From the Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021
Received for publication, December 26, 2002, and in revised form, January 24, 2003
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ABSTRACT |
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Fcp1 is an essential protein serine phosphatase
that dephosphorylates the C-terminal domain (CTD) of RNA
polymerase II. By testing the effects of serial N- and C-terminal
deletions of the 723-amino acid Schizosaccharomyces pombe
Fcp1, we defined a minimal phosphatase domain spanning amino acids
156-580. We employed site-directed mutagenesis (introducing 24 mutations at 14 conserved positions) to locate candidate catalytic
residues. We found that alanine substitutions for Arg223,
Asp258, Lys280, Asp297, and
Asp298 abrogated the phosphatase activity with either
p-nitrophenyl phosphate or CTD-PO4 as
substrates. Structure-activity relationships were determined by
introducing conservative substitutions at each essential position. Our
results, together with previous mutational studies, highlight a
constellation of seven amino acids (Asp170,
Asp172, Arg223, Asp258,
Lys280, Asp297, and Asp298) that
are conserved in all Fcp1 orthologs and likely comprise the
active site. Five of these residues (Asp170,
Asp172, Lys280, Asp297, and
Asp298) are conserved at the active site of T4
polynucleotide 3'-phosphatase, suggesting that Fcp1 and T4 phosphatase
are structurally and mechanistically related members of the
DXD phosphotransferase superfamily.
The C-terminal domain
(CTD)1 of the largest subunit
of RNA polymerase II (pol II) is composed of a tandemly repeated
heptapeptide motif (consensus sequence YSPTSPS). The CTD undergoes a
cycle of extensive phosphorylation and dephosphorylation at positions Ser5 and Ser2, which is coordinated with the
transcription cycle and is responsive to stress and developmental cues.
A general picture of CTD phosphorylation dynamics has emerged in which
dephosphorylated pol II is recruited to the preinitiation complex and
CTD phosphorylation ensues shortly after the elongation complex is
established (1). The phosphorylation state of the CTD is apparently
remodeled during its transit along the gene, the purpose of which may
be to control the ingress and egress of the mRNA processing
assemblies that modify the nascent transcript (1-3). Wholesale
dephosphorylation of the CTD may be required to recycle pol II for a
new round of initiation.
The enzyme Fcp1 is the major protein serine phosphatase responsible for
removing phosphates from the CTD (4-11). Fcp1 orthologs are present in
all known eukaryal proteomes, and the enzyme is essential for
cell viability in budding and fission yeast (7,10). Fcp1 catalyzes the
metal-dependent hydrolysis of phosphoserine from the CTD in
the context of the pol II elongation complex or isolated pol II and, in
the case of Schizosaccharomyces pombe Fcp1, from synthetic
CTD phosphopeptide substrates. Fcp1 also hydrolyzes the nonspecific
substrate p-nitrophenyl phosphate. The mechanism of the Fcp1
reaction is not known.
We are undertaking via site-directed mutagenesis and biochemical
methods to dissect the domain structure of S. pombe Fcp1 and
localize the essential constituents of the active site. We initially
characterized catalytically active deletion mutants Fcp1(140-723) and
Fcp1(1-580) and identified two acidic side chains, Asp170
and Asp172, as essential for phosphatase activity (11).
Here we delineate a minimal phosphatase catalytic domain,
Fcp1(156-580). To map the active site, we introduced alanine and
conservative mutations at 14 residues of S. pombe Fcp1 that
are invariant in Fcp1 orthologs from other eukarya (see Fig. 1). We
thereby identified five new residues that are essential for catalysis
and likely comprise the phosphatase active site. Our results
consolidate the hypothesis that Fcp1 belongs to the
DXDXT family of phosphotransferases (9, 11, 12).
A plausible catalytic mechanism for Fcp1 is suggested based on the
concordance of mutational data for Fcp1 and the polynucleotide 3'-phosphatase domain of bacteriophage T4 polynucleotide kinase.
Fcp1 Mutants--
Amino acid substitution mutations (and
diagnostic restriction sites) were introduced into the
fcp1+ cDNA by the two-stage overlap
extension method as described previously (11). pET-Fcp1 was used as the
template for the first-stage amplification. The mutated full-length
cDNAs generated in the second-stage amplification were digested
with NcoI and BamHI and then inserted into
pET16m. The inserts of the resulting pET-Fcp1* plasmids were sequenced
completely to confirm the desired mutations and exclude the acquisition
of unwanted changes during amplification or cloning. Deletion mutants
Fcp1(140-580), Fcp1(148-580), Fcp1(156-580), and Fcp1(162-580) were
constructed by PCR amplification with sense-strand primers that
introduced an NcoI restriction site and a methionine codon
in lieu of the codons for Gly139, Glu147,
Asn155, or Gln161 and an antisense-strand
primer that introduced a stop codon in lieu of the codon for
Pro581 and a BamHI site 3' of the new stop
codon. Additional C-terminal deletions were constructed by PCR
amplification with antisense primers that introduced stop codons in
place of the codons for Val505 or Trp561 and a
BamHI site 3' of the new stop codon. The PCR products were digested with NcoI and BamHI and then inserted
into pET16m. The pET-Fcp1* and pET-Fcp1 Phosphatase Assay--
Reaction mixtures (100 µl) containing
50 mM Tris acetate (pH 5.5), 10 mM
MgCl2, 10 mM p-nitrophenyl phosphate
(pNØP), and Fcp1 as specified were incubated for 30 min at 37 °C.
The reactions were quenched by adding 900 µl of 1 M
sodium carbonate. Release of p-nitrophenol (pNØ) was
determined by measuring A410 and interpolating the value to a pNØ standard curve.
CTD Phosphatase Assay--
Reaction mixtures (25 µl)
containing 50 mM Tris acetate (pH 5.5), 10 mM
MgCl2, 25 µM CTD-PO4 peptide (an
N-terminal biotinylated 28-aa CTD phosphopeptide composed of four
tandem YSPTSPS repeats containing phosphoserines at positions 2 plus
5), and Fcp1 as specified were incubated for 60 min at 37 °C. The
reactions were quenched by adding 1 ml of malachite green reagent
(BIOMOL GREEN reagent, purchased from BIOMOL Research Laboratories,
Plymouth Meeting, PA). Phosphate release was determined by measuring
A620 and interpolating the value to a phosphate
standard curve.
Alanine Scanning Mutagenesis of Fcp1--
A goal of the present
study was to extend the map of the active site of Fcp1 by identifying
individual amino acid functional groups required for the phosphatase
reaction. To do this, we introduced single alanine changes at 14 amino
acids of the S. pombe Fcp1 polypeptide that are invariant in
the putative phosphatase catalytic domains of other Fcp1 orthologs
(Fig. 1). We focused in particular on
basic and acid residues, which we viewed as potential ligands for the
phosphoserine substrate or the divalent cation cofactor. We also
mutated the two invariant histidines within the phosphatase domain,
His177 and His240, which we considered as
possible general acid catalysts. The pH optimum of 5.5 for the
phosphatase activity of S. pombe Fcp1 and the sharp
decrement in activity seen at
Full-length wild-type Fcp1 and the 14 Fcp1-Ala mutants were produced in
bacteria as His10-tagged fusions and purified from soluble
bacterial extracts by nickel-agarose chromatography. SDS-PAGE analysis
of the imidazole eluate fractions showed that the preparations were
highly enriched with respect to the His-Fcp1 polypeptide (Fig.
2A).
Alanine Mutational Effects on Phosphatase Activity--
Initial
characterization of the phosphatase activity of the purified
recombinant Fcp1-Ala proteins was performed using 10 mM
pNØP as a substrate. The pNØ reaction product was detected via its
absorbance at 410 nM. The extent of conversion of pNØP to
pNØ was directly proportional to the concentration of the recombinant wild-type protein, which released 22 nmol of pNØ per µg of protein in 30 min (Fig. 2B). We defined a significant mutational
effect as one that elicits at least a 10-fold decrement in specific
activity compared with wild-type Fcp1. The specific activities of the
R223A, K280A, D297A, and D298A mutants were <0.5% of the activity of wild-type Fcp1, whereas the D258A mutant was 3% as active as wild-type Fcp1 (Fig. 2B and Table
I). We conclude that the
Arg223, Asp258, Lys280,
Asp297, and Asp298 side chains are essential
for Fcp1 activity. Other Fcp1-Ala proteins either retained near
wild-type phosphatase activity (H177A, H240A, R271A, R299A, and D301A)
or displayed modest reductions in activity (E238A, R267A, and D272A)
that did not meet our threshold of significance (Fig. 2B and
Table I). We surmise that the His177, Glu238,
His240, Arg267, Arg271,
Asp272, Arg299, and Asp301 side
chains do not contribute significantly to catalysis of the phosphatase
reaction. A notable finding was that the D323A mutation resulted in a
4-fold increase in phosphatase activity over wild-type Fcp1
(Fig. 2B and Table I). This stimulation was reproducible for
multiple independent preparations of the D323A protein (data not
shown). Determination of the steady state kinetic parameters for
wild-type Fcp1 (Km = 18 mM pNØP;
kcat = 3.7 s Mutational Effects on CTD Phosphatase Activity--
CTD
phosphatase activity of the Fcp1-Ala mutants was measured by the
release of Pi from a synthetic 28-aa phosphopeptide
consisting of four tandem repeats of the CTD heptad sequence (YSPTSPS)
in which all Ser2 and Ser5 residues are
Ser-PO4 (11). The enzyme preparations were assayed in
parallel; the reaction mixtures contained 25 µM CTD
phosphopeptide and 2.5 µg of input Fcp1, an amount sufficient for
saturating levels of Pi release by wild-type Fcp1 (see Fig.
3B). Conducting the screening
assays in this fashion highlighted the most severe mutational effects
on CTD phosphatase activity (Fig. 3A). The four Fcp1 mutants
that were grossly defective in CTD dephosphorylation, R223A, K280A,
D297A, and D298A, are the same mutants that displayed <0.5% of
wild-type activity in hydrolyzing pNØP. D258A also displayed reduced
CTD phosphatase activity in the screening assay (Fig. 3A). A
titration experiment showed that the specific activity of D258A in
dephosphorylating the CTD phosphopeptide was 3% of the specific
activity of wild-type Fcp1 (Fig. 3B). This value is in
accord with the effects of the D258A mutation on hydrolysis of the
nonspecific substrate pNØP (Table I). The nine Fcp1-Ala proteins that
displayed near wild-type CTD phosphatase activity in the screening
assay were the same as those that retained activity in hydrolyzing
pNØP. We infer that: (i) a single active site is responsible for
catalyzing the hydrolysis of pNØP and the phospho-CTD and (ii)
Arg223, Asp258, Lys280,
Asp297, and Asp298 are likely to be involved
directly in catalysis.
The specific activity of wild-type Fcp1 on the CTD phosphopeptide
substrate (8.2 nmol of Pi released in 60 min per µg of
protein; Fig. 3B) translates into a turnover number of 0.19 s Structure-Activity Relationships at Essential Residues--
To
further evaluate the contributions of Arg223,
Asp258, Lys280, Asp297, and
Asp298 to the phosphatase reaction, we tested the effects
of conservative substitutions. Aspartate was replaced by asparagine and
glutamate, arginine by lysine and glutamine, and lysine by arginine and
glutamine. The R223K, R223Q, D258E, D258N, K280R, K280Q, D297E, D297N,
D298E, and D298N proteins were purified from soluble bacterial extracts by nickel-agarose chromatography (Fig.
4A).
Hydrolysis of pNØP was measured as a function of enzyme concentration
for wild-type Fcp1 and the 10 conservative mutants (Fig. 4B). We found that R223K and R223Q were just as defective as
the alanine mutant, with <1% of wild-type phosphatase activity. Thus, arginine is specifically required at position 223. K280R and K280Q were
as defective as K280A (<0.5% activity), indicating a stringent requirement for lysine at position 280. Conservative replacement of
Asp297 with Asn or Glu elicited a severe catalytic defect
comparable to that seen with D297A (<1% of wild-type activity). These
data establish a requirement for a carboxylate residue at position 297, and they indicate a steric constraint on the distance from the main
chain to the carboxylate, whereby the additional methylene group of
glutamate is detrimental. Replacing Asp298 with Asn
resulted in a modest gain of function (to 5% of wild-type activity)
compared with the D298A mutant (<0.5%), but substitution by Glu had
no salutary effect. These results indicate the importance of the
carboxylate at position 298 and a steric constraint on the main chain
to carboxylate distance. Glutamate substitution for Asp258
revived phosphatase activity to above our threshold of significant mutational effect (to 18% activity, compared with 3% for D258A), whereas the D258N mutant was still significantly compromised, with 7%
of wild-type activity. Thus, the carboxylate at position 258 is
essential, and the enzyme is flexible in its accommodation of the
glutamate side chain.
Defining a Minimal Fcp1 Phosphatase Domain--
We showed
previously that deletion mutants Fcp1(140-723) and Fcp1(1-580)
retained full phosphatase activity in vitro (11). To
delineate a minimal catalytic domain, we combined the N- and C-terminal
deletions to generate the doubly truncated derivative Fcp1(140-580).
Purified recombinant Fcp1(140-580) displayed full activity in
hydrolyzing pNØP (Fig. 5B)
and in dephosphorylating the synthetic CTD phosphopeptide (Fig.
3B). To finely delineate the N-terminal margin of the
catalytic domain, we produced and purified a new set of three
incrementally truncated derivatives: Fcp1(148-580), Fcp1(156-580),
and Fcp1(162-580). SDS-PAGE analysis revealed the expected serial
increases in their electrophoretic mobility (Fig. 5A). The
recombinant proteins were assayed for phosphatase activity with pNØP
(Fig. 5B). We found that deletion of amino acids 140-147
reduced phosphatase-specific activity to 72% that of Fcp1(140-580)
(Fig. 5B). Further removal of amino acids 148-155 reduced
the phosphatase activity to 28% of Fcp1(140-580). Because these
truncations did not meet our 10-fold threshold, we surmise that the
segment from 140 to 155 is not strictly essential for catalysis.
However, the next incremental deletion mutant, Fcp1(162-580), was only
1.7% as active as Fcp1(140-580), indicating that the segment from 156 to 161 is essential. These results delineate a requirement for ~15
amino acids upstream of the first essential aspartate
(Asp170) of the conserved DXDXT
motif.
We showed previously that the C-terminal deletion mutant Fcp1(1-486)
was catalytically inert (11). To better delineate a distal margin for
the catalytic domain of Fcp1, we introduced new C-terminal deletions
into the N Here we employed site-directed mutagenesis to define a minimal
catalytic domain and identify five new essential amino acids of the
S. pombe CTD phosphatase Fcp1. As discussed in detail below, our results suggest structural and mechanistic similarities between Fcp1 and the 3'-phosphatase domain of T4 polynucleotide kinase (13-16), and they consolidate the suggestion (9, 11, 12) that Fcp1 is
a member of a phosphatase superfamily defined by the
DXDXT motif.
Deletion analysis showed that large segments could be deleted
simultaneously from the N and C termini of S. pombe Fcp1 without loss of phosphatase activity. The apparent
minimal catalytic domain is Fcp1(156-580), which consists of an Fcp1
homology domain (shown in Fig. 1) linked to a downstream BRCT domain.
An earlier in vivo deletion analysis of S. cerevisiae Fcp1 showed that removal of the N-terminal region
upstream of the Fcp1 homology domain did not affect cell viability nor
did removal of the C-terminal segment downstream of the BRCT domain.
However, simultaneous deletion of both N- and C-terminal segments was
lethal in vivo (17). The present biochemical analysis of
S. pombe Fcp1 shows clearly that the flanking N- and
C-terminal domains are not essential for phosphatase activity, either
with pNØP or CTD-PO4. It is therefore likely that the
seemingly redundant in vivo roles of the N- and C-terminal
domains of yeast Fcp1 have more to do with ancillary functions, such as
protein-protein interactions (17, 18) and the pol II
elongation-stimulation activity of Fcp1 (9, 19), than with chemical
catalysis or CTD-PO4 recognition.
Fcp1 orthologs from diverse species display extensive sequence
similarity in the region spanning S. pombe Fcp1 residues
140-326 (Fig. 1). A short conserved peptide motif
(167LIVDLDQTII176 in S. pombe Fcp1)
located near the N-terminal margin of the minimal catalytic domain
corresponds to the signature sequence of the DXDXT family of metal-dependent
phosphohydrolases and phosphotransferases (20-22). Several
DXDXT family members have been shown to act via an acyl-phosphoenzyme intermediate in which the phosphate is linked to
the first aspartate in the DXDXT motif (20,
23-25). Given that the two aspartates in the
DXDXT element of Fcp1 were found previously to be
essential for Fcp1 phosphatase activity (11, 12), it was suggested that
Fcp1 is a member of the DXDXT superfamily. However, a DXD motif is also a defining feature of the
"Toprim" domains found in another class of phosphoryl transfer
enzymes (including DNA topoisomerases IA, and II and DNA primases),
which do not form acyl-phosphate intermediates (26-28). In the Toprim enzymes, the DXD motif coordinates a divalent cation.
In the absence of an atomic structure for Fcp1, we rely on primary
structure and mutational analysis for clues to which family Fcp1 might
belong. However, amino acid sequence searches reveal little similarity,
exclusive of the DXD motif itself, between Fcp1 and the
several DXDXT family members for which atomic
structures are available. Nonetheless, instructive clues emerged from a
comparison of the mutational results for Fcp1 to those obtained for the
polynucleotide 3'-phosphatase domain of T4 polynucleotide kinase (Pnk),
which also contains a signature DXDXT motif (Fig.
1). The active site of the Pnk phosphatase domain has been mapped by
extensive site-directed mutagenesis. The 10 side chains essential for
catalysis include six aspartates, two arginines, one lysine, and one
serine (13-15).2 The amino
acid composition of the Pnk active site is generally similar to the
constellation of essential residues of S. pombe Fcp1, which
consists, to date, of five aspartates, one arginine, and one lysine.
The most upstream essential residues in the Pnk and Fcp1 sequences are
the DXDXT motif aspartates
(165DVDGT169 in Pnk;
170DLDQT174 in S. pombe Fcp1).
Indeed, the N termini of Pnk and Fcp1 can be deleted to within ~15
amino acids of the first catalytic aspartate without significant loss
of phosphatase activity (15). The distal ends of the Pnk and Fcp1
active sites map to a conserved motif defined by two vicinal essential
aspartates, which are preceded by a hydrophobic cluster and then
followed by an conserved arginine and a second cluster of hydrophobic
residues (Fig. 1). The sequence of this "DD" motif in Pnk is
274LAIDDRTQVVEM285, and its putative equivalent
in Fcp1 is 294VVIDDRGDVWDW305 (Fig. 1). Note
that the length of the polypeptide segment separating the
DXD and DD motifs is quite similar in Pnk and Fcp1. The
occurrence of two signature motifs with similar order and spacing plus
the concordant mutational findings for the two aspartates of the DD motif of Fcp1 and Pnk (14)2 leads us to posit that
the Fcp1 and Pnk phosphatase domains are structurally and
mechanistically related. The crystal structure of Pnk shows that the
fold of the Pnk1 phosphatase domain resembles that of the exemplary
DXDXT family member, Methanococcus
jannaschii phosphoserine phosphatase (PSP)
(16).3 Thus, we infer that
Fcp1 is also a bona fide member of the
DXDXT superfamily.
Elegant crystallographic snapshots have been obtained for PSP at
sequential intermediate states of the catalytic cycle, which reveal the
role of the DXD motif in catalysis (24, 29). The proximal
Asp plays two key functions: O The PSP crystal structure highlights a lysine side chain that interacts
with the phosphate in the ground state and stabilizes the
pentacoordinate phosphorane transition state (24, 29). The
equivalent essential lysine in the phosphatase domain of Pnk (Lys258) is located 19 aa upstream of the DD motif of Pnk.
As shown here, Fcp1 also has an essential lysine (Lys280)
situated 17 aa upstream of its DD motif. The structure-activity relationships at Pnk Lys258 and Fcp1 Lys280 are
concordant with each other (i.e. neither arginine nor
glutamine could replace the essential lysine in Pnk or Fcp1), and the
functional data are consistent with the monovalent phosphate contact
seen for the catalytic lysine in the PSP crystal.
It is notable that PSP and most other proteins classified as
DXDXT phosphatases have no precise counterpart in
their primary structures of the DD motif present in Pnk and Fcp1.
Rather, the structural superposition of PSP and Pnk shows that two
aspartate side chains of PSP within a
167DXXXD171 motif occupy positions
at the active site that overlay with the two aspartates of the DD motif
of Pnk (16). Note that the proximal Asp277 of the
277DD278 dipeptide of Pnk superimposes on the
distal Asp171 of the PSP
167DXXXD171 motif, while
the distal Asp288 of Pnk DD overlies the
proximal Asp167 of the PSP
167DXXXD171 element. The
DXXXD motif was recognized as a signature feature, along
with the upstream DXDXT element, of the proteins
that were initially felt to comprise the phosphatase superfamily
(21, 22). Yet, the structural and mutational data for Pnk and Fcp1 suggest that they belong to a different subgroup of phosphatases within
the DXDXT superfamily, which is defined by their
DD motifs. The PSP structure shows that the two aspartates of the
DXXXD motif comprise part of the divalent cation
coordination complex in the active site via direct and water-mediated
contacts between the carboxylate oxygens and the metal (24, 29). Thus,
we infer that the essential 297DD298 dipeptide
of Fcp1 is also concerned with metal binding.
In summary, we have defined seven essential hydrophilic amino acids of
S. pombe Fcp1 that are conserved in all known Fcp1 orthologs
and likely comprise the phosphatase active site. We identify an
essential DD motif that is characteristic of Fcp1 and T4 Pnk. Plausible
mechanistic roles are proposed for five of the seven essential side
chains of Fcp1, either in nucleophilic attack on Ser-PO4
(Asp170), general acid-base catalysis (Asp172),
transition state stabilization (Lys280), or metal
coordination (Asp297, Asp298). The functions of
the other two essential Fcp1 side chains (Arg223 and
Asp258) cannot be surmised with confidence, but, by analogy
with Pnk, they may be involved in positioning one or more of the other
catalytic side chains at the active site via a network of
hydrogen-bonding interactions (14,16). Definitive evaluation of the
mechanism proposed here will hinge on crystallization of Fcp1.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
plasmids were introduced
into Escherichia coli BL21(DE3)-RIL, and the mutant Fcp1
proteins were purified from soluble bacterial lysates
nickel-nitrilotriacetic acid-agarose affinity chromatography as
described previously for wild-type Fcp1 (11). Protein concentrations
were determined by using the BioRad dye reagent with bovine serum
albumin as the standard.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
pH 7.5 had suggested the existence of a
protonated functional group with a pKa near
neutrality (11).
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Fig. 1.
Conservation of Fcp1 primary structure.
The amino acid sequence of S. pombe Fcp1 from residues
140-326 is aligned to the sequences of Fcp1 orthologs from
Saccharomyces cerevisiae (Sce),
Leptosphaeria maculans (Lma), Neurospora
crassa (Ncr), Aspergillus nidulans
(Ani), Candida albicans (Cal),
Xenopus laevis (Xla), Drosophila
melanogaster (Dme), Anopheles gambiae
(Aga), Homo sapiens (Hsa),
Caenorhabditis elegans (Cel), Dictyostelium
discoideum (Ddi), and Encephalitozoon
cuniculi (Ecu). Gaps in the alignment are indicated by
dashes. The essential Asp170 and
Asp172 residues of the DXDXT
phosphatase motif of S. pombe Fcp1 are shaded. The 14 amino
acids targeted for mutational analysis in the present study are
indicated by dots ( ). Newly defined essential residues of
S. pombe Fcp1 are shaded. Included at the
bottom of the alignment are two conserved motifs that comprise
the 3'-phosphatase active site of bacteriophage T4 Pnk.
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Fig. 2.
Purification of Fcp1-Ala mutants and effects
on phosphatase activity. A, aliquots (7 µg) of the
nickel-agarose preparations of wild-type (WT) Fcp1 and the
indicated Fcp1-Ala mutants were analyzed by SDS-PAGE. Polypeptides were
visualized by staining the gels with Coomassie Brilliant Blue dye. A
scan of the stained gels is shown. The positions and sizes (in kDa) of
marker proteins are indicated on the left. B,
reaction mixtures containing 50 mM Tris acetate (pH 5.5),
10 mM MgCl2, 10 mM pNØP, and WT or
mutant Fcp1 proteins as specified were incubated for 30 min at
37 °C. pNØ release is plotted as a function of input protein.
1) and the D323A
mutant (Km = 7.1 mM pNØP;
kcat = 8.3 s
1) showed that the
removal of the Asp323 side chain elicited ~2-fold
increases in the Km of Fcp1 for pNØP and the
turnover number for pNØP hydrolysis. The
kcat/Km ratio for the D323A
mutant was 5.6-fold greater than that of wild-type Fcp1.
Summary of mutational effects on Fcp1 phosphatase activity
P is the average of two independent titration experiments. The
activities are normalized to that of wild-type Fcp1 (defined as 100%).
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Fig. 3.
CTD phosphatase activity. A,
reaction mixtures containing 25 µM CTD-PO4
peptide and 2.5 µg of the indicated Fcp1 protein were incubated for
60 min at 37 °C. B, reaction mixtures containing 25 µM CTD-PO4 peptide and wild-type or mutant
Fcp1 proteins as specified were incubated for 60 min at 37 °C.
Pi release is plotted as a function of input enzyme.
1. Parallel titration of the D323A mutant showed that it
was 74% as active as wild-type Fcp1 with the CTD phosphopeptide
substrate (Fig. 3B). The nearly wild-type CTD phosphatase
activity of D323A contrasts with its 410% activity with pNØP (Fig.
2B). We surmise that Asp323 is not a genuine
modulator of Fcp1 activity but rather that the carboxylate is a
specific impediment to the binding and hydrolysis of pNØP, which is a
nonphysiological substrate with low affinity for the Fcp1 active site.
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Fig. 4.
Effects of conservative substitutions on
phosphatase activity. A, aliquots (7 µg) of the
indicated nickel-agarose Fcp1 preparations were analyzed by SDS-PAGE. A
scan of the Coomassie Blue-stained gel is shown. The positions and
sizes (in kDa) of marker proteins are indicated on the left.
B, reaction mixtures containing 50 mM Tris
acetate (pH 5.5), 10 mM MgCl2, 10 mM pNØP, and WT or mutant Fcp1 proteins as specified were
incubated for 30 min at 37 °C. pNØ release is plotted as a function
of input protein.
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Fig. 5.
Deletion analysis defines a minimal Fcp1
phosphatase domain. A, aliquots (7 µg) of the
indicated Fcp1 deletion mutants were analyzed by SDS-PAGE. A Coomassie
Blue-stained gel is shown. The positions and sizes (in kDa) of marker
proteins are indicated on the left. B, reaction
mixtures containing 50 mM Tris acetate (pH 5.5), 10 mM MgCl2, 10 mM pNØP, and either
WT or mutant proteins as specified were incubated for 30 min at
37 °C. pNØ release is plotted as a function of input protein.
139 Fcp1 derivative. We found that purified recombinant
Fcp1(140-505) had no detectable phosphatase activity (Fig. 5). A
longer derivative, Fcp1(140-560), was also catalytically inert (not
shown). We surmise that the protein segment from amino acids 560-580
is essential.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 attacks the phosphorus of the
substrate to form the acyl-phosphate intermediate, while O
2 is part
of the octahedral coordination complex of the essential divalent cation
cofactor. The distal aspartate of the DXD motif of PSP acts
as a general acid-base catalyst: it donates a proton to the serine
leaving group during the first phosphoryl transfer step, and it
abstracts a proton from the attacking water during the second
hydrolytic step. We infer that Fcp1 employs a similar mechanism of
acid-base catalysis via an acyl-phosphate intermediate. We had inferred
previously from the pH profile of S. pombe Fcp1 the
existence of at least two different functional groups at which the
protonation state has a strong impact on phosphatase activity (11). We
would now attribute the falloff in catalysis between pH 5.5/5.0 and pH
4.5 to protonation of the proximal Asp nucleophile of the
DXD active site motif. To explain the decline in activity as
the pH was increased above neutrality, we had invoked a requirement for
protonated functional group on Fcp1 enzyme and discussed a potential
histidine general acid with a presumptive pKa of
~6.5. The present mutational analysis vitiates the idea that either
of the two conserved Fcp1 histidines might play such a role. Rather,
the PSP structure and the sum of the mutational data focus attention on
Asp172 of the Fcp1 DXD motif as a potential
source of the activity decrement of S. pombe Fcp1 at
alkaline pH, i.e. via deprotonation of the Asp general acid.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM52470.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.
To whom correspondence should be addressed. Tel.: 212-639-7145;
Fax: 212-717-3623; E-mail: s-shuman@ski.mskcc.org.
Published, JBC Papers in Press, January 28, 2003, DOI 10.1074/jbc.M213191200
2 H. Zhu and S. Shuman, unpublished data.
3 L. Wang, S. Shuman, and C. Lima, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: CTD, C-terminal domain; pol II, polymerase II; pNØP, p-nitrophenyl phosphate; pNØ, p-nitrophenol; aa, amino acid; Pnk, polynucleotide kinase; PSP, phosphoserine phosphatase; WT, wild type.
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