Rat guanidinoacetate methyltransferase. Effect of site-directed alteration of an aspartic acid residue that is conserved across most mammalian S-adenosylmethionine-dependent methyltransferases.

Most mammalian S-adenosylmethionine (AdoMet)-dependent methyltransferases have a conserved aspartate residue in a sequence oDso (o denotes a hydrophobic amino acid and s denotes a small neutral amino acid). Rat guanidinoacetate methyltransferase has two aspartate residues (Asp-129 and Asp-134) conforming to the motif in close proximity to Tyr-136 that is photoaffinity-labeled by AdoMet (Takata, Y., and Fujioka, M. (1992) Biochemistry 31, 4369-4374). In order to investigate the role of these residues, we prepared variant forms of the enzyme by oligonucleotide-directed mutagenesis. Conversion of Asp-129 to asparagine or alanine resulted in a functional enzyme. Alteration of Asp-134 to glutamate (D134E) and asparagine (D134N) decreased activity, and replacement with alanine (D134A) led to inactivation. Decreases of 3- and 120-fold were found for kcat values of D134E and D134N, respectively. The Km values of D134E for AdoMet and those for guanidinoacetate were increased about 160- and 80-fold over the respective values of wild type. The corresponding increases in D134N were 800- and 50-fold, respectively. Conservative changes of the residues flanking Asp-134 had little effect on activity. Guanidinoacetate methyltransferase obeys an ordered Bi Bi mechanism in which AdoMet binds first. Thus, the large decreases in kcat/Km values for AdoMet indicate that Asp-134 is crucial for binding AdoMet. Spectroscopic studies indicated that the amino acid substitutions of Asp-134 resulted in no significant changes in the secondary and tertiary structures, and urea denaturation experiments showed that the altered enzymes were not destabilized.

found ubiquitously and in abundance in the livers of all vertebrates and is believed to be a major enzyme involved in the metabolic conversion of AdoMet to AdoHcy in these organisms. Rat liver guanidinoacetate methyltransferase is a simple, monomeric protein with M, 26,000 (Ogawa et al., 1983). The enzyme has been produced recombinantly in large amounts in Escherichia coli (Ogawa et al., 1988), and its structural and functional features have been studied using chemical modification, site-directed mutagenesis, and limited proteolysis. These studies have revealed that Cys-15, Cys-90, and Cys-219 occur spatially close together (Fujioka et al., 1988;Takata et al., 1991) and that the region around residues 19-24 is highly exposed to the solvent and flexible (Takata and Fujioka, 1990;Fujioka et al., 1991). Whereas disulfide cross-linking of Cys-15 with either Cys-90 or Cys-219 leads to a large loss of activity and removal of the N-terminal eicosapeptide results in an inactive enzyme, the portion comprising the 3 cysteines as well as the N-terminal region are apparently distant from the active site. The first indication of an active site residue has been obtained recently by photoaffinity labeling; UV irradiation of the enzyme in the presence of AdoMet resulted in covalent attachment of the compound to Tyr-136, showing characteristics of affinity labeling (Takata and Fujioka, 1992). Ingrosso et al. (1989) showed that many methyltransferases as well as other AdoMet-utilizing enzymes shared three regions of sequence similarity (regions I, 11, and I11 from the Nterminal side). Of these, region 111, which has a sequence motif L(R/K)PGGXL (X represents any amino acid), is unique to and is conserved among most mammalian methyltransferases (Gomi et al., 1992). A previous study, however, demonstrated that this region was not involved in binding of AdoMet in guanidinoacetate methyltransferase (Gomi et al., 1992). Region 11, which is located 20-30 residues upstream of region 111, has an aspartate residue preceded by a hydrophobic amino acid and followed successively by a small neutral and a hydrophobic residue (Table I). In guanidinoacetate methyltransferase, the sequences around Asp-129 and Asp-134 conform to the motif. Since these aspartates are close to Tyr-136, it may be considered that either one of the aspartatecontaining segments forms part of the AdoMet-binding site. In order to test this possibility and to explore the role of the conserved aspartate residue, we introduced amino acid changes to Asp-129, Asp-134, and neighboring residues. In this article, we show that alteration of Asp-134 only exerts profound effects on the catalytic properties of the enzyme.

EXPERIMENTAL PROCEDURES
Materials-AdoMet (chloride salt), AdoHcy, and adenosine deaminase (type VI) were obtained from Sigma, and sinefungin was 5537 purchased from Calbiochem. The amino acid calibration mixture, phenyl isothiocyanate, and ultrapure urea were purchased from Wako (Osaka, Japan). Standardphenylthiohydantoin-derivatives were from Pierce Chemical Co., and a-chymotrypsin and TPCK-trypsin were from Worthington. S-Adenosyl-~-[carbo~ytyl-"C]methionine ([carboxyl-"CIAdoMet) (51.4 mCi/mmol) was obtained from DuPont NEN. AdoMet was purified by passage through a Cla cartridge (Sep-Pak; Waters Associates) as described previously (Fujioka and Ishiguro, 1986), and iodoacetic acid (Merck) was purified by recrystallization from hot chloroform. Other chemicals were of the highest purity available from commercial sources and were used without further purification.  (Yanisch-Perron et al., 1985) transformed with plasmid pUCGAT9-1 that contained the coding region of rat guanidinoacetate methyltransferase cDNA linked to the lilc promoter (Ogawa et al., 1988). The cells were grown in 2 X YT medium containing 35 mg/liter ampicillin at 36 "C. When the cell turbidity measured at 600 nm reached an absorbance of about 0.2, isopropyl-1-thio-8-D-galactopyranoside was added to a concentration of 1 mM, and culture was continued for an additional 16 h. Cells harvested by centrifugation were lysed by treatment with lysozyme, and the recombinant enzyme was purified to homogeneity by the procedure described previously (Ogawa et al., 1988) except that Sephacryl S200 rather than Sephadex G-100 was used in the gel filtration step. The recombinant enzyme lacks the N-terminal acyl group present in the liver enzyme but shows the same kinetic and physical properties as the liver enzyme (Ogawa et al., 1988). Molar concentrations of the enzyme were determined either spectrophotometrically using t = 5.98 X lo' M-' cm" at 280 nm or from protein concentration using M, = 26,000 (Ogawa et al., 1988). Protein concentrations were determined by the method of Lowry et al. (1951) with recombinant guanidinoacetate methyltransferase as the standard.
Site-directed Mutagenesis-Oligonucleotide-directed mutagenesis was used to prepare cDNAs encoding variant forms of guanidinoacetate methyltransferase. Variants are designated by the one-letter symbol of the residue being changed, followed by the sequence number and the symbol of the substituted amino acid. Oligonucleotide primers used are shown in Fig. 1. Primers were synthesized on a Cyclone Plus DNA synthesizer (MilliGen/Biosearch) and purified by HPLC on a Cosmosil 5C18 300 (Nacalai Tesque, Kyoto, Japan) column. Sitedirected mutagenesis was carried out based on the method of Kunkel et al. (1987) using a Mutan-K site-directed mutagenesis kit (Takara Shuzo, Kyoto). Clones containing the desired mutation were identified by nucleotide sequence analysis across the mutation site by the dideoxy chain termination method (Sanger et al., 1977) using Sequenase (U. s. Biochemical Corp.).
Purification of Variant Enzymes-E. coli JM109 transformed with pUC118 plasmid containing each mutated sequence was grown, and the variant enzyme was induced by isopropyl-l-thio-@-D-galactopyranoside as described for wild type guanidinoacetate methyltransferase. All variant enzymes were found in the soluble fractions of E. coli extracts and were purified to electrophoretic homogeneity by the same procedure as wild type guanidinoacetate methyltransferase.
Enzyme Assay-To avoid product inhibition by AdoHcy, the assay of guanidinoacetate methyltransferase activity was carried out in the presence of excess AdoHcy hydrolase and adenosine deaminase, monitoring the formation of inosine. Guanidinoacetate methyltransferase was incubated with various concentrations of AdoMet and guanidinoacetate at 30 'C in 0.1 ml of 0.1 M potassium phosphate (pH 8.0) containing excess recombinant rat AdoHcy hydrolase (Gomi et al., 1989) and calf intestinal mucosa adenosine deaminase. The reaction was terminated by addition of 10 pl of 5 N perchloric acid. After removal of the precipitate by centrifugation, the supernatant was diluted 2-fold with water, and a suitable aliquot thereof was subjected to reverse-phase HPLC on a TSK ODS 120T column (0.46 X 25 cm) (Tosoh, Tokyo, Japan) as described previously (Gomi et al., 1989). Under these conditions, AdoMet appeared at 4.5 min, inosine at 8.5 min, and AdoHcy at 11.0 min. Peak area measurements were made with a Shimadzu C-MA data processor.
For initial velocity studies, the guanidinoacetate methyltransferase activity was determined spectrophotometrically by following the decrease of absorbance at 265 nm due to the conversion of adenosine to inosine (Fujioka et al., 1988). The assay mixture, in a volume of 2 ml, contained the same components as above.
Product inhibition experiments with AdoHcy as an inhibitor were carried out by measuring the rate of formation of [carboxyl-"C] AdoHcy from [~arboxyl-'~C]AdoMet ( [14C]AdoMet). Guanidinoacetate methyltransferase was incubated with ["CIAdoMet (1,500 dpm/ nmol) and guanidinoacetate in the presence of several concentrations of AdoHcy and in the absence of coupling enzymes in 0.2 ml of 0.1 M Tris-HC1 (pH 8.0). When the concentration of AdoMet was being varied, the concentration of guanidinoacetate was held constant at 1 mM, and when guanidinoacetate was the variable substrate, the concentration of AdoMet was 10 p~. The reaction was terminated by adding trichloroacetic acid to a concentration of 1.5%. After addition of carrier AdoHcy, the mixture was subjected to chromatography on DEAE-cellulose paper (Whatman DE81) with 5 mM KzHP04 as solvent. AdoMet and AdoHcy were located by UV light. AdoHcy (RF = 0.35) was well separated from AdoMet (RF = 0.95). The portion of the paper corresponding to the AdoHcy spot was cut out, and the radioactivity was determined by scintillation counting. Several aliquots (25 pl) were removed at time intervals from each reaction mixture to obtain initial velocities.
HPLC Peptide Mapping-Wild type and variant guanidinoacetate methyltransferases were treated with dithiothreitol and then S-carboxymethylated with iodoacetate at pH 8.0 in the presence of 6 M guanidine hydrochloride (Darbre, 1986) prior to proteolytic digestion. The carboxymethylated proteins were dialyzed extensively against water and lyophilized. The lyophilized samples were suspended in 0.1 M NH4HC03 and digested with TPCK-trypsin (substrate/protease ratio, 100:1, w/w) for 16 h at 37 "C. Each digest was fractionated on a TSK ODS 120T column (0.46 X 25 cm) using a gradient of CH&N in 0.05% trifluoroacetic acid as described previously (Takata and Fujioka, 1992). Subdigestion of isolated peptides with chymotrypsin (substrate/protease ratio, 200:1, w/w) was carried out in 0.1 M NH4HC03 for 5 h at 37 "C, and the resulting peptides were separated by HPLC as above. Identification of peptides was carried out by amino acid analysis (Takata and Fujioka, 1992).
Urea Denaturation-Stock urea solutions were prepared daily. The guanidinoacetate methyltransferase proteins were incubated in 0.1 M Tris-HC1 (pH 7.5) containing 5 mM dithiothreitol and various concentrations of urea for 30 min at 25 "C. Denaturation was followed by monitoring the change in intrinsic protein fluorescence. Wild type and altered guanidinoacetate methyltransferases had virtually identical fluorescence spectra with emission maxima at 337 nm (excitation at 280 nm). Upon denaturation with 8 M urea, the emission maximum was shifted to 351 nm with a slight reduction in fluoresence intensity. A wavelength of 326 nm, which shows the greatest fluorescence difference between the native and denatured states, was used to follow denaturation. The reversibility of denaturation was ascertained by the recovery of original spectra upon dialysis against 0.1 M Tris-HC1 (pH 7.5), containing 5 mM dithiothreitol.
Other Analytical Methods-Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed according to Laemmli (1970). The amino acid sequence was determined on an Applied Biosystems 470A/ 120A analyzer. Spectrophotometric and absorbance measurements were made with a Hitachi 320 recording spectrophotometer. Fluorescence spectra were recorded with a Hitachi fluorescence spectrophotometer F-3010 and CD spectra with a Jasco J-500C spectropolarimeter equipped with a DPdOON data processor.

Characterization of Variant Enzymes-Variant proteins
were expressed in E. coli JM109 and purified to electrophoretic homogeneity as described under "Experimental Procedures." Yields of purified proteins ranged from -10 mg/liter (D129A) to -20 mg/liter (D129N, Y133F, D134E, D134N, D134A, T135A, and Y136F). Preliminary activity measurements under the standard assay conditions (20 NM AdoMet and 0.1 mM guanidinoacetate) indicated that the proteins with substitutions at positions 129,133,135, and 136 retained considerable activity, whereas little or no activity was found for Asp-134 variants. To ascertain that the latter proteins contain only the desired and no other substitutions, they were subjected to tryptic peptide mapping. The HPLC elution profiles of the tryptic digests from variants were identical to the profile of the wild type digest except for one peptide in each case (data not shown). The peptides differing in retention times were isolated and further digested with chymotrypsin. The digestion yielded six major peptides in each case, five of which showed identical chromatographic behaviors. Table  I1 shows amino acid compositions of the peptides unique to each enzyme. The composition is compatible with residues 134-142 with or without amino acid substitution. The presence of asparagine rather than aspartate in the peptide from D134N was confirmed by sequence analysis (data not shown). These results establish that single amino acid alterations at position 134 lead to inactivation.
All variant guanidinoacetate methyltransferases prepared had UV absorption spectra and far-and near-UV CD spectra indistinguishable from those of wild type (data not shown). Fluorescence emission spectra (hmm = 337 nm; excitation at 280 nm) were also identical except for D134A that had the same X , but a 7.5% decreased fluorescence intensity. Kinetic Mechanism of Guunidinoacetate Methyltransferme-To facilitate interpretation of effects of amino acid changes on kinetic parameters, we first determined the kinetic mechanism of guanidinoacetate methyltransferase. Doublereciprocal plots of initial velocities against concentrations of AdoMet or guanidinoacetate at different fixed levels of the other substrate gave a series of straight lines intersecting to the left of the vertical axis (Fig. 2). The initial velocity patterns exclude the ping-pong or equilibrium ordered mechanism and indicate either a steady-state ordered or an equilibrium random mechanism for the guanidinoacetate methyltransferasecatalyzed reaction. To distinguish between ordered and random mechanisms, it is necessary to perform product and/or dead-end inhibition studies. Guanidinoacetate methyltransferase is not inhibited by creatine and analogues of guanidinoacetate (guanidinopropionate, guanidinosuccinate, glycine, @-alanine, and y-aminobutyrate) (Fujioka et al., 1988), and compounds to be used as inhibitors are limited to AdoHcy and AdoMet analogues. The product inhibition by AdoHcy was linear competitive versus AdoMet and linear noncompetitive versus guanidinoacetate (not shown). Sinefungin, an analogue of AdoMet, gave the same inhibition patterns (not shown). The observed patterns are consistent with an ordered Bi Bi mechanism in which AdoMet binds first. If the reaction

Mutagenesis of Guanidinoacetate Methyltransferase
follows a rapid equilibrium random mechanism, AdoHcy and sinefungin should be competitive inhibitors with respect to both substrates. In this mechanism, however, a noncompetitive pattern with respect to variable guanidinoacetate could be observed if E. AdoHcy guanidinoacetate or E. sinefungin. guanidinoacetate complex is formed. It was shown previously using equilibrium dialysis that free guanidinoacetate methyltransferase bound AdoMet, but guanidinoacetate became bound only in the presence of sinefungin (Konishi and Fujioka, 1991). Thus, consistent with the available data is an ordered steady-state mechanism in which AdoMet is the first reactant to bind. The values of kinetic constants obtained from the initial velocity and inhibition studies are shown in Table 111.
Kinetic Properties of Variant Enzymes- Table IV  to asparagine resulted in further reduction in knt, but the K,,, values were not greatly different from those of D134E. Binding of AdoMet to D134A was not detected by equilibrium dialysis study. Urea Denaturation of Asp-134 Variants-The difference in conformational stability between wild type and Asp-134 variants was assessed by analyzing urea denaturation curves. The reversible unfolding of the proteins in the presence of urea was followed by the decrease of intrinsic protein fluorescence. Between urea concentrations of 2.5 and 4 M, large changes in fluorescence were observed. The urea denaturation curves were similar to each other but were not superimposable. Assuming that no intermediate exists in the transition from native to denatured state, the free energy change of unfolding (AGO) at each urea concentration was calculated. As shown in Fig. 3, plots of AGD versus urea concentrations were linear, and linear extrapolation to zero urea concentration (Pace, 1986) gave AGDHZo values of 7.5, 7.0, 10.0, and 10.7 kcal/mol for wild type, D134E, D134N, and D134A, respectively.

DISCUSSION
With an exception of mouse erythroleukemia cell DNA methylase, all mammalian AdoMet-dependent methyltransferases sequenced to date have a conserved aspartate residue forming a sequence motif oDso (0 denotes a hydrophobic amino acid, and s denotes a small neutral amino acid) at 20-30 residues upstream of the highly conserved L(R/K)PGGXL motif ( Table I). The three-dimensional structure is not avail- able for any methyltransferase, and relevance of the conserved aspartates in the structure and function of methyltransferases remains to be explored. In the present study we probed the role of the conserved aspartate of guanidinoacetate methyltransferase by site-directed mutagenesis. Guanidinoacetate methyltransferase has two aspartates satisfying the sequence and positional requirements at positions 129 and 134. Conversion of Asp-129 to asparagine or alanine resulted in a functional enzyme, whereas alteration of Asp-134 exerted dramatic effects on catalytic properties (Table IV). Amino acid changes introduced to the residues flanking Asp-134 have minor effects on activity. Asp-134 is in the immediate vicinity of Tyr-136 that is photoaffinity-labeled with AdoMet (Takata and Fujioka, 1992). Thus, it appears that Asp-134 lying within the AdoMet-binding site plays a specific and crucial role in the function of guanidinoacetate methyltransferase.
The simplest kinetic mechanism for the guanidinoacetate methyltransferase reaction may be represented as, In this mechanism, ~,/K,AdoM" represents the association rate constant of AdoMet and guanidinoacetate methyltransferase. Thus, addition of only one extra methylene to or deprivation of negative charge from the residue at position 134 strongly interferes with the interaction between guanidinoacetate methyltransferase and AdoMet. As calculated from the magnitudes of changes in kat/K, values, a change of Asp-134 to glutamate is accompanied by a loss of binding energy of about 4 kcal/mol, and a change to asparagine causes a loss of about 7 kcal/mol. Guanidinoacetate methyltransferase would provide multiple sites of interaction, and these values suggest that something more than merely a salt bridge or hydrogen bonding interaction is lost when Asp-134 is changed to glutamate or asparagine.
Site-specific alteration of Asp-134 also reduced the knt/    KmCM value greatly. The degree of reduction is comparable with that of $.,/KmAdoMet and far exceeds that of kcat. The expression for kat/KmGM includes the unimolecular rate constant for isomerization of the central complex ( k s ) . We do not know which of the chemical step or product release step is normally rate-limiting and which of these steps is affected by amino acid substitutions. If ks is much greater than 4 and the amino acid change affects only the product release step, the decrease in kat/KmCM is due to a decreased affinity of guanidinoacetate to the E . AdoMet  the chemical step determines the rate of catalytic turnover and is influenced by amino acid substitution, the observed changes in kc,, and kat/KmGM suggest that the amino acid alterations exert large effects on k3 and/or k4 and decrease affinity. Thus, in the absence of knowledge about the ratedetermining step in wild type and variants, it is not possible to determine whether alteration of Asp-134 to glutamate or asparagine interferes with binding of guanidinoacetate. The most straightforward way to examine this would be to compare the values of inhibition constants of competitive inhibitors of guanidinoacetate between wild type and variants. Such compounds are not available at present, however. Electronic absorption, fluorescence, and CD spectra of the Asp-134 variants were virtually indistinguishable from those of wild type. Also, the urea denaturation curves were similar for all enzymes. These results indicate that the amino acid substitutions do not result in significant changes in the secondary and tertiary structures and do not destabilize the enzyme. Thus, Asp-134 appears to be a critical residue for the catalytic functioning of guanidinoacetate methyltransferase. The weakening of binding of AdoMet by amino acid substitutions, which is more than can be accounted for by loss of a single hydrogen bond or electrostatic interaction, suggests that the presence of an aspartate at this position is important in forming an adequate structure for substrate binding and catalysis.