(Received for publication, October 26, 1994; and in revised form, March 28, 1995)
From the
Site-specific mutagenesis was performed on the structural gene
for Escherichia coliS-adenosylmethionine (AdoMet)
synthetase to introduce mutations at cysteines 90 and 240, residues
previously implicated by chemical modification studies to be
catalytically and/or structurally important. The AdoMet synthetase
mutants (i.e. MetK/C90A, MetK/C90S, and MetK/C240A) retained
up to
S-adenosylmethionine synthetase (E.C.2.5.1.6,
ATP:L-methionine S-adenosyltransferase) catalyzes the
reaction of ATP and L-methionine to yield S-adenosylmethionine (AdoMet), pyrophosphate, and
orthophosphate. AdoMet serves in numerous metabolic roles, acting as
the methyl donor in methylation of DNA, RNA, and proteins, as the
propylamine donor in polyamine synthesis, and as a noncovalent
corepressor of the methionine biosynthetic regulon in Escherichia
coli and Salmonella typhimurium(1, 2) .
This laboratory has been engaged in the structural and mechanistic
characterization of the E. coli metK isozyme, which is a
tetramer of identical 383-residue polypeptide
chains(3, 4, 5, 6, 7, 8, 9, 10) .
Each active site binds two divalent metal ion activators (e.g. Mg Twelve AdoMet synthetase sequences have been reported,
which are all highly
homologous(11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21) .
A cysteine analogous to Cys-90 of the E. coli enzyme is
present in all 12 reported sequences, while at the equivalent of
position 240 other residues (e.g. alanine or threonine) have
been found. Of all the AdoMet synthetases that have been purified and
characterized, the E. coli enzyme is unique in having a
cysteine at position 240, and it also has a specific activity at least
10-fold higher than the enzymes from other sources, suggesting a
potential role for Cys-240 in enzyme function. To explore the
functionalities of these residues, we have constructed and
characterized site-directed mutants at positions 90 and 240. The
results clarify the roles of these residues in catalysis and subunit
interactions. L-methionine, ATP, KCl, MgCl
Initially, pT7KC90A and pT7KC240A were transformed
into the E. coli host strain DM50 for expression. MetK/C240A
expression in this strain accounted for 20-25% of total cellular
protein as estimated by SDS-polyacrylamide (SDS-PAGE) analysis, which
is comparable with cloned wild type expression levels in the same host.
However, MetK/C90A expression in DM50 was much lower, <5% of total
cellular protein. Subsequently, pT7KC90A was transformed into
RSR15(DE3), and following
isopropyl-1-thio- A standard purification protocol was used to
isolate wild type AdoMet synthetase and MetK mutants(3) . The
purification protocol consisted of preparation of crude cell extract,
streptomycin sulfate precipitation of nucleic acids, ammonium sulfate
fractionation, and sequential steps of chromatographic resolution on
phenyl-Sepharose (Sigma), hydroxylapatite (Bio-Rad), and
aminohexyl-Sepharose 4B (Pharmacia Biotech Inc.) as described
previously(3) . During the purification both the C90A and C90S
variants eluted as two active peaks on phenyl-Sepharose chromatography.
These two forms were purified separately in subsequent steps.
Typically, 50 g, wet cell weight, of cells were used in the isolation
of each of the mutants.
Substrate
saturation studies of the AdoMet synthetic activity of the MetK mutants
were performed to determine K K
Thermostability of the AdoMet
Synthetase Mutants-The stability of the mutant enzymes
relative to wild type AdoMet synthetase was examined. Enzymes were
diluted to a final concentration of 2.0 mg/ml with 50 mM Tris
Upon finding
that the use of BL21(DE3) as a host strain solved the problem of
expression of MetK/C90A, selection of a BL21(DE3) derivative with
reduced expression of chromosomal wild type MetK was necessary. This
was accomplished by the ethionine resistance selection protocol of
Hafner et al.(23) . The selected metK mutant
of BL21(DE3) was subsequently named RSR15(DE3) and used for the
expression of other MetK mutants. Crude extracts of this strain have no
detectable AdoMet synthetase activity, although an appropriately sized
protein, which cross-reacts with anti-AdoMet synthetase antibodies is
observed in immunoblots.
Figure 1:
A, native
PAGE 8-25% gradient gel of tetrameric and dimeric MetK/C90S. B, SDS-PAGE 8-25% gradient gel of denatured, reduced
tetrameric and dimeric MetK/C90S. In both gels, samples are (from left to right): molecular mass standards, wild type
MetK (tetramer), tetrameric MetK/C90S, and dimeric
MetK/C90S.
Figure 2:
Thermal inactivation of wild type MetK and
MetK mutants at 70 °C. Enzymes at 2.0 mg/ml in 50 mM Tris
While AdoMet activates the tripolyphosphatase reaction for the
mutants as well as the wild type enzyme, differences were observed
among the enzymes with respect to the concentration of AdoMet required
for maximal activation as well as the extent of activation. Maximal
AdoMet activation was observed at AdoMet concentrations of 30
µM for MetK/C90A and MetK/C90S and 40 µM for
MetK/C240A and wild type MetK. The kinetic parameters obtained in the
presence and absence of AdoMet are summarized in Table 3. The
results indicate that all four tetrameric enzymes have V
Figure 3:
A,
inactivation of wild type MetK and MetK mutants by NEM. Enzymes at 1.0
mg/ml (23 µM subunit) were incubated with 1.0 mM NEM in 50 mM HEPES
The conversion of Cys-90 and Cys-240 to alanine residues in
AdoMet synthetase did not yield an inactive form of AdoMet synthetase.
Thus inactivation by NEM is not the result of modification of a
catalytically essential residue; NEM modification at or near the active
site may prevent productive substrate binding as well as disrupt the
quaternary structure of the protein. The C240A mutant differed from
wild type MetK in having a 90% reduction in V The physical basis for the
thermal stabilization observed for the Cys-90 mutants is unclear.
Replacement of amino acid residues with alanine within While the specific activities of dimeric and
tetrameric MetK/C90A have decreased by 99 and 86%, respectively, the
most dramatic kinetic change observed is the >20-fold increase in K In the
presence or absence of AdoMet the tripolyphosphatase activity of
tetrameric MetK/C90A is reduced by 50% relative to wild type enzyme.
Likewise, in the presence or absence of AdoMet, the tripolyphosphatase
activity of dimeric MetK/C90A is decreased 20-fold relative to
tetrameric MetK/C90A. Since the K Because alterations in MetK/C90A may be
attributable to an inability to hydrogen bond, the C90S mutant was
prepared. Like the C90A mutant, the C90S mutant was also isolated as a
mixture of dimers and tetramers. With both dimeric and tetrameric
MetK/C90S, the apparent affinity for either L-methionine or
ATP is similar to that of the wild type enzyme, consistent with the
idea that the hydrogen bonding ability of residue 90 might be important
for ATP binding. However, the production of both dimeric and tetrameric
MetK/C90S as well as the significantly reduced specific activity of
MetK/C90S, show that the C90S mutation must still introduce a
significant perturbation within the folded enzyme. Like the
tripolyphosphatase activity of tetrameric MetK/C90A, in the absence of
AdoMet the tripolyphosphatase activity of tetrameric MetK/C90S is
Tetrameric
MetK/C90A and MetK/C90S lose activity upon incubation with NEM with the
incorporation of 1 equivalent of NEM/subunit. However, the half-time
for inactivation is significantly longer than for wild type enzyme, and
neither enzyme is completely inactivated; MetK/C90A retains 15% of
initial activity, and MetK/C90S retains 11% of initial activity after 1
h of incubation with NEM. Analysis of the NEM
incorporation-inactivation process reveals that one equivalent of NEM
is incorporated rapidly (within 10 min), while the inactivation process
occurs over 60 min. Native PAGE analysis of NEM-modified MetK/C90A or
MetK/C90S after a 60-min incubation indicates that the sample contains
tetramer, dimer, and monomer. Thus, it appears that the NEM
incorporation is not solely responsible for inactivation but promotes
the dissociation of the tetramer to dimers and subsequently inactive
monomers. This combination of fast NEM incorporation and slow enzyme
inactivation observed with the Cys-90 mutants may relate to the NEM
inactivation of wild type enzyme. Perhaps with the incorporation of 2
equivalents of NEM into a subunit of wild type enzyme the inactivation
is more synchronous with the NEM incorporation. Overall, the results
of these studies demonstrate that neither Cys-90 nor Cys-240 is
essential for enzyme function; Cys-90 apparently does have an important
conformational role, consistent with the conservation of cysteine at
this position in all reported AdoMet synthetase sequences.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
10% of wild type activity, demonstrating that neither
sulfhydryl is required for catalytic activity. Mutations at Cys-90
produced a mixture of noninterconverting dimeric and tetrameric
proteins, suggesting a structural significance for Cys-90. Dimeric
Cys-90 mutants retained
1% of wild type activity, indicating a
structural influence on enzyme activity. Both dimeric and tetrameric
MetK/C90A had up to a
70-fold increase in K
for ATP, while both dimeric and tetrameric MetK/C90S had K
values for ATP similar to the wild type
enzyme, suggesting a linkage between Cys-90 and the ATP binding site.
MetK/C240A was isolated solely as a tetramer and differed from wild
type enzyme only in its 10-fold reduction in specific activity,
suggesting that the mutation affects the rate-limiting step of the
reaction, which for the wild type enzyme is the joining of ATP and L-methionine to yield AdoMet and tripolyphosphate. Remarkably
all of the mutants are much more thermally stable than the wild type
enzyme.
) and a monovalent cation activator (e.g. K
). Attempts to identify residues important in
substrate binding or catalysis revealed that only a few chemical
modification reagents affect the enzymatic
activity(3, 9) . Treatment of the enzyme with N-ethylmaleimide results in the modification of two cysteine
residues (Cys-90 and Cys-240) per enzyme subunit and conversion of the
enzyme from an active tetramer to an inactive dimer(9) . The
modification studies implicated cysteines 90 and 240 as catalytically
and/or structurally important residues. These studies showed that a
single rate constant characterized the rate of modification at both
sites and the accompanying loss of activity. Thus the results could not
discriminate whether one or both modifications were required for enzyme
inactivation and/or conversion of the enzyme from a tetramer to a
dimer. Additionally, since an active dimer of this enzyme had not been
isolated, it was unclear whether the tetrameric state was required for
activity.
, Tris,
HEPES, 2-mercaptoethanol, NEM, (
)isopropyl-1-thio-
-Dgalactopyranoside,
tripolyphosphate, and AdoMet were purchased from Sigma. Glycerol was
purchased from Baxter Scientific. [1,2-
H]NEM was
purchased from DuPont NEN. Ecoscint scintillation fluid was purchased
from National Diagnostics. Phosphocellulose P81 filters (2.5 cm) were
purchased from Whatman. The Mutagene site-specific mutagenesis kit was
purchased from Bio-Rad.
Cell and Plasmids
E. coli strain DM50 (an E. coli K12 derivative) or RSR15(DE3) (an E. coli B
derivative) was used. DM50 is a metK mutant that expresses
5% of the wild type level of AdoMet synthetase
activity(10) . RSR15(DE3) is a spontaneous metK mutant
selected as an ethionine-resistant derivative of
BL21(DE3)(22) ; extracts of this strain have no detectable
AdoMet synthetase activity (<1%). Ethionine-resistant mutants of
BL21(DE3) were obtained as described by Hafner et
al.(23) . Several ethionine-resistant colonies were grown
to stationary phase in LB media; cell extracts were then prepared by
sonication and assayed for AdoMet synthetase activity. RSR15(DE3) was
chosen for further use since it uniquely exhibited no AdoMet synthetase
activity in cell-free extracts, although presumably AdoMet synthetase
has some activity in vivo since this is an essential
enzyme(10) . Phagemids pTZ18U and pTZ19U were purchased as part
of the Mutagene kit from Bio-Rad. Plasmid pT7-6 was a gift from
Dr. S. Tabor (Department of Biological Chemistry, Harvard Medical
School)(24) .
Site-directed Mutagenesis
Mutagenic
oligonucleotides were prepared in the Core Facility at Fox Chase Cancer
Center. Mutagenesis was performed on the plasmid pTZK, which consists
of the metK gene inserted between the PstI and EcoRI sites of pTZ18U. The uracil enrichment method of Kunkel
was used(25) . Following mutagenesis, plasmids were transformed
into E. coli strain MV1190 for propagation. Plasmid DNA was
subsequently extracted using the QIAGEN Plasmid Prep (QIAGEN Inc.,
Chatworth, CA) for restriction digestions and nucleotide sequencing.Screening and Nucleotide Sequencing of pTZKC90A,
pTZKC90S, and pTZKC240A
The oligonucleotides used in the
mutagenesis were designed such that along with encoding the nucleotide
sequence to generate the desired mutation, whenever possible they also
exploited codon degeneracy to encode a unique or additional restriction
endonuclease recognition site to facilitate identification. It was not
feasible to introduce a unique or additional restriction site into
pTZKC90S. Transformants containing pTZKC90A and pTZKC240A were
identified by restriction digest (SacII and NaeI,
respectively) of the purified plasmid DNA. Transformants containing
pTZKC90S were initially identified by preparation of crude cell
extracts from isolated colonies of pTZKC90S/MV1190 and assaying for
reduced AdoMet synthetic activity relative to crude extracts prepared
from wild type pTZK/MV1190. The complete nucleotide sequence was
determined for each putative mutant using the procedure of Sanger et al.(26) with the Sequenase kit (U.S. Biochemical
Corp.). The sequence showed that only the desired mutation was
introduced. In the course of sequencing the control pTZK plasmid a few
errors were found in the originally reported metK
sequence(12) . The corrected metK sequence will be
deposited in GenBank.
Expression and Purification of AdoMet Synthetase
Mutants
The metK mutant coding sequences were removed
from the pTZK plasmids by digestion with EcoRI and PstI and inserted into the EcoRI-PstI sites
of pT7-6. AdoMet synthetase expression from the pT7-6
vector was at least 10-fold higher. These plasmids are denoted as
pT7K(mutation).-Dgalactopyranoside induction, MetK/C90A
expression accounted for 20-25% of total cellular
protein(27) . pT7KC90S was also transformed into RSR15(DE3),
and following isopropyl-1-thio-
-Dgalactopyranoside
induction, MetK/C90S protein expression accounted for 20-25% of
total cellular protein.
PAGE
PAGE analysis of AdoMet synthetase mutants
was performed on a Pharmacia Phast System. The purification of AdoMet
synthetase mutants was monitored by PAGE on 10-15% gradient gels
containing SDS.Subunit Molecular Mass Determination of Wild Type AdoMet
Synthetase and AdoMet Synthetase Mutants
Matrix-assisted laser
desorption mass spectrometry (Finnigan, model 2000) was performed on
wild type MetK and the MetK mutants. Proteins at 3.0-5.0 mg/ml
were embedded in an -hydroxycinnamic acid matrix for analysis.
AdoMet Synthetase Assays
AdoMet synthetase
activity was determined by retention of the
[C]AdoMet formed from L-[methyl-
C]methionine by the cation
exchange filter binding method previously described(3) .
Conditions were varied depending on whether the assay was being used to
monitor enzyme purification or being used for kinetic characterization
of the AdoMet synthetase mutants. During an enzyme preparation, the
assays were performed at 25 °C in a mixture containing 53 mM HEPES
KOH, pH 8.0, 53 mM KCl, 21 mM
MgCl
, 10.5 mM ATP, 0.55 mML-[methyl-
C]methionine, specific
activity 1.93 mCi/mmol.
Kinetic Characterization of AdoMet Synthetase
Mutants
K and V
determinations for the mutant AdoMet synthetases were made by
modification of the protocol described above. Potassium ion activation
was evaluated in samples consisting of 0.5 mML-methionine/L-[methyl-
C]methionine
at a specific activity of 3.7 mCi/mmol, 10 mM ATP
(Tris
form), 20 mM MgCl
, 100
mM HEPES
(CH
)
N
at pH 8.0, and 5.8 pmol of MetK mutant (10% glycerol was included
in studies of MetK/C90A and MetK/C90S to enhance stability during
kinetic analysis). The potassium chloride concentration was varied
between 0 and 50 mM. Magnesium ion activation was evaluated as
described above. The KCl concentration was fixed at 50 mM, and
MgCl
was varied between 0 and 100 mM.
values for
ATP and L-methionine as well as V
.
Samples were incubated at 25 °C for a sufficient time to allow
approximately 10% of the L-methionine to be consumed, and
[
C]AdoMet was determined by the filter binding
method (3) . The amount of product formed was linear with time.
and V
values
for the tripolyphosphatase activity of the mutants were determined in
the presence and absence of AdoMet by measuring phosphate
production(28) . AdoMet activation of the tripolyphosphatase
was evaluated for wild type MetK and the MetK mutants. Samples
consisted of 0-500 µM AdoMet and 100 µM
tripolyphosphate (Na
form) in 50 mM
HEPES
(CH
)
N
at pH 7.8
with 10 mM MgCl
and 4.2 nmol of wild type MetK,
MetK/C240A, tetrameric MetK/C90A, or tetrameric MetK/C90S (8.4 nmol of
dimeric MetK/C90A or MetK/C90S). The concentration of AdoMet used for
evaluation of AdoMet-activated tripolyphosphatase activity is that
concentration at which maximal activation was observed for wild type
MetK and the MetK mutants. Substrate saturation data were evaluated
using the kinetic programs of Cleland(29) .
NEM Sensitivity of Mutant Enzymes
Inactivation of
the AdoMet synthetase mutants with NEM was evaluated as described
previously(9) . Incorporation of [H]NEM
(DuPont NEN) into the mutant proteins was also determined as described
previously (9) .
HCl at pH 8.0 containing 50 mM KCl, 10%
glycerol, and 0.1% 2-mercaptoethanol and incubated at 37, 50, 60, and
70 ± 1 °C. At various times aliquots were removed and
assayed for AdoMet synthetase activity.
Expression of AdoMet Synthetase Mutants
Preliminary electrophoretic analysis of crude extracts of
pT7KC90A and pT7KC240A transformed into DM50 revealed that while
MetK/C240A constituted approximately 20-25% of the total cellular
protein, MetK/C90A constituted <5% of the total cellular protein.
This finding suggested that MetK/C90A might have unusual structural
properties and is subsequently degraded by a housekeeping protease such
as the lon protease. To test this hypothesis pT7KC90A was
subsequently transformed into BL21(DE3), an E. coli B strain
that is lon- and ompT-deficient (30, 31, 32, 33, 34, 35) . Electrophoretic analysis of crude extracts prepared from
pT7KC90A/BL21(DE3) revealed increased MetK/C90A expression, which
approximated 20-25% of total cellular protein.
Behavior of AdoMet Synthetase Mutants during
Purification
During purification each of the Cys-90 mutants behaved
predictably in the steps prior to chromatography. However, anomalous
behavior was observed for each of the Cys-90 mutants in the
chromatographic steps, suggesting structural differences relative to
the wild type enzyme. For example, MetK/C90A separated into two
distinct pools of activity upon elution from phenyl-Sepharose; the
first pool (32% of the total activity), subsequently shown to be
tetrameric MetK/C90A, eluted at the end of the ammonium sulfate
gradient (as is the case for the elution of wild type MetK from
phenyl-Sepharose), while the second pool (68% of the total activity),
which was subsequently shown to be dimeric MetK/C90A, required a higher
glycerol concentration (20%) to effect elution. The two forms of
MetK/C90A were kept separate and subsequently chromatographed on DEAE52
and aminohexyl-Sepharose. As with MetK/C90A, MetK/C90S separated into
two distinct pools of activity during phenyl-Sepharose chromatography;
these again were subsequently shown to be dimeric and tetrameric forms.
The two forms of MetK/C90S were kept separate and were subsequently
chromatographed on hydroxylapatite and aminohexyl-Sepharose. The
majority of MetK/C90S was the dimeric species. The MetK/C240A mutant
behaved like the wild type enzyme throughout the purification.Differential Stability of Mutant Enzymes
Manipulation of the purified MetK mutant enzymes showed
pronounced differences among them with respect to stability. MetK/C240A
can be concentrated and/or buffer-exchanged using either a pressure
cell or centrifugal ultrafiltration, like wild type enzyme.
Concentrated samples of MetK/C240A (10 mg/ml) in 50 mM
HEPES
KOH at pH 8.0 with 50 mM KCl are stable for periods
of
6 months when stored at -70 °C or for periods of
8 h at 0 °C; however, upon standing at room temperature for
25 min MetK/C240A aggregates with concomitant loss of activity, in
contrast to wild type enzyme. Tetrameric MetK/C90S can be concentrated
and/or buffer-exchanged in ultrafiltration concentrators with no
apparent loss of enzyme activity; however, dimeric MetK/C90S is readily
inactivated when concentrated and/or buffer-exchanged in a pressure
cell or by ultrafiltration. Further evidence for the instability with
respect to pressure of both the MetK/C90S dimer and tetramer was
obtained when a molecular weight determination was attempted by gel
filtration on Superose 12 at 145 p.s.i. Both the MetK/C90S dimer and
tetramer were inactivated, and chromatograms revealed formation of a
higher molecular weight aggregate. In contrast, dimeric MetK/C90A and
tetrameric MetK/C90A can be concentrated and/or buffer-exchanged using
either a pressure cell or centrifugal ultrafiltration as with wild type
enzyme. Susceptibility to hydrostatic pressure inactivation has been
observed with rabbit muscle glycogen phosphorylase A and E. coli phosphofructokinase(36, 37) ; however, it has not
been observed with wild type AdoMet synthetase. These observations
suggest that there are structural differences among the mutants.
Attempts to obtain diffraction quality crystals of the mutants have
thus far been unsuccessful.
Physical Behavior of AdoMet Synthetase Mutants
Behavior during Native PAGE Analysis
SDS-PAGE
analysis of MetK mutants revealed that they all behave
indistinguishably from wild type MetK. Matrix assisted laser desorption
mass spectrometry of the wild type enzyme and both forms of the C90S
mutant gave indistinguishable subunit masses (<0.2% difference)
verifying the absence of proteolysis. Native PAGE of the MetK mutants
on a 8-25% gradient gel showed that MetK/C240A comigrated with
tetrameric wild type MetK, whereas the separate pools of MetK/C90A and
MetK/C90S were electrophoretically distinct. While one pool of both
MetK/C90A and MetK/C90S comigrated with tetrameric wild type MetK, the
other pool of both MetK/C90A and MetK/C90S had a higher mobility and
was thus of lower M, assuming that migration
distance on a native gel reflects molecular weight, since for identical
proteins in different oligomeric states net charge and conformation are
approximately constant (Fig. 1). While native PAGE is typically
used qualitatively for studying the composition and structure of native
proteins, it can be used quantitatively for molecular weight
measurements(39) . (
)Using mobility data from a
native PAGE 8-25% gradient gel, the smaller species was
calculated to be 55% of the molecular weight of wild type enzyme for
both the MetK/C90A and MetK/C90S, consistent with a dimeric state.
Thermal Stability of AdoMet Synthetase
Mutants
Because of the instability of the AdoMet synthetase
mutants encountered during experimental manipulation, the possibility
that the mutations may have altered thermal stability was examined. The
thermal stability of MetK/C240A, tetrameric MetK/C90A, and tetrameric
MetK/C90S was evaluated at temperatures of 37, 50, 60, and 70 °C.
Thermal inactivation of wild type MetK is a pseudo-first-order process
as previously reported(3) . Similarly, the thermal inactivation
of the AdoMet synthetase mutants is also pseudo-first-order (Fig. 2). MetK/C240A, like wild type MetK, shows progressively
increasing rates of inactivation at 50, 60, and 70 °C (Table 1). However, tetrameric MetK/C90A and MetK/C90S are not
inactivated significantly at temperatures below 70 °C (Table 1), indicating enhanced thermal stability relative to wild
type MetK and MetK/C240A. Native PAGE analysis of thermally inactivated
wild type MetK and MetK mutants indicates that the inactivation
corresponds to a conversion of tetrameric protein to a lower molecular
weight species, which appears to be a monomer. No active monomeric
AdoMet synthetase has been reported.
HCl/pH 8.0 with 50 mM KCl, 10% glycerol, and
0.1% 2-mercaptoethanol were incubated at 70 °C, and at various
times aliquots were removed for assay of AdoMet synthetase
activity.
Kinetic Characterization of AdoMet Synthetase
Mutants
Substrate Saturation
Preliminary monovalent and
divalent cation (K and Mg
,
respectively) activation studies revealed that while the K
for KCl was 2.0 mM for wild
type MetK as well as the MetK mutants, the K
for MgCl
for MetK/C90A and MetK/C90S was 6.0
mM, twice that of wild type MetK and MetK/C240A. The kinetic
parameters of the MetK mutants are summarized in Table 2. The
C240A mutant had
11% of the V
of the wild
type enzyme with no alteration in the K
value for ATP and a
4 fold decrease in the K
for L-methionine. In contrast,
both forms of the C90A mutant had a >20-fold increase in the K
for ATP but smaller changes in the K
for L-methionine as well as
decreased V
values. Interestingly, both C90S
variants had K
values for both substrates
that are close to wild type but V
values similar
to the C90A form. These results demonstrate that neither sulfhydryl is
essential for enzymatic activity. Both Cys-90 mutants show >20 fold
higher activity in the tetrameric state.
Tripolyphosphatase Activity of AdoMet Synthetase
Mutants
Following AdoMet formation, AdoMet synthetase hydrolyzes
the tripolyphosphate derived from ATP to yield pyrophosphate and
orthophosphate; the hydrolysis occurs prior to the release of AdoMet
from the enzyme. In addition, AdoMet synthetase catalyzes the
hydrolysis of exogenously added tripolyphosphate to pyrophosphate and
orthophosphate. The presence of AdoMet stimulates tripolyphosphate
hydrolysis (3, 40, 41, 42) . values within 2-fold of one another and K
values within
3-fold. As was seen
with the overall reaction the dimeric Cys-90 mutants have much lower
activity than the tetrameric forms (Table 3).
NEM Modification of AdoMet Synthetase Mutants
Fig. 3depicts the results of treatment of wild type
MetK, MetK/C240A, tetrameric MetKC90A, and tetrameric MetK/C90S with
NEM as a function of time. The stoichiometry of NEM modification of
wild type MetK and the MetK mutants was determined using
[ethyl-1,2-H]NEM, and the results are presented
in Table 4. Wild type MetK, tetrameric MetK/C90A, and tetrameric
MetK/C90S are inactivated by NEM, and loss of activity follows
pseudo-first-order kinetics. Incubation of MetK/C240A with NEM results
in the incorporation of 1 equivalent of NEM/enzyme subunit, presumably
at Cys-90. However, although NEM modifies MetK/C240A, no inactivation
is seen through 60 min of incubation. Native PAGE analysis of
NEM-modified MetK/C240A indicates that the enzyme remains tetrameric
upon incorporation of 1 equivalent of NEM/enzyme subunit. While the
inactivation of wild type MetK is associated with incorporation of 2
equivalents of NEM/enzyme active site, inactivation of tetrameric
MetK/C90A and tetrameric MetK/C90S correlates with incorporation of 1
equivalent of NEM/enzyme active site. It is worthy of note that the
rate of inactivation of wild type MetK is approximately twice as fast
as the rate of inactivation of tetrameric MetK/C90A and MetK/C90S.
Furthermore, through 60 min of incubation with NEM, neither tetrameric
MetK/C90A nor tetrameric MetK/C90S is completely inactivated, and both
retain
10% residual activity. The rate of
[
H]NEM incorporation in tetrameric MetK/C90A or
MetK/C90S is significantly greater than the rate of enzyme
inactivation, whereas with wild type enzyme both rates are similar. The
results for NEM modification of tetrameric MetK/C90S are depicted in Fig. 3B. These results suggest that in the wild type
enzyme Cys-90 and Cys-240 react with NEM with fortuitously the same
rate and that inactivation results from modification of Cys-240,
perhaps as the result of tetramer dissociation.
KOH at pH 8.0 with 50 mM KCl and 10 mM MgCl
at 25 °C. At various
times, 10-µl aliquots were diluted into 40 µl of assay mix
containing 2 mM 2-mercaptoethanol for estimation of residual
AdoMet synthetase activity. B, time course for the
incorporation of [
H]NEM into tetrameric
MetK/C90S. Reactions were initiated by the addition of
[
H]NEM, and at various times aliquots were
removed and diluted into buffer containing a 10-fold excess of
2-mercaptoethanol with respect to NEM. Time point samples were assayed
for residual activity and incorporation of [
H]NEM
into tetrameric MetK/C90S as trichloroacetic acid-precipitable
radioactivity.
for the AdoMet synthetic reaction. The tripolyphosphatase
half-reaction is only 30% reduced in V
, and the K
for tripolyphosphate is increased
3-fold. However, in the presence of AdoMet, the tripolyphosphatase
activity of MetK/C240A is identical to that of wild type enzyme.
Assuming that the AdoMet-stimulated tripolyphosphatase activity is an
accurate reflection of the efficacy of the tripolyphosphatase
associated with AdoMet formation (i.e. the role of
tripolyphosphatase in the overall reaction), then one can conclude that
the C240A mutation has primarily affected the V
for AdoMet formation. These results are consistent with a
functional role for a Cys residue at position 240 of the E. coli enzyme. When this residue was changed to an alanine residue (i.e. C240A), the specific activity decreased 10-fold and
consequently is comparable with the specific activity of AdoMet
synthetase isozyme from other sources, all of which contain either
alanine or threonine at the equivalent position. While the mutation of
Cys-240 to alanine produced a highly functionally conserved AdoMet
synthetase mutant, mutation of Cys-90 to either alanine or serine
yielded a protein that is dramatically different from wild type protein
both physically and catalytically. The Cys-90 mutants exist as dimers
and tetramers in contrast to wild type AdoMet synthetase, which is a
tetramer with a dissociation constant <10
M(9) . NEM modification studies of AdoMet
synthetase suggested the involvement of either Cys-90 or Cys-240 in the
oligomeric state of the enzyme, because NEM incorporation produced an
inactive dimeric enzyme(9) . The properties of the Cys-90
mutants support an important role for the side chain of Cys-90 in
tetramer stability. X-ray crystallographic studies of the wild type
AdoMet synthetase indicate that the tetramer consists of identical
subunits related by 222 symmetry. The overall shape shows that the
enzyme is a dimer of dimers. (
)
-helices of
T4 lysozyme have been shown to introduce various degrees of enhanced
stability within that protein(43) , however, crystallographic
studies indicate that Cys-90 is not a component of an
-helix
within AdoMet synthetase but rather is at the beginning of a
-sheet.
Analysis of thermally inactivated samples of
wild type and mutant AdoMet synthetases indicated that inactivation is
correlated with dissociation of tetrameric enzyme into inactive
monomeric protein. If the rate-limiting step in thermal inactivation is
breakdown of the oligomeric structure of the enzyme, then conversion of
Cys-90 to alanine or serine may stabilize the tetramer by removing a
potentially charged sulfhydryl group from a predominantly hydrophobic
subunit interface.
for ATP in both the dimer and tetramer.
Residue Cys-90 lies relatively close to the sequence
Gly-Ala-Gly-Asp-Gln-Gly, which contains the
Gly-X-Gly-X-X-Gly motif found in a number of
nucleotide binding proteins(44) . However, crystallographic
studies show that the side chain of Cys-90 is not directly in the
active site, implying that the changes in the K
for ATP result from structural alterations.
for
tripolyphosphate with the wild type enzyme is not a good reflection of
the dissociation constant due to the slow rate of tripolyphosphate
dissociation, a detailed interpretation of changes in K
values for the mutants relative to wild
type enzyme is not possible.
56% of the specific activity of wild type enzyme. In the presence
of AdoMet, the tripolyphosphatase activity of tetrameric MetK/C90S is
stimulated 30-fold, yielding a specific activity that is approximately
equivalent to that of wild type enzyme. Thus, it appears that the C90S
mutation primarily affects the rate of AdoMet formation.
We are grateful to Dr. S. Tabor for providing us with
the plasmid pT7-6. We are also grateful to J. C. Taylor and C.
Satishchandran for help and constructive discussions pertaining to the
preparation and characterization of the site-specific mutants of AdoMet
synthetase. We also thank A. Pomenti, A. Schmidt, and Dr. A. Zweidlerof
the Protein Analysis Facility for performing mass spectrometry analysis
of wild type MetK and the MetK mutants.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.