(Received for publication, October 27, 1994)
From the
The enzyme, 10-formyltetrahydrofolate dehydrogenase (10-FTHFDH)
(EC 1.5.1.6) catalyzes both the NADP-dependent
oxidation of 10-formyltetrahydrofolate to tetrahydrofolate and CO
and the NADP
-independent hydrolysis of
10-formyltetrahydrofolate to tetrahydrofolate and formate. The
COOH-terminal domain of the 10-FTHFDH (residues 417-902) shows a
46% identity with a series of NAD
-dependent aldehyde
dehydrogenases (EC 1.2.1.3). All known members of the aldehyde
dehydrogenase family and 10-FTHFDH have a strictly conserved cysteine
(Cys-707 for 10-FTHFDH), which has been predicted to be at the active
site of these enzymes. Rat liver 10-FTHFDH was expressed in a
baculovirus system, and site-directed mutagenesis has been used to
study the role of cysteine 707 in the activity of 10-FTHFDH. 10-FTHFDH
with alanine substituted for cysteine at position 707 had no
dehydrogenase activity, while hydrolase activity and binding of
NADP
were unchanged. Light scattering analysis
revealed that wild type and mutant 10-FTHFDH exist as tetramers. We
conclude that cysteine 707 is directly involved in the active site of
10-FTHFDH responsible for dehydrogenase activity, and there is a
separate site for the hydrolase activity.
10-Formyltetrahydrofolate dehydrogenase (10-FTHFDH) ()(EC 1.5.1.6) catalyzes both the
NADP
-dependent oxidation of 10-formyltetrahydrofolate
(10-HCO-H
PteGlu) to tetrahydrofolate and CO
and
the NADP
-independent hydrolysis of
10-HCO-H
PteGlu to tetrahydrofolate and
formate(1, 2, 3) . The amino-terminal domain
of rat liver 10-FTHFDH (residues 1-203) is 24-30% identical
to a group of glycinamide ribonucleotide transformylases (EC 2.1.2.1)
from different species(4) . The carboxyl-terminal domain
(residues 417-902) of 10-FTHFDH has 46% identity with a series of
NAD
-dependent aldehyde dehydrogenases (EC 1.2.1.3),
and the enzyme is able to oxidize aldehydes(4) .
All known
aldehyde dehydrogenases contain a conserved cysteine, which, in the
case of 10-FTHFDH, is Cys-707(4, 5) . The conserved
cysteine is presumed to act as a nucleophile in the formation of an
enzyme-linked thiohemiacetal intermediate(6, 7) . A
number of different approaches may be used to identify the amino acid
at the active site of an enzyme. Using an affinity label, Blatter et al.(7) found that Cys-302 formed a covalent
intermediate with the substrate in human aldehyde
dehydrogenase(7) . Site-directed mutagenesis confirmed that
Cys-302 is essential for enzyme activity of rat liver mitochondrial
aldehyde dehydrogenase, whereas Cys-49 and Cys-162 can be changed to
alanine without altering enzyme activity(6) . Additionally,
site-directed mutagenesis experiments have shown that serine 74 is also
important for enzyme activity(6) . This observation was
unexpected because this serine is conserved only in mammalian aldehyde
dehydrogenases(5) . It has been shown recently that serine 74
is not involved in aldehyde oxidation but may be involved in
NAD binding(8) . In the present study we
expressed rat 10-FTHFDH in a baculovirus system and used site-directed
mutagenesis to elucidate the role of the highly conserved Cys-707 in
the activity of the enzyme.
Figure 1:
Construction of the vector for
expression of rat 10-FTHFDH. A, subcloning 10-FTHFDH cDNA from
pBS KS II (4) to pVL 1393 baculovirus vector; B, removal of the XbaI-NcoI 5`-end of the
cDNA including entire uncoding sequence; C, insertion of XbaI-NcoI PCR-generated fragment including coding
sequence only.
Aldehyde dehydrogenase activity was assayed
using propanal essentially as described by Lindahl and
Evces(23) . The reaction mixture contained 60 mM sodium pyrophosphate buffer, pH 8.5, 5 mM propanal, 1
mM NADP, and enzyme in a total volume of 1
ml. Activity was estimated from the increase in absorbance at 340 nm.
Figure 2:
Activity of wild type and mutant
10-FTHFDH. HYD, hydrolase activity; DH, dehydrogenase
activity; TOTAL, total activity (measures both dehydrogenase
and hydrolase activity). The assays were performed as described under
``Experimental Procedures.'' A,
10-HCO-HPteGlu used as a substrate; B, 10-FDDF
used as a substrate.
It has been shown that 10-FTHFDH is able to
oxidize a series of aldehyde substrates in the presence of
NADP(4) . Kinetic analysis of propanal oxidation by recombinant
10-FTHFDH showed a K of 638 µM and an
estimated V
of 245 nmol of NADP min
mg
. These parameter were similar to those of
the natural rat liver enzyme. (
)The C707A mutant enzyme was
unable to oxidize propanal. These results show that the C707A mutant
10-FTHFDH has completely lost dehydrogenase activity.
Analysis of
activity of mutant 10-FTHFDH in the absence of NADP showed that the C707A mutant was able to catalyze the hydrolase
reaction. Hydrolase activity of the C707A mutant 10-FTHFDH was
comparable to the activity of wild type recombinant enzyme for both
10-HCO-H
PteGlu and 10-FDDF (Table 1).
Figure 3:
Fluorescence titration of 10-FTHFDH with
NADP. Curves 1 (opencircles), wild type recombinant 10-FTHFDH; curves2 (darkcircles), C707A mutant
10-FTHFDH. Insertion shows the fluorescence data plotted in linear
form. The value (1 - F/F
)
was
plotted against the inverse of NADP
concentration(25) . This is a modified form of the
classical Stern-Volmer plot, which relates the drop in fluorescence to
the concentration of a collisional quencher (see (26) for
review). F is intrinsic fluorescence observed at an
NADP
concentration; F
is
fluorescence in the absence of NADP
. The slope of the
line (least squares fit) gave a K
that
was approximately 0.3 µM for both wild type and the C707A
mutant 10-FTHFDH. The protein was excited at 291 nm and the emission
monitored at 340 nm. 9.0 nM of each enzyme was used for the
analysis, and concentration of NADP
was varied from
0.02 µM to 10.0 µM. Variation of the measured
values was about 5%.
The sequence identity between 10-FTHFDH and the group of
aldehyde dehydrogenases suggested that Cys-707 could be the active site
nucleophile. In order to test this hypothesis, Cys-707 in 10-FTHFDH was
modified. The closest amino acid substitution for cysteine is serine.
Serine can, however, function as a nucleophile in place of cysteine in
some enzyme-catalyzed reactions(27) . Therefore we replaced
Cys-707 with alanine. Such a substitution is used often in
site-directed mutagenesis to study the role of cysteine residues. In
our experiments mutation of Cys-707 of 10-FTHFDH to Ala resulted in
complete loss of dehydrogenase activity of the enzyme for both the
native substrate 10-HCO-HPteGlu and 10-FDDF. The C707A
mutant also lost the ability to oxidize propanal, while wild type
recombinant 10-FTHFDH oxidized propanal as well as the native rat liver
enzyme. Measurement of binding of the coenzyme, NADP
,
by fluorescence titration showed no difference between the mutant and
wild type enzyme, indicating that loss of dehydrogenase activity was
not due to any change in the coenzyme binding site.
Substitution of
a single amino acid residue can sometimes result in a decrease of
enzyme activity even if this residue is not involved in the active site
due to changes in the higher order protein structure(28) . Such
critical residues play a basic role in protein folding and/or in
supporting correct protein structure(29, 30) .
Replacement of such residues could lead to decreased protein stability (31) and also to a decreased level of expression due to higher
accessibility of mutant proteins to proteases(32) . On the
other hand, many amino acid substitutions do not have large effects on
protein stability(33, 34) . Even when a replaced
residue is essential for protein conformation, protein structures
adjust to compensate for changes in sequence(29) . Our study
did not reveal any changes in stability of the C707A mutant in
comparison with the wild type protein and both displayed a similar
level of expression. Moreover, the K values for
NADP
were similar for both enzymes. The behavior of
the mutant during the purification procedure was also identical to that
of the wild type recombinant protein, indicating no gross alteration of
the native structure(33) . It is known that native 10-FTHFDH
forms oligomers(2, 3, 16) ; therefore, we
also determined the oligomeric structure of the wild type recombinant
and the mutant enzymes. For this purpose, we used a laser-light
scattering technique. This technique involves analysis of the temporal
fluctuations in the intensity of light scattered by the Brownian motion
of macromolecules. It provides a rapid, accurate, and noninvasive
method of determining the translational diffusion coefficient of
macromolecules that can be related to an effective size(18) .
The analysis revealed that both wild type and mutant proteins existed
as tetramers. The fact that the entire population of the active wild
type recombinant 10-FTHFDH was organized as a tetramer indicates that
this is the functional form of the enzyme. Since the C707A mutant
10-FTHFDH also forms a tetramer, this suggests they have similar
conformations. Based on all these observations, we assume that the
substitution of an alanine for the cysteine did not change protein
conformation and stability. However, conservative mutations that
dramatically reduce activity without changes in protein stability
strongly suggest that a residue is important for substrate recognition
or activity.
The mutation of Cys-707 to Ala did not produce any changes in the hydrolase activity of 10-FTHFDH. This indicates that the enzyme has two different catalytic sites. This observation is not surprising since Rios-Orlandi et al.(2) showed that both dehydrogenase and hydrolase activities can take place simultaneously. The existence of two different functional domains was predicted when the sequence of the enzyme was determined(4) . A recent study by Schirch et al.(35) where differential scanning calorimetry and proteolytic digestion were used has also shown the hydrolase and dehydrogenase activities of rabbit liver 10-FTHFDH reside in different domains.
The results show that cysteine 707 is a key residue of the dehydrogenase active site of 10-FTHFDH. Similar results were obtained for aldehyde dehydrogenase from rat liver mitochondria(6) , which has 46% homology with the carboxyl-terminal domain of rat 10-FTHFDH(4) . Aldehyde dehydrogenase with the corresponding C302A mutation was devoid of activity(6) . At the same time substitution an alanine for 2 other cysteines in positions 49 and 162 of aldehyde dehydrogenase did not result in changes of enzyme activity(6) . Apparently, the dehydrogenase catalytic site of 10-FTHFDH is structurally similar to that of aldehyde dehydrogenase with a cysteine as a catalytic nucleophile residue and supports the role of a thiohemiacetal intermediate in the reaction mechanism.