From the
Prolyl 4-hydroxylase (EC 1.14.11.2) catalyzes the formation of
4-hydroxyproline in collagens. The vertebrate enzyme is an
Prolyl 4-hydroxylase (EC 1.14.11.2) catalyzes the formation of
4-hydroxyproline in collagens and related proteins by the hydroxylation
of proline residues in - X-Pro-Gly- sequences. This
modification plays a central role in the synthesis of all collagens, as
4-hydroxyproline residues are essential for the folding of the newly
synthesized collagen polypeptide chains into triple-helical molecules
(for reviews, see Refs. 1-3).
Prolyl 4-hydroxylase requires
Fe
Complete cDNA-derived amino acid sequences have now been reported
for the
Experiments on the
inactivation of prolyl 4-hydroxylase by diethyl pyrocarbonate and the
prevention of this inactivation by cosubstrates of the reaction have
suggested that histidine residues are functional at the catalytic sites
of the enzyme, probably at the Fe
In order to elucidate the
roles of histidine residues of the
It has also been
suggested that cysteine residues may be functional at the
Fe
The human
To study the effect of the various
histidine mutations on the prolyl 4-hydroxylase activity of the
tetramer, Triton X-100 extracts from cell homogenates were assayed by a
method based on measurement of the hydroxylation-coupled
decarboxylation of 2-oxo[1-
Three histidine to serine mutations
were found to have a marked effect on prolyl 4-hydroxylase activity in
the resulting tetramer (). A mutation in position 412 or
483 totally eliminated the enzyme activity, whereas a mutation in
position 501 reduced it to about 4% (). None of the
mutations involving the 3 nonconserved histidines that did not inhibit
tetramer assembly, residues 63, 141, and 296, had any effect on enzyme
activity of the tetramer. The two mutations of conserved histidines
that appeared to have a minor inhibitory effect on the amount of enzyme
tetramer, residue 165 and 221 (see above), produced a reduction of
about 70 or 50%, respectively, in enzyme activity per unit extractable
cell protein relative to that obtained with the wild-type
Further
investigations into the properties of the histidine 501 to serine
mutant enzyme showed no differences in the K
Because prolyl 4-hydroxylase becomes bound to
poly(L-proline) even in the absence of Fe
The present data demonstrate that there are 3 conserved
histidine residues that play a major role in the catalytic activity of
prolyl 4-hydroxylase. Mutation of either histidine 412 or 483 to serine
completely inactivated the enzyme, whereas mutation of histidine 501 to
serine reduced its activity to about 5%. These results agree with
previous data indicating that the enzyme is inactivated by diethyl
pyrocarbonate, an effect which could be partially prevented by the
presence of Fe
The Fe
The most likely role for all
three critical histidines identified here is that they provide the
three ligands involved in the coordination of Fe
The
finding that the histidine 412, 483, and 501 mutant enzymes all became
bound to poly(L-proline) clearly demonstrates that none of
these histidines is critically involved in a poly(L-proline)
binding site. This finding also suggests that none of these mutations
caused any marked overall changes in the structure of the enzyme, as
the sites involved in poly(L-proline) binding appear to have
retained their native-type conformations. The data indicating that none
of these three histidine mutations inhibited assembly of the prolyl
4-hydroxylase tetramer likewise suggest that these mutations have
caused no major structural changes.
A histidine-containing motif
showing homology to that around histidine 483 in the
Three histidine to serine
mutations appeared to influence the tetramer assembly. Surprisingly,
mutation of one of the 4 nonconserved histidines, residue 324,
completely prevented tetramer formation, indicating that although this
position can be occupied by either histidine or lysine
(11) ,
its occupation by serine will prevent tetramer assembly. Mutation of
two conserved histidines, residue 165 or 221, had more minor effects on
tetramer assembly or stability. The amount of enzyme activity obtained
with these two mutants decreased to an even larger extent than the
amount of tetramer, suggesting that the mutations caused some minor
structural changes. The cysteine 150 to serine mutation may also have
caused minor structural changes, as the amount of tetramer was
consistently slightly lower, whereas the two other cysteine mutations
studied, those involving residues 486 and 511, totally eliminated
tetramer assembly. This result differs from those obtained in
experiments on assembly of the prolyl 4-hydroxylase tetramer in a
cell-free system, in that mutation of either cysteine 486 or 511
reduced the amount of tetramer formed but did not totally prevent
tetramer assembly
(21) . The data obtained both here and in a
cell-free system
(21) suggest that these 2 cysteines play a
major role in maintaining the native-type structure of the
The functions of the
asparagine-linked carbohydrate units present in the
All values have been
determined in Triton X-100 extract from cells expressing either the
wild-type or mutant tetramer as the source of the enzyme. The
K
For polypeptide nomenclature and
measurement units, see Table I.
We thank Eeva Lehtimäki and Riitta Polojärvi
for their expert technical assistance.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
tetramer in which the
subunits
contribute to most parts of the two catalytic sites. To study the roles
of histidine and cysteine residues in this catalytic activity we
converted all 5 histidines that are conserved between species, 4
nonconserved histidines, and 3 conserved cysteines of the human
subunit individually to serine and expressed the mutant
subunits
together with the wild-type
subunit in insect cells by means of
baculovirus vectors. Mutation of any of the 3 conserved histidines,
residues 412, 483, and 501, inactivated the enzyme completely or
essentially completely, with no effect on tetramer assembly or binding
of the tetramer to poly(L-proline). These histidines are
likely to provide the three ligands needed for the binding of
Fe
to a catalytic site. Mutation of either of the
other 2 conserved histidines reduced the amount of enzyme tetramer by
20-25% and the activity of the tetramer by 30-60%. Mutation
of the nonconserved histidine 324 totally prevented tetramer assembly,
whereas mutation of the 3 other nonconserved histidines had no effects.
Two of the 3 cysteine to serine mutations, those involving residues 486
and 511, totally prevented tetramer assembly under the present
conditions, whereas the third, involving residue 150, had only a minor
effect in reducing tetramer assembly and activity. The data do not
support previous suggestions that cysteine residues are involved in
Fe
binding sites. Additional mutagenesis experiments
demonstrated that the two glycosylated asparagines have no role in
tetramer assembly or catalytic activity.
, 2-oxoglutarate, O
, and ascorbate. The
active enzyme from vertebrates is an
tetramer in which the
subunits contribute to most parts of
the two catalytic sites
(1, 2, 3) . A catalytic
site appears to comprise a set of separate locations for binding of the
peptide substrate and the various cosubstrates. The Fe
is probably coordinated with the enzyme by three side chains
(1, 2, 3, 4) . The 2-oxoglutarate
binding site can be divided into distinct subsites; subsite I is
probably a positively charged side chain of the enzyme that ionically
binds the C-5 carboxyl group of 2-oxoglutarate, whereas subsite II
consists of two cis-positioned coordination sites for the
enzyme-bound Fe
which is chelated by the C-2 oxo and
C-1 carboxyl functions
(1, 2, 3, 4, 5) . Binding of
O
and decarboxylation of 2-oxoglutarate is assumed to lead
to the formation of ferryl ion, which subsequently hydroxylates a
proline residue
(1, 3, 4) . Ascorbate acts as an
alternative oxygen acceptor in the uncoupled decarboxylation cycles, in
which the 2-oxoglutarate is decarboxylated without subsequent
hydroxylation of the peptide substrate
(6, 7) . The
ascorbate binding site also contains the two cis-positioned
coordination sites for the enzyme-bound iron and is thus partially
identical to the binding site of 2-oxoglutarate
(8) .
subunit of prolyl 4-hydroxylase from man
(9) ,
chick
(10) , and Caenorhabditis elegans (11) and for the
subunit from several species
(2, 12, 13, 14, 15, 16) .
Surprisingly, the
subunit was found to be identical to the
protein disulfide-isomerase (EC 5.3.4.1) and to have protein
disulfide-isomerase activity even when present in the prolyl
4-hydroxylase tetramer
(12, 13, 17) . This
protein disulfide-isomerase/
subunit has subsequently been found
to be a highly unusual multifunctional polypeptide
(2, 3, 15, 16) .
binding sites
(18) . A search for conserved amino acids within the sequences
of several 2-oxoglutarate dioxygenases demonstrated a weak homology
within 2-histidine-containing motifs located about 50-70 amino
acids apart, the histidines concerned being residues 412 and 483 in the
human
subunit sequence
(18) .
subunit in the catalytic
activity of prolyl 4-hydroxylase, we converted all 5 histidines that
are conserved between species from man to C. elegans and 4
additional nonconserved histidines individually to serine and expressed
the mutant human
subunits together with the wild-type protein
disulfide-isomerase/
subunit in insect cells
(19, 20) by means of baculovirus vectors.
binding sites of prolyl 4-hydroxylase
(3) .
Two cysteine residues of the
subunit have been reported to form
an intrachain disulfide bond that is essential for tetramer assembly
(21) . We studied here the possible roles of the 3 other
cysteine residues in catalytic activity. In addition, the roles of the
2 glycosylated asparagine residues were studied.
Site-directed Mutagenesis
Histidines 63, 141,
165, 221, 296, 324, 412, 483, and 501 (codon CAC or CAT) and cysteines
150, 486, and 511 (codon TGC or TGT) in the human subunit were
converted individually to serine (codon TCC). The 2 asparagines,
residues 96 (codon AAC) and 242 (codon AAT), which serve as the
N-glycosylation sites, were converted to glutamines (codon
CAG) in a double mutant
subunit. The site-directed mutagenesis
steps were performed in a pBluescript (Stratagene) vector containing
the full-length cDNA clone (PA-59) of the human
subunit at the
SmaI site
(9) . The mutagenesis was carried out using
an oligonucleotide-directed in vitro mutagenesis system based
on the unique site elimination procedure (Pharmacia Biotech Inc.),
after which the plasmid was digested with PstI and
BamHI, the cleavage sites for which closely flank the
SmaI site. The resulting PstI- PstI and
PstI and BamHI fragments containing 61 bp of the
5`-untranslated sequence, the whole coding region, and 551 bp of the
3`-untranslated sequence were then cloned into the
PstI- BamHI site of the baculovirus transfer vector
pVL1392
(22) . The sequences were verified by Sanger
dideoxynucleotide sequencing
(23) .
Generation of Recombinant Baculoviruses
The
recombinant baculovirus transfer vectors were cotransfected into
Spodoptera frugiperda Sf9 insect cells with a modified
Autographa californica nuclear polyhedrosis virus DNA
(PharMingen) by calcium phosphate transfection, and the recombinant
viruses were selected
(24) . The resultant viruses encoding the
mutant subunit sequences were termed
-H63S,
-H141S,
-H165S,
-H221S,
-H296S,
-H324S,
-H412S,
-H483S,
-H501S,
-C150S,
-C486S,
-C511S, and
-N96Q,N242Q.
Analysis of Recombinant Proteins in Insect
Cells
The insect cells (Sf9 or High Five, Invitrogen) were
cultured in TNM-FH medium (Sigma) supplemented with 10% fetal bovine
serum (BioClear) at 27 °C. The cells seeded at a density
10/ml were infected at a multiplicity of 5 with any of the
viruses coding for the mutant
subunit together with a virus
coding for the protein disulfide-isomerase/
subunit
(19) .
The cells were harvested 72 h after infection, washed twice with a
solution of 0.15 M NaCl and 0.02 M phosphate, pH 7.4,
homogenized in a 0.1 M NaCl, 0.1 M glycine, 10
µM dithiothreitol, 0.1% Triton X-100, and 0.01 M
Tris buffer, pH 7.8, and centrifuged at 10,000
g for
20 min. Aliquots of the supernatants were analyzed by denaturing 8%
SDS-PAGE
(
)
or nondenaturing 8% PAGE and assayed
for enzyme activity.
Binding of Mutant Enzymes to
Poly(L-proline)
Binding of prolyl 4-hydroxylase
tetramers containing some of the mutant subunits to
poly(L-proline) was studied by mixing 0.5-ml aliquots of the
supernatants of cell homogenates expressing the wild-type enzyme or a
mutant enzyme tetramer with 0.5 ml of a suspension containing
poly(L-proline) coupled to agarose
(25) . After gentle
stirring for 6 h at 4 °C, the poly(L-proline)-agarose was
separated out by centrifugation at 10,000
g for 5 min,
and the resulting supernatants were analyzed by nondenaturing 8% PAGE
and Coomassie Brilliant Blue staining.
Purification of Prolyl 4-Hydroxylase
The prolyl
4-hydroxylase tetramer containing the double mutant N96Q,N242Q
subunit was purified by a procedure consisting of
poly(L-proline) affinity chromatography and DEAE-cellulose
chromatography
(26) .
Other Assays
Prolyl 4-hydroxylase activity was
assayed by a method based on the hydroxylation-coupled decarboxylation
of 2-oxo[1-C]glutarate
(25) .
K
values were determined as described
previously
(27) . Protein concentrations were determined with a
Bio-Rad protein assay kit (Bio-Rad). The levels of expression of the
wild-type and mutant prolyl 4-hydroxylase tetramers were compared by
densitometry of the Coomassie Brilliant Blue-stained bands in
nondenaturing PAGE using a bioimage (BioImage, Millipore).
Selection of Residues for Mutagenesis
The
subunit of human prolyl 4-hydroxylase has 12 histidine residues
(Fig. 1). One of them is located in the alternatively spliced
region in which the sequence coded by exon 9 contains a histidine in
position 349, whereas the sequence coded by exon 10 contains it in
position 360
(28) . A recently cloned prolyl 4-hydroxylase
subunit isoform, termed the
(II) subunit, has been found to form
an (
(II))
tetramer with the protein
disulfide-isomerase/
subunit
(29) . The
K
values of this tetramer for the
cosubstrates and the peptide substrate are very similar to those of the
tetramer studied here, but the
sequence of the
(II) subunit contains an arginine in the position
corresponding to histidine 349
(29) . Thus histidine 349/360 can
be regarded as a nonconserved residue. Of the remaining 11 histidines,
only 5 ( circled in Fig. 1) are conserved between the
human and C. elegans
subunits
(9, 11) .
All these, together with 4 nonconserved histidines (these 9 histidines
are underlined in Fig. 1) were converted individually to
serine.
Figure 1:
Schematic
representation of the subunit of human prolyl 4-hydroxylase.
Numbering of the amino acids begins with the first residue in the
processed
subunit, the signal sequence being indicated by a
shaded box and the alternatively spliced region by a black
box. All histidine and cysteine residues and the 2 glycosylated
asparagine residues are shown. Conserved residues (see text) are
circled, and residues subjected to mutation in the present
experiments are underlined. This numbering differs from that
used initially (9) in that the initial numbering began with the first
residue of the signal peptide.
All 5 cysteine residues (Fig. 1) are conserved in the
subunit from man to C. elegans (9, 10, 11) . Recent work on assembly of
the prolyl 4-hydroxylase tetramer in a cell-free system
(30) has demonstrated that cysteines 276 and 293 form an
intrachain disulfide bond that is essential for tetramer assembly
(21) . Cysteines 486 and 511 may form another intrachain
disulfide, but they are not essential for tetramer assembly. Cysteine
150 does not seem to be involved in any disulfide bonding
(21) .
The possible catalytic roles of cysteines as 150, 486, and 511 were
studied here by converting these individually to serine.
subunit contains 2 asparagine residues that act as attachment
sites for oligosaccharide units (asparagines 96 and 242, Fig. 1).
These vary greatly in the extent of their glycosylation, the
polypeptide being present in diglycosylated, monoglycosylated, and
nonglycosylated forms
(19, 31) . Both these asparagines
were converted to glutamines in a double mutant
subunit.
Histidine to Serine Mutants
In order to study
whether any of the 9 histidine mutations inhibits assembly of the
prolyl 4-hydroxylase tetramer, each of the mutant subunits was
expressed in insect cells together with the wild-type protein
disulfide-isomerase/
subunit. The cells were harvested 72 h after
baculovirus infection, homogenized in a buffer containing 0.1% Triton
X-100, and centrifuged. The Triton X-100-soluble proteins were then
analyzed by PAGE performed under nondenaturing conditions and Coomassie
staining (Fig. 2). A distinct band corresponding to the enzyme
tetramer
(11, 19, 20) was seen in the Triton
X-100-soluble fraction from cells infected with a virus coding for
either the wild-type
subunit or any of 8 out of the 9 histidine
to serine mutant
subunits. The only mutant
subunit that did
not form a tetramer was that containing the histidine to serine
mutation in position 324. Surprisingly, this is one of the 4
nonconserved histidines studied here, this position being occupied by
lysine in the C. elegans
subunit
(11) . Repeated
control experiments indicated that this result was not due to a
methodological artifact. As the goal of this work was to identify
residues critical for catalytic activity rather than tetramer assembly,
this aspect was not pursued any further.
Figure 2:
Nondenaturing PAGE analysis of prolyl
4-hydroxylase tetramer formation from wild-type or histidine to serine
mutant human subunits and the wild-type human protein
disulfide-isomerase/
subunit expressed in insect cells by means of
baculovirus vectors. The samples were extracted with a buffer
containing 0.1% Triton X-100 and analyzed by nondenaturing PAGE and
Coomassie staining. Lanes 1 and 12, extracts from
cells infected with the wild-type
subunit-coding virus and
wild-type protein disulfide-isomerase/
subunit-coding virus;
lanes 2-10, extracts from cells infected with a mutant
subunit-coding virus and the wild-type protein
disulfide-isomerase/
subunit-coding virus, the
subunits
having the following mutations: H63S ( lane 2), H141S ( lane
3), H165S ( lane 4), H221S ( lane 5), H296S
( lane 6), H324S ( lane 7), H412S ( lane 8),
H483S ( lane 9), and H501S ( lane 10). Lane
11, extract from cells infected with the protein
disulfide-isomerase/
subunit-coding virus alone. The samples in
lanes 1-6 and 10-12 were run in the same
experiment and those in lanes 7-9 in a second
experiment. The arrows indicate the enzyme tetramer
(
) and the nonassociated protein
disulfide-isomerase/
subunit (
). The nonassociated
subunit is insoluble in 0.1% Triton X-100 and is not
seen.
Densitometric tracings of
the Coomassie-stained bands corresponding to the enzyme tetramer from
at least four experiments in the case of each mutant indicated that
there are no major differences in the amounts of tetramer formed per
unit extractable cell protein except in the case of the histidine 324
mutation described above. However, two of the mutant
subunit-coding viruses, those with histidine 165 or 221 mutation,
consistently produced about 20-25% less tetramer than the other
viruses (details not shown).
C]glutarate
(25) . All values were expressed per 10 µg of extractable
cell protein ().
subunit
(). Since the amount of tetramer was reduced by about
20-25%, however (see above), the actual reduction in the activity
of the tetramer was about 60 or 30%. The K
values for Fe
, 2-oxoglutarate, ascorbate, and
the peptide substrate with these two mutant tetramers were identical to
those with the wild-type enzyme (details not shown).
values for Fe
, ascorbate, or the peptide
substrate relative to the wild-type enzyme, but the
K
for 2-oxoglutarate was consistently
about 3-fold (). In agreement with this minor difference,
the K
for pyridine-2,4-dicarboxylate,
which acts as a competitive inhibitor with respect to 2-oxoglutarate,
was about 2.5-fold (). The main difference, however, was
that the V
determined from the kinetic plots was
consistently less than about 5% of that of the wild-type enzyme
(details not shown).
and other cosubstrates
(3, 25, 32) , it
seemed possible to determine whether the three histidine mutations that
had a marked effect on enzyme activity had any effect on binding to
poly(L-proline). Small aliquots of Triton X-100 extracts from
cells expressing either the wild-type enzyme or an enzyme containing
any of the three mutant
subunits with a histidine 412, 483, or
501 to serine substitution were mixed with poly(L-proline)
linked to agarose. The recently discovered
(
(II))
tetramer (see above), which
differs from the
tetramer studied
here in that it does not bind to poly(L-proline)
(29) ,
served as a control. All three mutant enzymes became bound to
poly(L-proline) as efficiently as the wild-type enzyme,
whereas no binding was detected with the
(
(II))
tetramer (Fig. 3). These
data indicate that none of the three histidine mutations with a marked
effect on enzyme activity had any effect on the binding of the enzyme
to poly(L-proline).
Figure 3:
Nondenaturing PAGE analysis of the binding
of prolyl 4-hydroxylase tetramers containing certain wild-type or
histidine to serine mutant subunits to poly(L-proline)
linked to agarose. Small aliquots of Triton X-100 extracts from cells
expressing either a wild-type enzyme tetramer or a mutant tetramer were
mixed with poly(L-proline) linked to agarose. After gentle
stirring for 6 h at 4 °C, the poly(L-proline)-agarose was
separated out by centrifugation, and the resulting supernatants were
analyzed by nondenaturing PAGE and Coomassie staining. A,
samples analyzed before incubation with
poly(L-proline)-agarose; B, samples analyzed after
incubation. Lanes 1, extract from cells infected with the
wild-type
subunit-coding virus and wild-type protein
disulfide-isomerase/
subunit-coding virus; lanes 2,
extract from cells infected with the mouse type II
subunit (see
text) and human protein disulfide-isomerase/
subunit-coding
viruses; lanes 3-5, extracts from cells infected with a
mutant
subunit and wild-type protein disulfide-isomerase/
subunit-coding viruses, the
subunits having the following
mutations: H412S ( lane 3), H483S ( lane 4), H501S
( lane 5). The arrows indicate the enzyme tetramer
(
) and the nonassociated protein
disulfide-isomerase/
subunit (
).
Cysteine to Serine Mutants
As in the case of the
histidine mutants, each of the cysteine mutant subunits was
expressed in insect cells together with the wild-type protein
disulfide-isomerase/
subunit, and the Triton X-100-soluble
proteins were used for the analyses. Two out of the three cysteine
mutants, those involving residues 486 and 511, were found either to
prevent tetramer assembly or to destabilize thetetramer to such an
extent that no tetramer band was seen by PAGE performed under
nondenaturing conditions and Coomassie staining (Fig. 4). The
third cysteine mutant, that involving residue 150, had no major effect
on tetramer assembly (Fig. 4), but densitometric tracings of the
Coomassie-stained band indicated that the amount of tetramer formed was
slightly reduced, being about 80% of that seen with most of the other
viruses (details not shown). The amount of prolyl 4-hydroxylase
activity observed with the cysteine 150 mutant
subunit had
decreased to a slightly larger extent than the amount of tetramer
(I), suggesting that the specific activity of the mutant
tetramer was about 80% of that of the wild-type enzyme. The two other
cysteine mutants that appeared to prevent tetramer assembly generated
no prolyl 4-hydroxylase activity (I).
Figure 4:
Nondenaturing PAGE analysis of prolyl
4-hydroxylase tetramer formation from wild-type or mutant human
subunits and wild-type human protein disulfide-isomerase/
subunit
expressed in insect cells by means of baculovirus vectors. The samples
were extracted with a buffer containing 0.1% Triton X-100 and analyzed
by nondenaturing PAGE and Coomassie staining. Lanes 1 and
7, extracts from cells infected with the wild-type
subunit-coding virus and wild-type protein disulfide-isomerase/
subunit-coding virus; lanes 2-5, extracts from cells
infected with a mutant
subunit-coding virus and the wild-type
protein disulfide-isomerase/
subunit-coding virus, the
subunits having the following mutations: N96Q,N242Q double mutation
( lane 2), C150S ( lane 3), C486S ( lane 4),
and C511S ( lane 5). Lane 6, extract from cells
infected with the protein disulfide-isomerase/
subunit-coding
virus alone. The arrows indicate the enzyme tetramer
(
) and the nonassociated protein
disulfide-isomerase/
subunit (
).
Asparagines 96 and 242 to Glutamine Double
Mutant
An subunit containing both these mutations was
found to form a tetramer, as shown by analysis of the Triton
X-100-soluble proteins by PAGE performed under nondenaturing conditions
(Fig. 4). Densitometric tracings of the Coomassie-stained band
indicated no decrease in the amount of tetramer formed. The amount of
prolyl 4-hydroxylase activity observed with the double mutant
subunit was identical to that of the wild-type enzyme (I).
When the tetramer was purified by an affinity column procedure and
analyzed by SDS-PAGE under reducing conditions, the size of the double
mutant
subunit was distinctly smaller than that of either the
diglycosylated or monoglycosylated
subunit present in the
wild-type enzyme, the difference being consistent with loss of all the
carbohydrate (Fig. 5).
Figure 5:
SDS-PAGE analysis under reducing
conditions of purified prolyl 4-hydroxylase tetramers containing either
the wild-type or asparagine to glutamine double mutant subunit.
The wild-type enzyme is shown in lane 2 and the mutant enzyme
in lane 3. Molecular weight markers were run in lane
1. The gel was stained with Coomassie Brilliant Blue. The
arrows indicate the diglycosylated and monoglycosylated
wild-type
subunits (
-wt), the nonglycosylated
asparagines 96 and 242 to glutamine double mutant
subunit
(
-N96Q,N242Q), and the wild-type protein
disulfide-isomerase/
subunit (
).
and 2-oxoglutarate or Fe
and ascorbate but not by the peptide substrate, even though the
peptide becomes bound to the enzyme in the absence of Fe
and other cosubstrates
(18) . Protection by Fe
alone could not be studied, as Fe
will not
remain bound to the enzyme under nonturnover conditions
(3, 18) .
is probably
coordinated with a catalytic site of prolyl 4-hydroxylase by means of
three side chains
(1, 2, 3, 4) . Some
early results suggested that one or more of these side chains may be
cysteine residues
(3) . Nevertheless, the present data are not
consistent with this suggestion, as mutation of the only cysteine that
was not essential for tetramer assembly or stability, residue 150, had
practically no effect on the catalytic activity of the tetramer.
Analyses of another 2-oxoglutarate dioxygenase, isopenicillin N
synthase by a variety techniques, suggest that all three Fe
binding ligands are histidines
(33, 34, 35) .
with
a catalytic site. As the main binding site for both 2-oxoglutarate
(4, 5) and ascorbate
(8) consists of the
enzyme-bound iron atom, it is unlikely that mutation of a residue
contributing to an additional subsite of either a 2-oxoglutarate or
ascorbate binding site would lead to such a marked inactivation of the
enzyme. Although mutation of histidine 501 increased the
K
for 2-oxoglutarate and the
K
for pyridine-2,4-dicarboxylate about
3-fold, this increase is not consistent with the possibility that
histidine 501 may provide the positively charged side chain that
interacts with the negatively charged carboxyl group at C-5 of
2-oxoglutarate and is termed subsite I
(5) . The
K
of the wild-type enzyme for
pyridine-2,4-dicarboyxylate, which is able to bind at subsites I and II
(subsite II being Fe
), is less than 1/10 of that of
pyridine-2-carboxylate, which lacks a domain able to bind at subsite I
(5) . Accordingly, mutation of the positively charged subsite I
residue is likely to lead to a much more than a 3-fold increase in the
K
for 2-oxoglutarate and the
K
for pyridine-2,4-dicarboxylate.
subunit of
human prolyl 4-hydroxylase has recently been identified in bovine
aspartyl (asparaginyl)
-hydroxylase
(36) . When the
histidine present in this motif was converted to alanine, no enzyme
activity was detected in the resulting mutant enzyme
(36) .
Also, the mutant enzyme failed to bind
Fe
/2-oxoglutarate, suggesting that the histidine
residue is involved in the binding of either Fe
or
2-oxoglutarate. The data further suggested that no major structural
changes had taken place as a result of the mutation
(36) , the
results being in a complete agreement with those obtained here with
prolyl 4-hydroxylase. Sequence analyses of flavone 3
hydroxylase
from several sources also demonstrated the presence of three conserved
histidines which may be involved in the binding of Fe
to the catalytic site
(37) .
subunit, probably by intrachain disulfide bond formation, and thus
these 2 cysteines are not likely to be directly involved in the
catalytic mechanism of the enzyme.
subunit of
prolyl 4-hydroxylase are unknown
(3) , and it has been
demonstrated that enzymic removal of the carbohydrate from the tetramer
has little if any effect on the enzyme activity
(31) . The
present data demonstrate that mutation of both glycosylated asparagines
to glutamines causes no changes in tetramer assembly or in the specific
activity of the resulting tetramer. As the extent of glycosylation of
these 2 asparagines in the wild-type prolyl 4-hydroxylase is highly
heterogeneous, the carbohydrate-free tetramer produced here may be more
suitable than the wild-type tetramer for attempts to crystallize the
enzyme.
Table:
Prolyl 4-hydroxylase activity of Triton
X-100 extracts from cells expressing various histidine to serine mutant
subunits together with the wild-type protein
disulfide-isomerase/
subunit
Table:
Kvalues for cosubstrates
and the peptide substrate and K
value for
pyridine-2,4-dicarboxylate of the enzyme tetramer containing the
histidine 501 to serine mutant
subunit
obtained for 2-oxoglutarate with this
crude wild-type enzyme is about twice that obtained with the pure
enzyme (19).
Table:
Prolyl 4-hydroxylase activity of
Triton X-100 extracts from cells expressing various cysteine to serine
mutant subunits or the asparagine to glutamine double mutant
subunit together with the wild-type protein
disulfide-isomerase/
subunit
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.