(Received for publication, January 23, 1995; and in revised form, July 31, 1995)
From the
Pigeon liver malic enzyme was rapidly inactivated by micromolar
concentration of Fe in the presence of ascorbate at
neutral pH. The inactivated enzyme was subsequently cleaved by the
Fe
-ascorbate system at the chemical bond between
Asp
and Ile
(Wei, C. H., Chou, W. Y.,
Huang, S. M., Lin, C. C., and Chang, G. G.(1994) Biochemistry,
33, 7931-7936), which was confirmed by site-specific mutagenesis
(Wei, C. H., Chou, W. Y., and Chang, G. G.(1995) Biochemistry 34, 7949-7954). In the present study, at neutral pH,
Cu
was found to be more reactive in the oxidative
modification of malic enzyme and the enzyme was cleaved in a similar
manner as Fe
did. At acidic pH, however,
Fe
was found to be ineffective in oxidative
modification of the enzyme. Nevertheless, Cu
still
caused enzyme inactivation and cleaved the enzyme at
Asp
-Gly
,
Asp
-Pro
, or
Asp
-Asp
. Mn
and L-malate synergistically protect the enzyme from
Cu
inactivation at acidic pH. Cu
is
also a competitive inhibitor versus Mn
in
the malic enzyme-catalyzed reaction with K
value 70.3 ± 5.8 µM. The above results
indicated that, in addition to the previously determined Asp
at neutral pH, Asp
, Asp
, and
Asp
are also the coordination sites for the metal binding
of malic enzyme. We suggest that the mechanism of affinity modification
and cleavage of malic enzyme by the Cu
-ascorbate
system proceed in the following sequence. First, Cu
binds with the enzyme at the Mn
binding site
and reduces to Cu
by ascorbate. Next, the local oxygen
molecules are reduced by Cu
, thereby generating
superoxide or other reactive free radicals. These radicals interact
with the susceptible essential amino acid residues at the metal-binding
site, ultimately causing enzyme inactivation. Finally, the modified
enzyme is cleaved into several peptide fragments, allowing the
identification of metal site of the enzyme. The pH-dependent different
specificities of metal-catalyzed oxidation system may be generally
applicable for other enzymes or proteins.
Cytosolic malic enzyme ((S)-malate:NADP oxidoreductase (oxaloacetate-decarboxylating), EC 1.1.1.40)
catalyzes the divalent metal ion-dependent reversible oxidative
decarboxylation of L-malate to give CO
and
pyruvate, with concomitant reduction of NADP
to NADPH ().
The role of metal ion in the enzymatic reaction entails
providing a bridge between malate and the enzyme and functions as a
second sphere complex with the substrate (Hsu et al., 1976).
Therefore, identification of the ligands for metal binding is
ultimately important in understanding the structure-function
relationship of this enzyme. In most metalloproteins, the amino acid
residues involved in metal binding are dispersed along the complete
sequence (Regan, 1993). Without a three-dimensional crystal structure
available, affinity cleavage at the putative metal-binding site by the
metal-catalyzed oxidation system (MCO) ()may be the optimal
approach of reaching the above goal. Using this technique with the
Fe
-ascorbate system, Asp
is
successfully identified in our previous study as one of the
metal-binding sites (Wei et al., 1994), as confirmed by
site-directed mutagenesis (Wei et al., 1995). In that study,
some divalent metal ions were found to be capable of providing
protection of the enzyme against Fe
-induced
inactivation. Among the divalent metal ions, only Cu
was found to accelerate the
Fe
-ascorbate-induced enzyme inactivation rate.
In
this work, the inactivation of malic enzyme by
Cu-ascorbate system is investigated. We demonstrate,
for the first time, that at different pH values
Cu
-ascorbate system shows different specificities in
protein modification and peptide bond cleavage. Taking the advantage of
this selectivity, three more metal binding ligands of pigeon liver
malic enzyme are successfully identified. Novel sequence motifs for the
metal-binding site of malic enzyme are also deduced.
For detecting the peptide bond cleavage, the samples withdrawn from the reaction mixture were added to EDTA solution (4 mM) to prevent further reaction from occurring. Next, the protein samples were subjected to SDS-PAGE for separating the peptide fragments. The resulting gel pattern was quantified by densitometric analysis and the isolated peptide fragments were then transblotted to an Immobilon-P membrane and subjected to automatic amino acid sequence analysis as described previously (Wei et al., 1994).
where v is the observed enzyme activity, V is the maximum enzyme activity, K
denotes the Michaelis constants for
Mn
, and K
denotes
the inhibition constant for Cu
.
Figure 1:
Inactivation of pigeon liver malic
enzyme by Cu- or Fe
-ascorbate
systems at different pH values. The reaction mixture contained malic
enzyme (0.97 µM) in 66.7 mM sodium acetate (pH
5.0,
,
) or triethanolamine-HCl (pH 7.4,
,
)
buffer, 6 µM cupric nitrate (
,
) or 20
µM ferrous sulfate (
,
), and 20 mM ascorbate. Incubation was at 0 °C.
Inactivation
of malic enzyme by Cu required ascorbate in the
system; Cu
or ascorbate alone did not cause any
inactivation. Cu
was much less effective;
Cu
(6 µM)-ascorbate (20 mM)
caused only 10% inactivation in 1 h under conditions that caused
>95% inactivation by the Cu
-ascorbate system.
Figure 2:
Effect of pH on the
Cu-induced inactivation of pigeon liver malic enzyme.
Experimental conditions were the same as in Fig. 1with 6
µM Cu
, except that different buffers
were used (Bis-Tris for pH 4.0-7.0,
; Bis-Tris-propane for
pH 6.3-9.0,
). The enzyme activity lost after a 1-h
incubation was measured.
Figure 3:
Effect of Cu
concentration on the inactivation of pigeon liver malic enzyme.
Experimental conditions were the same as in Fig. 1at pH 5.0,
except that the Cu
concentrations used were:
,
2 µM;
, 3 µM;
, 4
µM;
, 5 µM;
, 6
µM.
Similar to
Fe-induced inactivation, Cu
-induced
inactivation could be stopped by EDTA (4 mM), which, however,
did not reverse the already inactivated enzyme activity.
Figure 4:
Protection of pigeon liver malic enzyme
from Cu-catalyzed inactivation by substrates.
Experimental conditions were the same as in Fig. 1with 6
µM Cu
and pH 5.0, but with the following
additions:
, none;
, 5 mML-malate;
, 4 mM Mn
;
, 5 mML-malate + 4 mM Mn
;
,
5 mML-malate + 4 mM Mn
+ 0.23 mM NADP
.
Figure 5:
Competitive inhibition of pigeon liver
malic enzyme by Cu with respect to
Mn
. In the routine assay mixture, the Mn
concentration was varied from 1 µM to 10 µM with all other components held at fixed concentration. Malic
enzyme activity was assay in the presence of various concentrations of
Cu
:
, none;
, 100 µM;
, 200 µM. Lines are computer fitted to .
For comparison, Cu was also tested
for inhibition of malic enzyme. Cu
as high as 160
µM showed no inhibition on the enzyme. At 169
µM, the inhibition was only 7%. These results strongly
suggest that only divalent Cu
has a high affinity
with malic enzyme.
Figure 6:
SDS-PAGE pattern of the metal-catalyzed
oxidized malic enzyme. A, lane1 is M standards (phosphorylase b, M
94,000; bovine serum albumin, M
67,000; ovalbumin, M
43,000; carbonic
anhydrase, M
30,000; trypsin inhibitor, M
20,000;
-lactalbumin, M
14,400). Lane2 is the unmodified native
enzyme. Lanes3 and 4 are enzymes modified
at pH 7.0. Lanes5 and 6 are enzymes
modified at pH 5.0. Lanes3 and 5 are
enzymes modified with Fe
-ascorbate system. Lanes4 and 6 are enzymes modified with
Cu
-ascorbate system. B, cleavage positions
of malic enzyme by the MCO systems identified by amino acid sequence
analysis of the peptide fragments.
The switch of specificity was examined by monitoring the protein cleavage pattern at various pH values. As the pH decreased from neutral to acidic, fragments I, II, III, VI, VII, and VIII gradually increased, while fragments IV and V decreased (data not shown).
Corresponding to peptide E` and E`
are peptides E`
and E`
, which contain the N-terminal peptides
Met
-Asp
and
Met
-Asp
, respectively, and should not
detect any sequence since the N terminus of malic enzyme is blocked
(Wei et al., 1994). When peptides E`
and E`
were subjected to amino acid sequence
analysis, multiple small peaks were detected and identifying any
reliable sequence was relatively difficult. When consulting the gel
pattern shown in Fig. 6, we can conclude that while peptides E`
-E`
represent the
major cleavage products, minor cleavage at other sites also occurred.
Peptide E`
contains sequence
Met
-Asp
and some other minor peptides.
This is also true for peptide E`
. The failure in
identifying some amino acid residues in E`
may be
due to the interference of other minor peptides. A sufficient quantity
of fragment I for sequence analysis was not collected in this study to
confirm that it is blocked at the N terminus.
Although the peptide
cleavage caused by Cu system is complicated at pH
5.0, three more metal ligand sites are unequivocally identified, i.e. Asp
, Asp
, and
Asp
. A comparison with the known amino acid sequences for
malic enzyme from various sources (Bagchi et al., 1987;
Börsch and Westhoff, 1990; Chou et al.,
1994; Hsu et al., 1992; Loeber et al., 1991, 1994;
Magnuson et al., 1986; Kobayashi et al., 1989;
Kulkarni et al., 1993; Rothermel and Nelson, 1989) (
)reveals that these aspartate residues are highly conserved (Fig. 7Fig. 8Fig. 9).
Figure 7:
Comparison of the putative Mn binding site Asp
of malic enzymes. Figure shows
amino acid sequences around Asp
of pigeon and duck,
Asp
of human, rat and mice, Asp
of human
liver mitochondria, Asp
of Ascaris suum mitochondria, Asp
of maize chloroplast, Asp
of dicotyledonous C4 plant Flaveria trinervia, and
Asp
of Bacillus stearothermophilus malic enzymes
were aligned to show the high conservation in this region. The cleavage
site of pigeon liver malic enzyme by the Cu
-ascorbate
was indicated by an arrow. The dashedunderlined sequence was confirmed by amino acid sequence analysis. The gray area showed identical amino acid residues between
different malic enzymes. The putative Mn
-binding
ligand Asp
was highlighted.
Figure 8:
Comparison of the putative Mn binding site Asp
of malic enzymes. Figure is same
as Fig. 7but with amino acid sequences around Asp
of pigeon and duck, Asp
of human, rat and mice,
Asp
of human liver mitochondria, Asp
of Ascaris suum mitochondria, Asp
of maize
chloroplast, Asp
of Flaveria trinervia, and
Asp
of Bacillus stearothermophilus malic
enzymes.
Figure 9:
Comparison of the putative Mn binding site Asp
of malic enzymes. Figure is same
as Fig. 7but with amino acid sequences around Asp
of pigeon, Glu
(may be Asp
) of duck,
Asp
of human, rat, and mice, Asp
of human
liver mitochondria, Asn
(may be Asp
or
Asp
) of Ascaris suum mitochondria, Glu
(may be Asp
) of maize chloroplast, Asp
of Flaveria trinervia, and Val
(may be
Glu
) of Bacillus stearothermophilus malic
enzymes.
Pigeon liver malic enzyme is among the most sensitive enzymes
toward MCO system. We have demonstrated previously that this enzyme was
rapidly inactivated by the Fe-ascorbate system at
neutral pH. The inactivation was rapid that incubation at 0 °C was
necessary to slow down the reaction rate. In this study, the
Cu
-ascorbate system was found to be more effective
than the Fe
system. Experimental results indicated
that the Cu
system produced an entirely different
cleavage pattern at pH 5.0 as compared to the pattern at pH 7.0. The
following criteria indicate that Cu
-ascorbate caused
affinity modification and cleavage of pigeon liver malic enzyme at the
putative metal-binding site. (a) Cu
is
essential for the process; Cu
is much less effective,
suggesting that Cu
must bind with the enzyme at the
divalent metal ion binding site before inactivation takes place. (b) Cu
is a competitive inhibitor versus Mn
for the enzyme, indicating that
Cu
and Mn
compete for the same
binding site. (c) Inactivation of the enzyme is prevented by
Mn
plus L-malate, indicating that
modification is at the active site.
Based on the above discussion,
the reaction sequence of oxidative modification and peptide bond
cleavage of pigeon liver malic enzyme by the
Cu-ascorbate system can be summarized in Fig. SI. First, Cu
binds with the enzyme at
the Mn
binding site (step 1). Second, ascorbate
reduces Cu
to Cu
(step 2), which, in
the presence of dissolved O
, generates reactive free
radicals (e.g. O
,
OH
) that in turn modify the essential amino acid
residue(s) nearby and forming the inactivated enzyme (E`)
(step 3). Finally, depending on pH of the solution, the enzyme molecule
is cleaved at four possible sites giving peptide fragments E`
+ E`
, E`
+ E`
, or E`
+ E`
at pH 5.0, or E`
+ E`
at pH 7.0 (step
4). In this manner, different specificities are achieved by
manipulating the reaction conditions. We suggest that this strategy can
be generally applied to other enzymes or proteins in elucidating the
metal-binding sites. We propose that the catalytically essential
carboxyl group of Asp
has a pK
value
of 6.7, as determined by chemical modification experiments (Chang et al., 1985). At pH 5.0, this carboxyl group is protonated
and loss its metal-binding ability and is not reactive toward oxidative
modification. Under this circumstance, Fe
is inactive
but other metal ligands Asp
, Asp
, or
Asp
, which might have pK
values near
4.7, are modified by the more reactive Cu
.
Figure SI:
Scheme IProposed reaction mechanism for
the oxidative modification and affinity cleavage of pigeon liver malic
enzyme by the Cu-ascorbate system. The N and C under each peptide fragment denote the N- or C-terminal half
before cleavage.
Interestingly, the metal ligands Asp,
Asp
, Asp
, and Asp
of malic
enzyme were all aspartate residue, which has been indicated to be the
major metal-binding ligand for many metal-proteins (Higaki et
al., 1992; Vallee and Auld, 1993; Traut, 1994). Furthermore,
isocitrate dehydrogenase, which catalyzes a similar oxidative
decarboxylation reaction as malic enzyme, was demonstrated to have
Asp
, Asp
from the same subunit, and
Asp
` from the other subunit as the metal coordinates
(Hurley et al., 1990); this enzyme from pig heart was also
sensitive to MCO system (Soundar and Coleman, 1993). Examination of the
sequences shown in Fig. 7Fig. 8Fig. 9reveals that
Asp
and Asp
, the major cleavage sites by
the Cu
system, are strictly conserved in all malic
enzyme with known amino acid sequences. Asp
is also
highly conserved; however, this region has higher variations among
malic enzyme of different origins. This site is a minor cleavage site.
Maximum alignment of other malic enzyme sequences with Asp
of pigeon enzyme reveals that Glu
of duck,
Asn
of ascaris, and Glu
of maize enzymes
may be the metal coordinates. Although Asn and Glu are found as metal
ligand in many metal-proteins (Villafranca and Nowak, 1992; Higaki et al., 1992; Vallee and Auld, 1993; Traut, 1994), an
observation of the nearby amino acid residues reveals that Asp
of duck, Asp
or Asp
of ascaris, and
Asp
of maize enzyme may be the authentic metal ligands.
These results enforce the critical value of aspartate residue as the
metal coordinate in proteins. Only bacillus malic enzyme was
nonconservatively substituted this Asp with Val
. The
actual metal ligand, however, may be Glu
, which is also a
conservative substitution.
Malic enzyme is a bifunctional enzyme. It catalyzes both the oxidoreduction and decarboxylation reactions. The reaction mechanism of the enzyme proceeds in two steps with hydride transfer preceding decarboxylation (Hermes et al., 1982). These two functions can be assessed separately by assaying the partial reactions with appropriate substrate (Hsu, 1982). One of the distinguishing features of the decarboxylase activity of malic enzyme is its pH optimum being at 4.5 (Salles and Ochoa, 1950). We propose that the proton released during dehydrogenase reaction provides a favorable local active site environment for the decarboxylation reaction, which involves metal ion-stabilized enolate anion transition state (Hsu et al., 1976; O'Leary, 1992). During the catalytic cycle, the enzyme might undergo an isomerization that favors the decarboxylation reaction. Results presented in this study support the hypothesis that the enzyme exists as different conformational isoforms at neutral or acidic environment. However, another possibility that the observed pH effects were due to changes in the catalytic cleavage rate of different sites rather than to global conformational changes was not ruled out (Kufel and Kirsebom, 1994).
Metal-catalyzed oxidation of proteins has been suggested to be the
marker for protein turnover in vivo (Stadtman and Oliver,
1991; Stadtman, 1992). According to this theory, the oxidized protein
molecules are unstable and prone to degradation by multicatalytic
proteases (proteasomes) in cells (Rivett, 1993). The normal copper
concentration in serum is 16-31 µM (Murray et
al., 1990); both iron and copper ions are normally presented in
the cells, which also contain various reducing compounds. If malic
enzyme is sensitive to only a few micromolar concentration of
Cu in the cytoplasm, an intriguing question is that
how could malic enzyme survive in vivo for a reasonable period
of time to perform its metabolic roles? Protection of the enzyme by
Mg
or other divalent cations and substrate is one of
the answers. Furthermore, cells contain other defense mechanisms
against oxidative damage. The endogenous antioxidant enzymes (catalase,
dismutase) are active in normal young tissues; a rapid physiological
response of the translational or transcriptional control of the
detoxification genes might play an important role when metal ion
concentrations exceed a dangerous threshold (O'Halloran, 1993).
These detoxification gene products may play important roles in cell
protection, e.g. the high Fe
content and
oxygen carrier function of red blood cells indicate the high oxidation
stress experience by red blood cells, which contains an abundant
natural killer enhancing factor that protects red blood cells from
oxidation injuries (Shau and Kim, 1994). These protecting proteins are
highly homologous with yeast thiol-specific antioxidant, which protects
yeast cells from oxidation insults (Chae et al., 1993). It is
these natural protecting mechanisms that prevent cell proteins and
other cellular components from experiencing oxidative damage.