©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Structural and Functional Roles of Cysteine 90 and Cysteine 240 in S-Adenosylmethionine Synthetase (*)

(Received for publication, October 26, 1994; and in revised form, March 28, 1995)

Robert S. Reczkowski George D. Markham (§)

From the Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 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.


INTRODUCTION

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) 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.

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.


MATERIALS AND METHODS

L-methionine, ATP, KCl, MgCl(2), Tris, HEPES, 2-mercaptoethanol, NEM, (^1)isopropyl-1-thio-beta-Dgalactopyranoside, tripolyphosphate, and AdoMet were purchased from Sigma. Glycerol was purchased from Baxter Scientific. [1,2-^3H]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).

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-beta-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-beta-Dgalactopyranoside induction, MetK/C90S protein expression accounted for 20-25% of total cellular protein.

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.

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 alpha-hydroxycinnamic acid matrix for analysis.

AdoMet Synthetase Assays

AdoMet synthetase activity was determined by retention of the [^14C]AdoMet formed from L-[methyl-^14C]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 HEPESbulletKOH, pH 8.0, 53 mM KCl, 21 mM MgCl(2), 10.5 mM ATP, 0.55 mML-[methyl-^14C]methionine, specific activity 1.93 mCi/mmol.

Kinetic Characterization of AdoMet Synthetase Mutants

K and V(max) 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-^14C]methionine at a specific activity of 3.7 mCi/mmol, 10 mM ATP (Tris form), 20 mM MgCl(2), 100 mM HEPESbullet(CH(3))(4)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(2) was varied between 0 and 100 mM.

Substrate saturation studies of the AdoMet synthetic activity of the MetK mutants were performed to determine K values for ATP and L-methionine as well as V(max). Samples were incubated at 25 °C for a sufficient time to allow approximately 10% of the L-methionine to be consumed, and [^14C]AdoMet was determined by the filter binding method (3) . The amount of product formed was linear with time.

K and V(max) 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 HEPESbullet(CH(3))(4)N at pH 7.8 with 10 mM MgCl(2) 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 [^3H]NEM (DuPont NEN) into the mutant proteins was also determined as described previously (9) .

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 TrisbulletHCl 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.


RESULTS

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) .^2 Electrophoretic analysis of crude extracts prepared from pT7KC90A/BL21(DE3) revealed increased MetK/C90A expression, which approximated 20-25% of total cellular protein.

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.^3

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 HEPESbulletKOH at pH 8.0 with 50 mM KCl are stable for periods of geq 6 months when stored at -70 °C or for periods of 8 h at 0 °C; however, upon standing at room temperature for geq 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(r), 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) . (^4)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.


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.



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.


Figure 2: Thermal inactivation of wild type MetK and MetK mutants at 70 °C. Enzymes at 2.0 mg/ml in 50 mM TrisbulletHCl/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(2) 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(max) 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(max) values. Interestingly, both C90S variants had K values for both substrates that are close to wild type but V(max) 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) .

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(max) 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-^3H]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 [^3H]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.


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 HEPESbulletKOH at pH 8.0 with 50 mM KCl and 10 mM MgCl(2) 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 [^3H]NEM into tetrameric MetK/C90S. Reactions were initiated by the addition of [^3H]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 [^3H]NEM into tetrameric MetK/C90S as trichloroacetic acid-precipitable radioactivity.






DISCUSSION

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(max) for the AdoMet synthetic reaction. The tripolyphosphatase half-reaction is only 30% reduced in V(max), 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(max) 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 <10M(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. (^5)

The physical basis for the thermal stabilization observed for the Cys-90 mutants is unclear. Replacement of amino acid residues with alanine within alpha-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 alpha-helix within AdoMet synthetase but rather is at the beginning of a beta-sheet.^3 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.

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 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.

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 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.

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 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.

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.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants GM-31186 and CA-06927 and an appropriation from the Commonwealth of Pennsylvania. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Inst. for Cancer Research, Fox Chase Cancer Center, 7701 Burholme Ave., Philadelphia, PA 19111.

^1
The abbreviations used are: NEM, N-ethylmaleimide; MetK/C240A, the alanine 240 AdoMet synthetase mutant; MetK/C90A, the alanine 90 AdoMet synthetase mutant; MetK/C90S, the serine 90 AdoMet synthetase mutant.

^3
R. S. Reczkowski and G. D. Markham, unpublished observation.

^5
S. Kamitori, S. Misaki, G. D. Markham, and F. Takusagawa, submitted for publication.

^2
Novagen Technical Bulletin for pET vectors.

^4
Pharmacia Phast System&#174; Separation Technique File Number 120.


ACKNOWLEDGEMENTS

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.


REFERENCES

  1. Cantoni, G. L. (1975)Annu. Rev. Biochem.44,435-451 [CrossRef][Medline] [Order article via Infotrieve]
  2. Tabor, C. W., and Tabor, H.(1976)Annu. Rev. Biochem.45,285-306 [CrossRef][Medline] [Order article via Infotrieve]
  3. Markham, G. D., Hafner, E. W., Tabor, C. W., and Tabor, H.(1980)J. Biol. Chem.255,9082-9092 [Abstract/Free Full Text]
  4. Markham, G. D. (1981)J. Biol. Chem.256,1903-1909 [Abstract/Free Full Text]
  5. Markham, G. D. (1984)Biochemistry23,470-478 [Medline] [Order article via Infotrieve]
  6. Markham, G. D. (1986)J. Biol. Chem.261,1507-1509 [Abstract/Free Full Text]
  7. Markham, G. D., and Leyh, T. S.(1987)J. Amer. Chem. Soc.109,599-600
  8. Markham, G. D., Parkin, D. W., Mentch, F., and Schramm, V. L.(1987)J. Biol. Chem.262,5609-5615 [Abstract/Free Full Text]
  9. Markham, G. D, and Satishchandran, C.(1988)J. Biol. Chem. 263,8666-8670 [Abstract/Free Full Text]
  10. Satishchandran, C., Taylor, J. C., and Markham, G. D.(1990)J. Bacteriol. 17,4489-4496
  11. Horikawa, S., Tshikawa, M., Ozasa, H., and Tsukada, K.(1989)Eur. J. Biochem. 184,497-501 [Abstract]
  12. Markham, G. D., DeParasis, J., and Gatmaitan, J.(1984)J. Biol. Chem. 259,14505-14507 [Abstract/Free Full Text]
  13. Satishchandran, C., Taylor, J. C., and Markham, G. D.(1993)Mol. Microbiol.9,835-846 [Medline] [Order article via Infotrieve]
  14. Thomas, D., and Surdin-Kerjan, Y.(1987)J. Biol. Chem.262,16704-16709 [Abstract/Free Full Text]
  15. Thomas, D., Rothstein, R., Rosenberg, N., and Surdin-Kerjan, Y.(1988)Mol. Cell. Biol.8,5132-5139 [Medline] [Order article via Infotrieve]
  16. Peleman, J., Saito, K., Cottyn, B., Engler, G., Seurinck, J., Van Montagn, M., and Inze, D. (1989)Gene (Amst.)84,359-369 [CrossRef][Medline] [Order article via Infotrieve]
  17. Peleman, J., Boerjan, W., Engler, G., Seurinck, J., Botterman, J., Alliotte, T., Van Montegu, M., and Inze, D.(1989)Plant Cell1,81-93 [Abstract/Free Full Text]
  18. Horikawa, S., Sasuga, J., Shimuzu, K., Ozasa, H., and Tsukada, K.(1990)J. Biol. Chem.265,13683-13686 [Abstract/Free Full Text]
  19. Horikawa, S., and Tsukada, K.(1991)Biochem. Int.25,81-90 [Medline] [Order article via Infotrieve]
  20. Horikawa, S., and Tsukada, K.(1993)FEBS Lett.312,37-41 [CrossRef]
  21. Larsen, P. B., and Woodson, W. R.(1991)Plant Physiol.96,997-999
  22. Studier, W. F., and Moffatt, B. A.(1986)J. Mol. Biol.189,113-130 [Medline] [Order article via Infotrieve]
  23. Hafner, E. W., Tabor, C. W., and Tabor, H.(1977)J. Bacteriol.132,832-840 [Medline] [Order article via Infotrieve]
  24. Tabor, S., and Richardson, C. C.(1985)Proc. Natl. Acad. Sci. U. S. A.82,1074-1078 [Abstract]
  25. Kunkel, T.(1985) Proc. Natl. Acad. Sci. U. S. A.82,488-492 [Abstract]
  26. Sanger, F., Nicklen, S., and Coulson, A. R.(1977)Proc. Natl. Acad. Sci. U. S. A.74,5463-5467 [Abstract]
  27. Maniatus, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, p. 432, Cold Spring Harbor, NY
  28. Baykov, A. A., Evtushenko, O. A., and Avaeva, S. M.(1988)Anal. Biochem.171,266-270 [Medline] [Order article via Infotrieve]
  29. Cleland, W. W. (1979)Methods Enzymol.63,103-138 [Medline] [Order article via Infotrieve]
  30. Moffatt, B. A., and Studier, F. W.(1987)Cell49,221-227 [Medline] [Order article via Infotrieve]
  31. Deleted in proof
  32. Bukhari, A. I., and Zipser, D.(1973)Nature243,238-241 [Medline] [Order article via Infotrieve]
  33. Donch, J., Chung, Y. S., and Greenberg, J.(1969)Genetics61,363-370 [Free Full Text]
  34. Phillips, T. A., Van Bogelen, R. A., and Neidhardt, F. C.(1984)J. Bacteriol.159,283-287 [Medline] [Order article via Infotrieve]
  35. Grodberg, J., and Dunn, J. J.(1988)J. Bacteriol.170,1245-1253 [Medline] [Order article via Infotrieve]
  36. Ruan, K., and Weber, G. (1993)Biochemistry32,6295-6301 [Medline] [Order article via Infotrieve]
  37. Deville-Bonne, D., and Else, A. J.(1991)Eur. J. Biochem.220,747-750
  38. Deleted in proof
  39. Peacocke, A. R., and Harrington, W. F. (eds) (1986) Electrophoresis: Theory, Techniques and Biochemical and Clinical Applications, Clarendon Press, Oxford
  40. Mudd, S. H.(1963) J. Biol. Chem.238,2156-2163 [Free Full Text]
  41. Chou, T-C. and Talalay, P.,(1972)Biochemistry11,1065-1073 [Medline] [Order article via Infotrieve]
  42. Chiang, P. K., and Cantoni, G. L.(1977)J. Biol. Chem.252,4506-4513 [Medline] [Order article via Infotrieve]
  43. Zhang, X-J., Baase, W. A., and Matthews, B. W.(1991)Biochemistry 30,2012-2017 [Medline] [Order article via Infotrieve]
  44. Wierenga, R. K., Terpstra, P., and Hol, W. G. J.(1986)J. Mol. Biol. 187,101-107 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.