 kDa. The amount of protein in each lane was
quantitated by the use of a Molecular Dynamics Laser Scanning Personal
Densitometer. Pronase E activity was measured using casein as a
substrate, following the protocol supplied by Sigma.
Mutagenesis
A mutant P. putida Glx I with
Asp-93 replacing Glu-93 (E93D) was prepared using plasmid pBTac1/glx I
described elsewhere
(8) . A 1.5-kilobase pair
HindIII-EcoRI restriction fragment containing the Glx
I gene was inserted into the HindIII/EcoRI site of
M13mp8. Recombinant M13bacteriophage was propagated in Escherichia
coli strain TG1, and single-stranded M13 DNA was
purified
(13) . Oligonucleotide-directed mutagenesis was
performed by the method of Eckstein
(14) , as described
elsewhere
(15) . Mutants were identified by single-base sequence
analysis and confirmed by performing a full sequencing reaction. The
Asp-93 Glx I mutant gene was isolated from M13mp8 on a 1.5-kilobase
pair HindIII-EcoRI fragment and served as a template
for polymerase chain reaction amplification to obtain a 535-base pair
fragment containing the Glx I gene. The upstream oligonucleotide placed
an NdeI restriction site right before the start codon of Glx
I. The downstream oligonucleotide placed a BamHI site 7
nucleotides from the stop codon (TAA) of Glx I. The E93D Glx I mutant
gene was isolated from the polymerase chain reaction mixture and
inserted into the NdeI/BamHI site of a pET-15b vector
(Novagen). Glx I was purified from E. coli BL21(DE3)
transformed with this vector, using methods described
elsewhere
(16) .
Figure D1:
Diagram 1.
Fluorescence and Circular Dichroism Measurements
In
order to further evaluate this hypothesis, the enediol analog
(
1) was tested for its ability to quench the intrinsic
fluorescence of human erythrocyte Glx I. Increasing concentrations of
1 progressively decrease protein fluorescence to about
89% of its original value (Fig. 1). Regression analysis of the
fluorescence intensity data gave an apparent dissociation constant
(K= 0.92 ± 0.32
µM) in approximate agreement with the competitive
inhibition constant of
1 with Glx I
(K
= 1.7 ± 0.1
µM(4) ). S-D-Lactoylglutathione
does not produce fluorescence quenching under saturating conditions, in
accordance with the original report of Mannervik and
co-workers
(17) . To the extent that 1 is a
reasonable analog of the enediol(ate) intermediate, the ligand-induced
fluorescence quenching effects are indicative of a catalytically
important change in enzyme conformation going from bound intermediate
to bound product. Nevertheless, this conformational change must be
subtle, since the circular dichroism spectrum of the enzyme is
relatively insensitive to the presence of saturating concentrations of
either
1 or of the aromatic enediol analog
4 (data not shown).
Figure 1:
Fluorescence intensity of an aqueous
solution of human erythrocyte Glx I (1 µM in active sites)
as a function of
[S-(N-methyl-N-hydroxycarbamoyl)glutathione(1)]:
= 295 nm,
= 346 nm.
The solidline through the data is the best fit line
obtained from regression analysis of the data (see ``Experimental
Procedures''). Inset, fluorescence emission spectrum
(
= 295 nm) of an aqueous solution of Glx I (1
µM in active sites) in presence (dottedline) and in the absence (solidline)
of a near-saturating concentration of 1 (7.73
µM). Conditions: 10 mM Tris-HCl (pH 7.8), 25
°C.
Proteolytic Susceptibility Measurements
The
movement of a flexible loop of contiguous amino acid residues near an
active site is one type of subtle conformational change that has been
proposed to accompany catalysis by such diverse enzymes as
phosphoglycerate mutase (18), lactate dehydrogenase
(19) , and
TIM
(20) . This prompted a search for an analogous peptide loop
near the active site of Glx I by determining the susceptibility of the
enzyme to proteolytic cleavage in the presence and in the absence of
enediol analogs. For example, the catalytically important peptide loop
near the active site of TIM is known to be susceptible to proteolytic
cleavage by subtilisin
(21) .
9.5 and
5.1 kDa. Near-saturating concentrations of
3 decrease the first order rate constant for loss of
Glx I from a value of 3.0
10
min
to a value of 9.3
10
min
(Fig. 3). Since Pronase E is not
inhibited by
3 at the concentration used in the
proteolytic digestion studies, the protective effect of
3 must be due to binding to Glx I. In contrast, near-saturating
concentrations of the product S-D-lactoylglutathione
actually increases the rate of loss of intact enzyme
(k
= 4.5
10
min
) ( Fig. 2and Fig. 3). Edman
analysis of the 9.5-kDa species showed that it was actually composed of
two peptides resulting from cleavage of the enzyme at the C-side of
Ala-92 and Leu-93 (Fig. 4). Analysis of the 5.1-kDa fragment
showed that it arose from cleavage of the enzyme at the C-side of
Ala-23, near to the N terminus of the untreated enzyme
(Ser-18).
(
)
No evidence could be found for
proteolytic cleavage of P. putida Glx I by Pronase E. This
appears to reflect some critical difference in the primary structures
of the human and bacterial enzymes at the sites of proteolytic cleavage
of the human enzyme (Fig. 4).
Figure 2:
SDS-PAGE gels showing the time course of
proteolytic digestion of human erythrocyte Glx I (443 µg/ml) by
Pronase E (44 µg/ml, 2.2 10
units) in
solutions containing no added ligand (panel A), containing a
near-saturating concentration of
S-D-lactoylglutathione (17.3 mM) (panel
B) and containing a near-saturating concentration of
S-(N-4-chlorophenyl-N-hydroxycarbamoyl)glutathione
(3) (19.3 µM) (panel C). Conditions: 30
mM Tris-HCl buffer (pH 7.5), 37
°C.
Figure 3:
First-order rate plots for the loss of
intact human erythrocyte Glx I (443 µg/ml), due to the action of
Pronase E (44 µg/ml, 2.2 10
units), in
solutions containing no added ligand (cir-cles), containing a
near-saturating concentration of
S-(N-4-chlorophenyl-N-hydroxycarbamoyl)glutathione
(3) (19.3 µM) (squares), and containing
a near-saturating concentration of
S-D-lactoylglutathione (17.3 mM)
(triangles). Conditions: 30 mM Tris-HCl buffer (pH
7.5), 37 °C.
Figure 4:
Amino acid sequences deduced from the
published gene structures of human colon/human leukocyte Glx I (6, 7)
(upper) and P. putida Glx I (8) (lower).
a, N terminus of purified human erythrocyte Glx I deduced from
Edman analysis. b, N terminus of the 5.1-kDa peptide
fragment generated from Pronase E digestion of human erythrocyte Glx I.
c, N termini of the two
9.5-kDa peptide fragments
generated from Pronase E digestion of human erythrocyte Glx I.
d, mutation of Glu-93 to Asp-93 in P. putida Glx I
dramatically reduces catalytic activity. The overlined region
shows significant sequence similarity to the catalytic loop of
TIM.
In native proteins, proteolytic
cleavage sites are normally observed at or near loops or turns
characterized by high flexibility. This was clearly demonstrated by
Fontana and co-workers
(22) when they established an exact
correspondence between the sites of proteolytic cleavage in thermolysin
and regions of high mobility in the enzyme protein, indicated by the
poor resolution of these regions in the x-ray structure of the
crystalline enzyme. Thus, Glx I probably has at least one such flexible
loop containing Leu-93 and Ser-94 (Fig. 4). With respect to the
minimum size of the loop, proteases generally bind at primary and
secondary sites of peptide segments containing 6-8 amino acid
residues
(23) . The partial protection from proteolysis afforded
by the enediol analogs can be explained by closure of the loop over
bound enediol analog. The fact that
S-D-lactoylglutathione stimulates proteolytic
cleavage implies that this ligand binds to and stabilizes a loop-open
form of Glx I that is more accessible to binding by Pronase E. These
observations are consistent with a model of catalysis in which a
``catalytic'' loop near the active site functions to
stabilize the bound enediol(ate) intermediate, and conversion to
S-D-lactoylglutathione is coupled to opening of the
loop in order to facilitate product release. An analogous role has been
proposed for the peptide loop near the active site of TIM
(20) .
Mutagenesis
Indeed, TIM and Glx I may be distantly
related.(
)
The sites of proteolytic cleavage at
Ala-92/Leu-93 in human Glx I are near a region(100-111) showing
significant sequence homology to the catalytic loop of TIM
(Fig. 5).
Figure 5:
Comparison of the catalytic loop region of
TIM with an analogous region in Glx I.
This region is one of several highly conserved
regions between human and P. putida Glx I, implying an
important role in the structure/function of the enzyme (Fig. 4).
The comparison with the TIM sequence also suggests that Glu-100 of
human Glx I and Glu-93 of P. putida Glx I might contribute the
active site base involved in the enediol-proton-transfer mechanism
proposed for Glx I (Fig. R1). These residues correspond in
position to Glu-165 of TIM. The carboxyl group of Glu-165 undoubtedly
catalyzes the proton-transfer reaction associated with isomerization,
on the basis of affinity labeling
(25, 26, 27) and site-directed mutagenesis studies
(28) .
-fold lower than that of wild-type
TIM
(28) . Indeed, E93D Glx I is catalytically inactive within
the limits of detection: specific activity
0.02% of wild-type
enzyme. The mutant enzyme appears to be fully folded in solution, as
the CD spectrum is similar to that of wild-type Glx I. These
observations are consistent with a catalytic role for Glu-93 in the
mechanism of action of P. putida Glx I.
Conclusions
Glx I appears to contain a flexible
peptide loop near the active site that is structurally and functionally
analogous to the catalytic loop of TIM. If correct, this would be
evidence for the long sought-after linkage between the glyoxalase and
glycolytic pathways, recently discussed by Vander Jagt
(2) .
24.5 kDa) was
observed to decrease to a mass of
19.5 kDa after overnight
dialysis of the column effluent. Nevertheless, the specific activities
of the 24.5- and 19.5-kDa species were found to be similar: 1,640 and
1,360 units/mg, respectively. These values are also close to the
literature value (1,000 units/mg), for which the molecular mass of the
human enzyme was reported to be
26 kDa (SDS-PAGE) (11).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.