mutagenic investigation of conserved functional amino acids in

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 269, No. 9, Issue of Mareh 4, pp. 6313-6319, 1994 Printed in U S A .

Mutagenic Investigation of Conserved Functional Amino Acids in Escherichia coli L-Aspartase”

(Received for publication, June 11, 1993, and in revised form, November 18, 1993)

A. Sami SanbagS, John F. Schindler, and Ronald E. Violail From the Department of Chemistry, The University of Akron, Akron, Ohio 44325-3601

The potential importance of several functional amino acids in the activity of L-aspartase from Escherichia coli has been examined by site-directed mutagenesis. Amino acids whose importance in enzyme activity was suggested by chemical modification and pH dependence studies were chosen as candidates for investigation. The selection of the particular amino acid targets was guided by homology comparisons among the other sequenced bacterial L-aspartases and by the broader com- parison among the fumarase-aspartase enzyme family. Substitution of the most highly conserved cysteine with either serine or alanine, or the most highly conserved histidine with leucine, had no significant effect on the activity of L-aspartase or on the sensitivity of these mutated L-aspartases to cysteine and histidine specific modifying reagents. However, alteration of each of the two conserved lysines to arginine did cause dramatic changes in the catalytic properties of the enzyme. Modi- fication of lysine 54 results in the complete loss of enzyme activity. However, this activity loss appears to be related to changes in the subunit association properties of the arginine 54 mutant. Lysine 326 appears to be involved in substrate binding. Modification of this residue causes a 5-fold increase in the K, for aspartic acid, a drastic decrease in kc, JK,,,, and a change in the divalent metal ion requirements of the enzyme.

L-Aspartase (L-aspartate ammonia-lyase, EC 4.3.1.1) is a bacterial enzyme which catalyzes the reversible deamination of L-aspartic acid to yield fumarate and ammonium ion. This enzyme is highly specific for its substrate, L-aspartate (l), and has been shown to require M$+ ions at pH values above 7.5 (2, 3). The gene encoding L-aspartase has been cloned and sequenced from several different bacterial sources (4-7). The Escherichia coli gene, aspA, encodes a 477 amino acid protein with a subunit molecular weight of 52 kDa @), and the catalytically active enzyme is a tetramer composed of identical subunits (9). “here is good sequence homology among L-aspartases, including 40- 50% sequence identity among three different bacterial species. This homology extends to functionally related enzymes such as the class I1 fumarases, with 72-76% identity among seven species ranging from E. coli to human (lo), the argininosuccinate lyases with 51% identity between two species (11-13), and a prokaryotic and eukaryotic adenylsuccinate lyase with 27% sequence identity (14). Recent studies have shown that 3-car-

* This work was supported by Grant GM34542 (to R. E. V.) from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Current address: Dept. of Biology, University of Pennsylvania, Philadelphia, PA 19104.

$3 To whom correspondence should be addressed. Tel.: 216-972-6065; Fax: 216-972-7370; E-mail: [email protected].

boxymuconate lactonizing enzyme is also a member of this homologous fumarase-aspartase family (15).

Chemical modification and pH studies have suggested that lysine, cysteine, and histidine residues may play a role in the catalytic activity of L-aspartase (16-18). “Wo specific cysteine residues, cysteine 140 and cysteine 430, were previously ear- marked as potentially important catalytic residues by using chemical modification and subsequent identification of the modified tryptic peptides (2). However, the same researchers later reported that site-directed mutagenesis of cysteine 430 to tryptophan did not result in a loss of enzyme activity, indicating that this residue is not directly involved in the catalytic reaction (19). In the absence of a high resolution structure of an enzyme, homology between structurally similar enzymes can aid in the selection of suitable functional amino acid targets for examination by site specific mutagenesis. Here, we report the results of the mutation of several conserved amino acid residues identified by this approach and the kinetic and structural characterization of the altered L-aspartases resulting from these mutations.

EXPERIMENTAL PROCEDURES Chemicals and Reagents-Restriction endonucleases were obtained

from following companies: U. S. Biochemical Corp., Promega, New Eng- land Biolabs, Pharmacia LKB Biotechnology Inc., and Stratagene. T4- ligase, and Magic Miniprep for DNA isolation were purchased from Promega. T7 DNA polymerase, deoxynucleotides, iaopropyl thiogalado- side, and the Sequenase DNA sequencing kit were obtained from U. S . Biochemical Corp. [d6S1dATP was purchased from DuPont NEN.

Bacterial Strains, Plasmids, and Bacteriophage-E. coli RZ1032 was obtained from Dr. J. J. Villafranca (Bristol-Myers Squibb). E. coli TGl (ecoK-) was purchased from Amersham Corp., and E. coli JRG 1476 (aspA-) strain (4) was a generous gift from Dr. J. R. Guest (University of Sheffield, Sheffield, United Kingdom). The pTZl8R vector was purchased from U. S. Biochemical Corp., and M13mp19 was purchased from Life Technologies, Inc. The pASP vector carrying the aspA gene was a giR from Dr. D. Rozzel (Genetic Institute, Cambridge, MA).

Construction of the pTZ18-ASP and MlJ-ASP(-) Vectors-An expression vector for L-aspartase was constructed by subcloning an aspA containing fragment into the T7 promoter containing pTZ18R plasmid (20). A 1.6-kilobase pair NdeYHindIII fragment was excised from the pASP plasmid, and the NdeI cohesive end was filled by dATP and dlTP using the Klenow fragment of DNA polymerase I. Blunt end ligation was then carried out in the SmaI site of the pTZ18R vector along with Hind111 site ligation to form the 4.6-kilobase pair pTZ18-ASP vector. M13- ASP(-) was constructed by cleaving a 1.55-kilobase pair KpnYHindIII fragment from pTZ18-ASP and subcloning this into M13mp19 bacteriophage in such a way that the single-stranded DNA of the M13-ASP vector will have the (-)-strand of the aspA gene.

Oligonucleotides-The unpurified oligonucleotide primers for mutagenesis were ordered from Operon Technology and were dissolved in sterile TE (Tris-EDTA) buffer (pH 7.3). These primers were either used without further purification or, in some cases, were purified by ammonium acetate precipitation (21). Each oligonucleotide was designed in such a way that screening of the mutants could be accomplished by restriction enzyme mapping. The C389S and C389A mutations resulted in the destruction of one of the two NsiI sites on the plasmid (Table I), with the subsequent loss of a 900-base pair fragment. Two mutations, H123L and K326R, created new restriction sites which did not previ-

6313

6314 Conserved Functional Amino Acids in L-Aspartase TABLE I

Oligonucleotides used in site-directed mutagenesis The mismatched bases are shown in boldface, and the restriction endonuclease sites are underlined and annotated.

Ser-389 oligonucleotide: (Ala-389) cDNA

Leu-123 oligonucleotide:

cDNA

Arg-326 oligonucleotide:

cDNA

5"G GAA AAA TCC ATT AAC G (0)

C CTT TTT ACG TAA TTG C N s i I

5"G ATG GGT CTC CAG AAA G B s a I

C TAC CCA GTG GTC T T T C

5"CAT GCC AGC TCG AGT AAA CCC G Xho I

GTA CGG TCG ATT TCA TTT GGG C

Arg-54 oligonucleotide: 5"GGT AAT GGT TCG AAA AGC CGC AG

cDNA B s t B I

CCA TTA CCA ATT TTT TCG GCG TC

ously exist. In the H123L mutation, conversion of the histidine codon to leucine was accomplished by a 2-base change to create aBsa1 restriction site, which generated 1709- and 2703-base pair restriction fragments to serve as a marker for mutagenesis (Table I). For K326R, a unique XhoI restriction site was created upon changing the lysine codon to arginine, resulting in the conversion of the circular vector to linear DNA upon XhoI treatment. In the K54R mutation, a second BstI site was introduced when the lysine codon was converted into arginine, generating a new 940-base pair fragment.

Site-directed Mutagenesis-The template containing the (-)-strand of the aspA gene was isolated &r transfecting RZ1032 cells with the M13-ASP(-) vector. Uracil-containing single-stranded M13-ASP(-)U was purified by established methods (21), to permit recovery with very high purity without any exogenous primers. The oligonucleotides were phosphorylated by using T4-polynucleotide kinase (21). The mutagenic oligonucleotide to template ratio was 20:l. Annealing reactions were carried out in 10 pl of buffer containing 20 m TriwHCl (pH 7.5), 10 m MgCl,, 50 m~ NaC1, and 1 m~ Dl"..' This oligonucleotide-template mixture was heated at temperatures ranging from 70 to 85 "C, depend- ing on the calculated melting temperature, and then cooled slowly over 30 min to allow the annealing. When the temperature reached <30 "C, the annealing mixture was placed on ice and mixed with 10 pl of 20 m Tris.HC1 (pH 7.5) containing 10 m MgCl,, 10 m Dl", 500 p~ dNTPs, 1 m~ ATP, 1-2 units of T4 DNA ligase, and 1-2 units of native T7 DNA polymerase. The mixture was incubated at 0°C for 5 min, at room temperature for 5 min, and finally a t 37 "C for 2 h. The reaction mixture (5 pl) was analyzed by using a 0.8% agarose gel to observe covalently closed circular DNA formation. This reaction mixture was either used directly to transform TG1 cells permeabilized by the CaCl, method (21) or was kept frozen at -20 "C until use. Six plaques were picked after each mutation and used to transfect exponential TG1 cells to obtain replicative form DNA. The infected cultures were grown in 5 ml of 2YT (16 glliter of bactotrypton, 10 g/liter of yeast extract, 5 g/liter of NaC1) media for 3-4 h. Then, the samples were centrifuged, the bacterial pellet was recovered, and the replicative form DNAs for each mutation were isolated using Promega's Magic Miniprep. The mutations were screened by restriction site analysis.

DNA sequencing reactions were carried out by the dideoxy chain termination method using a Sequenase kit and [ C ~ - ~ ~ S ] ~ A T P to confirm the mutations that had been created. Primer oligonucleotides (17-mer) were designed to be complementary to a position about 100 base pairs upstream from the site of mutation. The reaction mixtures were run on 8% Long Ranger polyacrylamide sequencing gels. The sequences were analyzed by exposure of the gels to x-ray film and subsequent develop- ment according to the manufacturers' guidelines.

Mutant Expression Vectors-Mutated aspA fragments were excised from the M13 vector by using a Hind111 and KpnI double restriction enzyme digestion an.d purified by the Geneclean method. The mutated aspA DNA was then ligated back into pTZ18R vector. The mutant vectors, pTZ18-S389, pTZ18-A389, pTZ18-R326, pTZ18-Ll23, and pTZ18- R54, were thus constructed. These vedors were then used to transform JRG 1476 (aspA-) cells by using the same CaC1, permeabilization method.

1 The abbreviations used are: Dl", dithiothreitol; DEP, diethylpyrocarbonate; DTNB, 5,5'-dithiobis(2-nitrobenzoic acid); NEM, N-ethylmaleimide; OPA, o-phthalaldehyde; YT, yeast tryptone growth media.

Isolation of the Mutated L-Aspartuses-JRG 1476 cells carrying these mutated plasmids were grown in 10 ml of 2YT medium supplemented with ampicillin (100 pg/ml) and tetracycline (20 &ml) overnight. One ml from each culture was then transferred into 100-200 ml of 2YT medium supplemented with ampicillin and tetracycline and grown for 4-5 h at 37 "C. The cultures were harvested by centrifugation. The pellets were resuspended in H E P E W s buffer (pH 7.0) containing 1 m~ Dm, 1 m EDTA, and 30 m magnesium acetate. After sonication, the cell debris was removed by centrifugation, and the clear supernatant was loaded onto a Red A affinity column which was equilibrated with the same buffer (22). The columns were washed with the same buffer until no protein was detected in the wash. The C389S. C389A. and H123L mutations were eluted from the column with the elution buffer containing 2 m L-aspartic acid, whereas the K326R and K54R mutations were eluted with a 0.05-0.35 M KC1 gradient.

Enzyme AssapL-Aspartase activity was determined in 50 m~ HEPES buffer (pH 7.0) containing 30 m~ L-aspartic acid and 5 m~ magnesium acetate at 30 "C in a Perkin-Elmer Lambda-1 spectropho- tometer, by measuring the formation of fumarate a t 240 nm. The protein concentration was determined by Bio-Rad assay. A unit of enzyme activity is defined as the production of 1 pmol of fiunarate/min/ml under these assay conditions. The temperature dependence of L-aspartase activity was measured by incubating the enzyme for 5 min at each temperature in 50 m HEPES buffer (pH 7.5). Aliquots were removed and assayed as described above, and activity is reported as micromoles of product formed per min. Aspartase activity was also examined in the direction of aspartic acid synthesis to allow a determination of the divalent metal ion and activator requirements for the various mutants. Assay mixtures, containing 20 m fumarate and 50 m~ NH4C1, were examined at pH 7.5 and above in the presence or absence of saturating levels of Mg2' (5 m) and various activators.

Chemical Modification Studies-Enzyme modification with o-phthalaldehyde (OPA) was carried out in 400 m borate (pH 8.5) on 0.2 mg of L-aspartase with 100 p~ reagent. Stock solutions of OPA were prepared fresh in 1% methanoUwater. The formation of the isoindole ring was followed spectrophotometrically by using an absorption coefficient of 7.66 m-l cm-' at 337 nm (23) or fluorometrically by excitation at 338 nm and observing changes in the emission spectrum at 420 nm. Pro- tection studies were conducted in the presence of saturating levels of fumarate (20 m), a-methylaspartate (10 m), and M e (5 m).

Enzyme modification with diethylpyrocarbonate (DEP) was conducted in 100 m~ phosphate buffer (pH 6.0). Stock reagent solutions were prepared fresh daily in acetonitrile. The low levels of acetonitrile ( 4 % ) that is introduced into the reaction mixtures by the addition of the varying levels of DEP (50 p~ to 1 m) was shown to have no effect on the activity of L-aspartase. The DEP stock solution concentrations were calibrated by reaction with standard imidazole solutions, with the resulting adduct having an extinction coefficient of 3.0 m-' cm" at 230 nm (24). The extent of L-aspartase modification was also measured spectrophotometrically and compared with the loss of enzyme activity as described above. The site of DEP reaction was confirmed to be at histidyl residues by subsequent reversal of the modification upon addition of hydroxylamine (24).

The number of reactive sulfhydryl groups in L-aspartase under non- denaturing conditions was determined by titration with from 5 to 20 p~ 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) according to the procedure of Ellman (25). The resulting absorbance changes after incubation with

Conserved Functional Amino Acids in L-Aspartase 6315 DTNB were followed at 412 nm, and the stoichiometry of modification was calculated by using an extinction coefficient of 13.6 m"l cm". The rate of enzyme inactivation was examined with the sulfhydryl reagent N-ethylmaleimide (NEM). Enzyme samples were incubated in 100 m~ phosphate (pH 6.5), with varying concentrations (0.2-2.0 m) of NEM. At fixed time intervals, aliquots were removed from this reaction mixture and assayed for catalytic activity as described above.

Gel Electrophoresis-Polyacrylamide gel electrophoresis were run on a Protean I1 Electrophoresis Apparatus (Bio-Rad). 7.5% denaturing gels were prepared in the presence of SDS, 10 pg of each protein was loaded in 50 m~ HEPES buffer (pH 7.0), and the samples were run at 36 mA constant current. Gels were stained with Coomassie Blue dye to visu- alize the protein bands. For the native gels SDS was omitted from the sample and the running buffers (26). The samples were first incubated in buffer (or buffer with 1.0 M guanidine hydrochloride) at mom temperature for 30 min. All native gel samples were diluted in 60 m~ Tris buffer (pH 6.8), containing 10% glycerol and 0.025% bromphenol blue, and were loaded onto the gel without heating. Native gels were run and stained as described above.

Fluorescence Spectroscopy-The fluorescence spectra of various L- aspartase mutants were measured in 50 m~ HEPES buffer (pH 7.0) at room temperature by using a Gilford Fluoro IV spectrofluorometer. The excitation wavelength was set at 280 nm, and the emission spectra were scanned from 400 to 280 nm. A low pass W filter was used to filter out the scattering that would be observed near the excitation wavelength. For the enzyme stability studies, aliquots of proteins were incubated at temperatures between 20 and 77 "C in HEPES buffer (pH 7.3), and the changes in the fluorescence spectra were measured as a function of increasing temperature.

RESULTS

Mutagenic Method-The initial attempts at site-directed mutagenesis of the aspA gene were carried out by using standard protocols. However, methods that have been designed to selectively enhance the production of mutated heteroduplex DNA, including the use of uracil-containing DNA templates (27-29), or the incorporation of phosphorothioates into the mutant-containing DNA strand (30, 31), were not successful in producing mutant colonies. Extensive purification of the template DNA and synthetic oligonucleotides, and subsequent subcloning of the aspA gene into an M13 vector, failed to alleviate the prob- lem. Addition of a single-stranded binding protein (T4 gene 32 protein) did improve the yield of dsDNA, presumably by reliev- ing DNA secondary structure (32,331; however, no mutant colonies were obtained. Repeating the cloning experiments with the antisense (-)-strand of the aspA gene led to a high yield of dsDNA using either sequenase (modified T7 DNA polymerase) or native T7 DNA polymerase. A representative sampling of the plaques from each of the plates were analyzed by restriction enzyme mapping. The plaques from the T7 DNA polymerase- mediated reaction mixture yielded the desired mutation (C389S) with 100% efficiency, whereas the sequenase-catalyzed DNA synthesis did not produce any mutant from the plaques that were analyzed. This experimental approach (34) was then tested by examining mutations at several additional regions of the aspA gene. In each case an oligonucleotide was designed to be complementary to the (-)-strand of the aspA gene. Even in the cases where the yield of dsDNA was low from the (-)-strand, only a few plaques were screened and at least one of the plaques carried the desired mutation.

Mutation of Cysteine and Histidine Residues-N-Ethylma- leimide modification of L-aspartase, and protection by aspartic acid, have suggested the involvement of a cysteine in the catalytic mechanism of L-aspartase (16). Among the 11 cysteines that are present in the E. coli enzyme, only 3 are conserved among the bacterial L-aspartases that have been sequenced. One of these conserved cysteines, cysteine 389, is the only cysteine that is also conserved among all of the members of this homologous fumarase-aspartase enzyme family. The cysteine at this position was replaced with a serine to test the role of the sulfhydryl group in the catalytic activity of L-aspartase from E.

TABLE I1 Kinetic parameters of the L-aspartase mutants

Assay conditions: 30 m~ HEPES, pH 7.0,lO m~ magnesium acetate, and varied concentrations of L-aspartic acid at 30°C. The data were fitted to the Michaelis-Menten equation to obtain the kinetic parameters.

Enzyme V d E t ::?$ LdKm (L-aapartate) K,

min" Native 61.4 f 2.2 88

min" m K 1 14.4 f 1.6

my

C389S 48.2 * 1.9 69 4.3 f 0.6

16.0 * 2.5 C389A 66.2 i 3.6 95 12.7 f 1.9 5.2 * 1.0

3.0 f 0.6

H123L 50.7 f 1.0 72 23.9 f 2.5 2.1 0.3 K326R 0.20 f 0.03 K54R

0.3 0.009 f 0.001 23.8 * 6.0 0.0

a Relative to the consensus V,JEt value of 70 min" for the freshly purified native enzyme under these assay conditions.

coli. No significant decrease was observed in the kinetic parameters of this C389S mutant (Table 11). Next, the cysteine at this position was replaced with an alanine. This nonconservative substitution also failed to alter the kinetic properties of the enzyme. To test the reactivity of this cysteine, the C389A mutant was incubated with varying amounts of NEM. A time- dependent loss of activity was observed. When examined in parallel experiments, the rate constant for activity loss (0.93

8-l) was found to be identical to that measured for the native enzyme. The addition of saturating levels of fumarate provided complete protection against NEM inactivation for both the native and the C389A enzymes. Titration of L-aspartase with DTNB revealed the presence of 1.7 reactive sulfhydryl groups per subunit for the native enzyme. The C389A enzyme was found to have 1.9 reactive cysteinyl residues under the same conditions. When these titrations were repeated in the presence of saturating levels of fumarate, a-methylaspartate, and M e , the stoichiometry of reactive sulfhydryl groups dropped to between 0.7 and 0.9 per subunit for both the native enzyme and the C389A mutant.

The putative role of a histidine in the activity of L-aspartase was also examined by site-directed mutagenesis. Chemical modification studies with diethylpyrocarbonate (17) and pH- dependent activity changes (18) have suggested the presence of an essential histidine in L-aspartase. However, none of the 8 histidines in E. coli L-aspartase are conserved throughout the entire fumarase-aspartase family of enzymes, and only one, histidine 123, is found in the corresponding position in each of the L-aspartases. Mutation of histidine 123 to leucine was carried out to yield an active enzyme which behaved similarly to the native enzyme during purification. The V,, for this mutant has decreased slightly from that of the native enzyme, whereas the kJK, value increased by a factor of two (Table 11). Treatment of the H123L mutant with DEP showed that this enzyme has the same sensitivity to this reagent as was observed for the native enzyme.

Mutation of Lysine Residues-Chemical modification and pH-dependent activity studies have also suggested the importance of a lysyl residue in L-aspartase (18). There are 27 lysines in the enzyme; however only 2 of these, lysine 54 and lysine 326, are completely conserved within the fumarase-aspartase enzyme family. Lysine 326 is also located in a highly homologous region that is found throughout this family of related enzymes and serves as a signature sequence for the recognition of members of this family (Fig. 1). Changing lysine 326 to arginine results in an enzyme that is expressed to high levels in aspartase-deficient (JRG 1476) cells, but which has very low catalytic activity. Purification of the K326R mutant was accomplished on a Red Aaffinity column, to which the enzyme showed similar high binding affinity to that observed for the native enzyme. However, in contrast to the native enzyme, no signifi-

6316 Conserved Functional Amino Acids in Aspartase

Species/enzyme 326

E C A s p A

P f A s p A

B s A s p A

EcFumC

R a t F u m

HumFum

YeaArs

HumArs

PfPcaB

hc. 1. A highly conserved region among the ~aepartaaes, fumarases, and argininosuccinate lyases. Species: Ec, E. coli; w, Pseudomonas fluorescens; Pp, Pseudomonas putida; Bs, Bacillus subti- lis; Hum, human; Yea, yeast. Enzymes: AspA, L-aspartase; Fum, fumarase; Ars, argininosuccinate lyase; PcaB, 3-carboxymuconate ladoniz- ing enzyme. The dark shaded region encompasses those residues that are identical between the E. coli L-aspartase and the other members of this enzyme family. The light shaded area encloses residues that are identical among the functionally related subfamilies, the fumarases, and the argininosuccinate lyases.

cant elution from this column was obtained with L-aspartic acid, even when added at ten times the usual eluant levels. This enzyme was eluted, with a high degree of purity, by using a 0.05-0.3 M KC1 gradient. The purified mutant enzyme has a V,,, which is only 0.3% that of the native enzyme and a k,,lK,,, which has decreased by 3 orders of magnitude (Table 11). The failure of L-aspartic acid to efficiently elute this enzyme during purification is probably due to the decreased affinity of K326R for its substrate, since the K,,, for L-aspartic acid has increased by a factor of five in this mutant.

When L-aspartase was incubated with OPA, a linear relation- ship was observed between the loss of enzymatic activity and changes in both the absorbance and the fluorescence spectra of the enzyme. This loss of activity is correlated with an increase in ahsorbance at 377 nm and the appearance of a peak in the fluorescence emission spectrum at 420 nm when this new absorbance band is excited. These absorbance and fluorescence changes are characteristic of isoindole ring formation resulting from the modification of adjacent lysine and cysteine residues (35). Complete protection against both inactivation and spec- tral modification was observed in the presence of saturating levels of a substrate (fumarate), an activator (a-methylaspartate), and a divalent metal ion ( M e ) , whereas protection was lost if any one of these components was omitted. The K326R mutant was found to be resistant to inactivation by OPA, even in the absence of added substrates or activators, and no spec- tral changes were observed even after prolonged incubation of this mutated L-aspartase with this reagent.

Lysine 54 is also absolutely conserved throughout this enzyme family and was therefore examined by site-directed mutagenesis. The K54R mutant that was constructed is overpro- duced in aspartase-deficient cells; however, no catalytic activity was observed in this cell line. A large fraction of this protein was not solubilized into the supernatant fraction when these cells were initially disrupted by sonication. The soluble protein fraction containing a portion of this mutant was purified by measuring the protein absorbance at 280 nm and by following the overexpressed band that was observed on SDS gels in the position corresponding to that of the native enzyme throughout the protein purification. The highly purified protein sample that was obtained was also observed to be devoid of measurable enzymatic activity.

Kinetic Characterization of the Mutated L-Aspartases-"he pH profiles of the mutated enzymes were examined from pH 6.0

100%

I \ - _

80% - I \

I \ , \ I \ , \

I \

60% - I \ , \

I \ , \ I \

4 280 300 320 340 360 330 400

nm

the K326R (. . . .), and the K54R (- - - -) mutated ~-aspartases are hc. 2. The fluorescence intenmities of ~aspartaaes. Native (-),

shown. In each case, 6 pg of protein was dissolved in 3 ml of 50 m HEPES buffer (pH 7.0). The excitation wavelength was 280 nm, and the emission spectra were scanned up to 400 nm.

to 9.5 and compared with that of the native enzyme. The C389A, C389S, and H123L mutations possess a similar pH dependence to that of the native L-aspartase $181, showing decreased activity below pH 8 and maximum activity near pH 8.5 (data not shown). These mutated enzymes were determined to also have the same M e dependence as has been observed in the native enzyme, with no requirement for Mg2' for catalytic activity at pH values below 7.0. The K326R mutation, which has only 0.3% of the activity of the native enzyme, produces a pH profile similar to that of wild type enzyme when M$+ ions are present. However, in contrast to the wild type or the other mutated L-aspartases, this mutated enzyme has no measurable activity at any pH value when M$+ ions are omitted.

The wild type enzyme has been shown to display a lag time prior to catalyzing the formation of L-aspartic acid, when incubated in a buffer at pH values above 7.5, even in the presence of sufficient levels of fumarate, ammonium ions, and Mg2' (1). This same lag time is observed for the mutations at Cys-389 and His-123. In contrast, the K326R mutation does not possess this lag time even at pH values of 8.0 and above, where the lag in the native enzyme can extend to several hours. The reverse reaction starts immediately upon addition of the mutated enzyme. Furthermore, the rate of the reaction catalyzed by K326R is not affected by activators, such as D-aspartate, L- aspartate, or a-methylaspartic acid, which were shown to eliminate the lag time in the reaction of the wild type enzyme (1).

Structural Characterization of the Mutated L-Aspartases-L- Aspartase does not contain any tryptophans, so the fluorescence emission measured for this enzyme results from tyrosine fluorescence. The fluorescence spectra of native L-aspartase was observed to be essentially the same as that of free L-tyrosine, when examined under similar conditions. The fluorescence spectra of the mutants, C389S and C389A, are also the same as that of L-tyrosine and the native enzyme. However, the spectra of several of the mutated L-aspartases do show some differences. The emission maxima for H123L is the same as that of the wild type enzyme. However, in the high wavelength region, an additional peak is observed at about 360 nm which is not present in the wild type spectrum or in that of the cysteine mutants (Fig. 2). The spectmm of the K326R mutation is also similar to that of H123L mutation, whereas the K54R mutation showed a dramatically different emission spectrum. This spec-

Conserved Functional Amino Acids in Aspartase 6317 I I , 1.0

e A 4 0.8

1 6% 20 30 40 50 60 70 80

24%

20%

6 0 0

0 0 B 1.0

1 6% 2 0 30 40 50 60 70 80

I I 90% -

80% -

70% -

60% -

50% -

40% - v

30% ; 20 30 40 50 60 70 80

Temperature (OC) FIG. 3. The temperature dependence of the fluorescence emis-

sion spectrum and catalytic activity of ~~aspartase. A, native L- aspartase; B, the C389S; and C, the K54R mutants of L-aspartase. The relative fluorescence at the emission maximum (-) and the enzyme activity (- - - -) in micromoles of produdmin were measured in 50 m~ HEPES buffer (pH 7.3-7.5). The filled symbols indicate the temperature above which turbidity is observed.

trum has a much broader peak which is shifted to higher wavelength by about 10 nm and shows a 4-fold increase in the fluorescence intensity (Fig. 2).

The stability of each of the mutated L-aspartases was analyzed by measuring the changes in fluorescence intensity at increasing temperatures. The fluorescence intensity of these proteins was observed to gradually decrease as the temperature was increasing, until the protein began to denature. At this point the solution became cloudy due to the denaturation of the protein, and the fluorescence intensity began to increase (Fig. 3A) . The mutated enzymes, C389A, H123L, and K326R, show a similar pattern to that of wild type enzyme. The denaturation of the wild type enzyme and each of these mutations, as measured by fluorescence intensity changes and the onset of turbidity, begins at a temperature of about 45-50 "C. The catalytic activity of these enzyme forms also decreases rapidly in this temperature range (Fig. 3A). The C389S fluorescence spectrum also decreases as the temperature rises; however, no cloudiness is observed in the solution until a temperature of 75 "C. There is also no break in the gradual decrease in fluorescence intensity, although the catalytic activity is lost in the same temperature range (40-50 "C) as the native enzyme (Fig. 3B). The K54R fluorescence spectrum also decreases as the temperature rises; however, the onset of turbidity in the solution is not observed until a temperature of 60-65 "C (Fig. 3). The fluorescence intensity continues to decrease more rapidly at increasing temperatures, reaching a value at the highest

1 2 3 4

oligomer +

tetramer +

various ~"partases. Approximately 10 pg of each enzyme in HEPES FIG. 4. Polyacrylamide gel electrophoresis-gel analysis of the

buffer (pH 7.0) were loaded onto a 7.5% gel which was run at constant current (36 mA). Lune 1, native enzyme; lane 2, K326R mutant; lane 3, K54R mutant; lane 4, C389S mutant.

temperatures measured that approaches that of the other temperature denatured baspartases.

The complete loss of catalytic activity and the striking changes in the fluorescence spectrum and thermostability of the K54R mutation suggests that this single lysine to arginine mutation has caused extensive changes in the structure of L- aspartase. As expected, SDS gels of K54R, and all of the other mutations of L-aspartase that have been produced, show that each of the resulting subunits are indistinguishable from those of the native enzyme (data not shown). However, native gels of the K54R mutation indicates a large increase in the size of this protein when compared with that of the native enzyme or to the Cys-389 or Lys-326 mutations (Fig. 4). Thus, the major structural alteration in K54R appears to be to the enzyme quater- nary structure. Treatment of the native enzyme with 1 M guanidine HCl had been shown previously to cause dissociation of L-aspartase into monomers and also to result in extensive un- folding of the enzyme subunit (36). Subjecting the Cys-389 and Lys-326 mutations to these conditions also resulted in subunit dissociation of the tetramer. However, no change was observed in the gel mobility of the K54R oligomer, suggesting that this oligomer remains intact under these dissociating conditions.

DISCUSSION

Homology among the Members of the Fumarase-Aspartase Family-There is about 38% overall sequence identity in the fumarase-aspartase enzyme family and a much higher degree of homology if conservative substitutions are included. There are also several well defined regions of high sequence identity (such as that shown in Fig. 1) which clearly establishes these enzymes as members of a homologous, structurally related

6318 Conserved Functional Amino Acids in Aspartase

family. This sequence homology has assisted in the selection of potential targets to examine by site-directed mutagenesis.

Mutagenesis-Attempts at oligonucleotide-directed mutagenesis of the aspA gene did not initially succeed in producing the desired mutations. The success of this modified method of site-directed mutagenesis (34), that is, reverse cloning into an M13 vector and the use of native T7 DNA polymerase, has led to high yields of mutant colonies, and this success has been confirmed at several loci in the aspA gene. This approach appears to be a useful alternative method to carry out site-directed mutagenesis of genes that are resistant to mutagenesis by the standard protocols.

The replacement of an amino acid located at a specific position in a protein has become, in principle, quite straightfor- ward. However, unless extraordinary measures are employed, the amino acid to be substituted is limited to one of the twenty amino acids specified by the genetic code. In many cases, the best candidates that are available for substitution can still result in dramatic alterations in the amino acid side chain properties. In these cases, a decision must be made about which property (charge or side chain volume) might be more critical for enzymatic activity. Among the changes that have been en- gineered into the amino acid sequence of L-aspartase are conservative changes that preserve the properties of the original amino acid (cysteine + serine), changes that preserve the side chain volume but eliminate the amino acid charge (histidine +

leucine), and changes that preserve the charge but increase the volume (lysine + arginine). A statistical analysis of exchanged amino acids in homologous protein families has provided some guidelines for amino acid substitutions that are least likely to disturb either the local or the overall protein structure (37). Although no pattern of tolerable amino acid substitutions for histidines was found among this group of proteins, arginine was found to be the most acceptable substitution for a lysine residue. These specific amino acid substitutions have been carried out to examine the role of some conserved functional amino acids in L-aspartase.

Properties of the Mutated L-Aspartases-The K,,, value of the H123L enzyme for L-aspartic acid is not appreciably altered, indicating that histidine 123 is probably not involved in direct interactions with the substrate. The V,,, of this mutated enzyme is reduced by about 30% when compared with the consensus value observed for the native enzyme. The reduction in catalytic activity can most likely be attributed to minor structural alterations. This view is supported by the small changes that have been observed in the fluorescence spectrum of this mutant. Replacement of this histidine with a leucine does not alter the sensitivity of the enzyme to histidine modifying reagents nor the metal ion dependence of the enzyme, thus elimi- nating this residue from consideration as a metal ligand at the enzyme activator site. Despite the kinetic and pH dependence evidence suggesting the importance of a histidyl group in L- aspartase, there are no other conserved histidines among the bacterial L-aspartases that are obvious candidates for either an active site functional group or a metal ion binding group. The highly conserved cysteine residue at position 389 also does not appear to be essential for binding or for catalytic activity. Re- placement with either a serine or an alanine has no effect on the activity of the enzyme. Chemical modification of L-aspartase with the sulfhydryl reagents DTNB and NEM was also unaffected by these amino acid replacements. Two reactive cysteines remain in the mutated enzyme, with only a single cysteine modified in the presence of substrate and activator. These studies continue to suggest a functional role in L-aspartase for a cysteinyl residue, the identity of which has not yet been resolved.

In contrast to the lack of significant kinetic consequences

when the most highly conserved cysteine or histidine residues were mutated, alteration of the conserved lysyl residues causes dramatic changes in the catalytic properties of L-aspartase. Lysine 326, located in the highly conserved signature sequence of the fumarase-aspartase family, appears to be directly involved in the binding of the substrate. The evidence in support of this putative role for lysine 326 includes the significant changes that are observed in the kinetic parameters for this mutated enzyme and the complete loss of reactivity with a reagent (OPA) that was shown to selectively modify a substrate protected lysyl residue. The most likely region of contact of lysine 326 with the substrate is at one of the two carboxylic acid groups of either L-aspartic acid or fumaric acid. The other members of this homologous enzyme family also have fumaric acid as a common reactant. The conserved lysine at the corresponding position probably plays a similar role in each of these enzymes. Substitution of this lysine with the bulkier, but still positively charged, arginine may hinder access of the substrate to the enzyme catalytic site or may result in the substrate binding in a distorted orientation. It has not been possible to directly test the regiospecificity of this interaction, since substrate structural analogues which are missing either the p-carboxyl group (alanine) or the a-carboxyl group @-alanine) do not bind to L-aspartase. The enzyme containing this lysine to arginine mutation at position 326 has also lost the pH-dependent metal ion activation that is observed with the wild type L-

aspartase. This K326R mutant has an absolute requirement for a divalent metal ion at all pH values and also shows no lag time even at higher pH. These changes are probably not the result of any direct metal ion interactions with this lysine, but may be a consequence of the change in communication between the active and activator sites upon mutation. High resolution structural characterization of L-aspartase will be required to address this question, and progress is being made toward this god (38).

Alteration of the other lysine residue has an even more dramatic effect on the enzyme. Substitution of lysine 54 with arginine causes the complete loss of L-aspartase catalytic activity. The 4-fold increase in fluorescence emission intensity measured with the K54R mutant is indicative of a more highly shielded tyrosine environment (less solvent quenching) when compared with the wild type enzyme or free tyrosine. This observation is consistent with the oligomerization of K54R that has been seen on native gels. The results of these experiments support the idea that lysine 54 may be located on the solvent accessible surface of the enzyme. However, this residue is probably not involved at the subunit interface, since further oligomerization of tetramer would require the interaction of a surface residue that is still exposed. The new contacts that result in protein aggregation with the arginine 54 substitution must be quite strong, despite the somewhat innocuous replacement of a single positively charged residue with another. The K54R mutant that is produced is isolated from E. coli predominately as an aggregated inclusion body. Conditions, such as increasing levels of guanidine HC1, that have been shown to separate the native tetramer into monomers appear to have no dissociative effect on the K54R oligomer.

Homology studies among sequence related protein families can frequently serve to identify potentially important amino acid residues. However, in L-aspartase the most highly conserved cysteine and histidine residues have been shown to play no direct role in the mechanism of action of this enzyme. Al- though it is necessary to preserve the functionality of an amino acid residue that plays an essential role in binding or in cataly- sis, the identification of a highly Conserved amino acid of this functional type in a related family of proteins does not appear to be sufficient evidence for essentiality.

Conserved Functional Amino Acids in L-Aspartase 63 19

Acknowledgments-The temperature-dependent activity studies were conducted by Maithri Jayasekera. We thank Drs. John Guest and Joseph Viafranca for providmg various bacterial strains that were used in this investigation. The generous gift of the aspA-containing plasmid by Dr. David Rozzel is also gratefully acknowledged.

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