Biochimica et Biophysica Acta, 789 (1984) 51-56 51 Elsevier

BBA 31955

PARTIAL AMINO-ACID SEQUENCE AND CYSTEINE REACTIVITIES OF CYTOSOLIC ASPARTATE AMINOTRANSFERASE FROM H O R S E HEART

FILIPPO MARTINI a, SEBASTIANA ANGELACCIO a DONATELLA BARRA a, SHAWN DOONAN b and FRANCESCO BOSSA a.,

a Istituto di Chimica Biologica, Universiti~ di Roma "La Sapienza" and Centro di Biologia Molecolare del C.N.R. Rome (Italy) and b Department of Biochemistry, University College, Cork (Republic of Ireland)

(Received December 29th, 1983)

Key words: Amino acid sequence," Aspartate aminotransferase; Isoenzyme; Chemical modification; Cysteine," (Horse heart)

Cytosolic aspartate aminotransferase (L-aspartate: 2-oxoglutarate aminotransferase, EC 2.6.1.1) from horse heart has five cysteine residues, two of which can be titrated with 5,5'-dithiobis(2-nitrobenzoid acid) in the native enzyme with no impairment of catalytic activity. The rate of modification is unaffected by the presence of substrates. Reaction with N-ethylmaleimide leads to loss of catalytic activity, the rate of inactivation being increased by the presence of substrates. Peptides containing 361 amino-acid residues (about 88% of the total number in the protein) have been isolated and aligned by comparison with the known sequence of the isotopic isoenzyme from pig heart. In the regions compared, 342 of the residues are identical. Hence, assuming that those regions are representative of the whole, then the cytosolic isoenzymes from horse and from pig have about 95% identity of structure. Uniquely among the mammalian cytosolic aspartate aminotransferases so far examined, the enzyme from horse heart is acetylated at the N-terminus.

Introduction

In a previous paper [1] we reported a nearly complete primary structure for mitochondrial aspartate aminotransferase (L-aspartate : 2-oxo- glutarate aminotransferase, EC 2.6.1.1) from horse heart. Comparison with the amino-acid sequence of the homotopic isoenzyme from pig heart re- vealed that the two proteins have 96.3% identity of primary structure. Among the features conserved in the two proteins are both the number and reactivity of sulphydryl groups. In particular, cy- steine-166, which is implicated in the process of uptake of the rat liver isoenzyme into mitochondria [2], is conserved in the isoenzyme from horse heart,

* To whom correspondence should be addressed: Istituto di Chimica Biologica, Universita di Roma, Citth Universitaria, 00185 Roma, Italy.

as has been shown also for all other mitochondrial isoenzymes so far analyzed.

Since statistical comparisons of amino-acid compositions of aspartate aminotransferase isoen- zymes suggested that the mitochondrial and cyto- solic forms have evolved at approximately equal rates [3], whereas the results obtained from im- munochemical studies [4,5] suggested a closer rela- t ionship be tween m i t o c h o n d r i a l a spa r t a t e aminotransferases than between the cytosolic iso- enzymes, availability of further sequence data on isoenzyme pairs seemed highly desirable in order to clarify these apparently contradictory findings. For this reason, and also to contribute to a fuller understanding of structure-function relationships in this class of proteins, we undertook a study of the primary structure and properties of sulphydryl groups of cytosolic aspartate aminotransferase from horse heart.

0167-4838/84/$03.00 © 1984 Elsevier Science Publishers B.V.

52

Materials and Methods

Cytosolic aspartate aminotransferase was pre- pared from horse heart according to a previously published scheme which yields also the mitochon- drial isoenzyme [6].

Titration of sulphydryl groups with 5,5'-di- thiobis(2-nitrobenzoic acid) in sodium dodecyl sulphate and determination of the reactivity of sulphydryl groups in the native protein toward this reagent or towards N-ethylmaleimide in the pres- ence or absence of the substrate pair were per- formed on freshly prepared samples of enzyme essentially according to the procedures used by Gehring and Christen [7] and by Petrilli et al. [8]. The total number of cysteinyl residues was de- termined by analysis of cysteic acid residues per- formed after acid hydrolysis of the protein in the presence of dimethylsulphoxide [9].

A sample (250 mg) of holoenzyme was reduced with NaBH4 and carboxymethylated with radioac- tive iodoacetate as previously described [10] and then digested with 5 mg of chymotrypsin (Worthington) in 0.1 M ammonium bicarbonate for 4 h at 37°C. Procedures adopted for peptide purification and analysis were the same as those used for the mitochondrial isoenzyme [1,11].

A peptide which was unresponsive to N-termi- nal analysis was deblocked by treatment with tri- fluoroacetic acid (25%, v /v ) for 2 h at 55°C, and then subjected to normal N-terminal analysis and manual dansyl-Edman degradation [12]. For isola- tion of the N-terminal blocked peptides from the intact protein two approaches were followed. In the first, 20 mg of carboxymethylated protein was digested with 1 mg of trypsin for 5 h at 37°C in 0.1 M ammonium bicarbonate and then with 0.8 mg of carboxypeptidase A for 2 h under the same conditions. The mixture of peptides after repeated lyophilization was loaded onto a column (2.5 x 24 cm) of SP-Sephadex C25 in the H + form and the column eluted with 0.1 M acetic acid. The eluted fraction was lyophilized and subjected to final purification by high-performance liquid chro- matography on a reverse-phase column (Brownlee Labs, RP-300, 10 /zm) with a gradient of 5% to 50% (v /v) acetonitrile in 0.2% trifluoroacetic acid generated in a Beckman model 420 instrument at a flow rate of 1.0 m l / m i n . The absorbance of the

effluent was monitored using a Beckman model 165 variable wavelength detector set at 220 nm. Peaks were collected at a point immediately after the detector flow-cell, lyophilized and subse- quently analyzed.

In the second approach, native enzyme (29 mg) in 13 ml of 10 mM ammonium bicarbonate buffer pH 7.8 was digested with 0.5 mg of the proteinase from S. aureus for 24 h at 37°C. Soluble peptides from the digest were dried, taken up in water and applied to a column (0.8 x 1.2 cm) of Dowex 50W-X4 (200-400 mesh) in the H + form. Unre- tarded material was eluted with water.

The nature of the blocking group on the N- terminus of the peptide isolated as described above was investigated by a modification of the method reported by Schmer and Kreil [13]. Anhydrous hydrazine was prepared by azeotropic distillation with toluene [14]. The peptide was dissolved in 0.5 ml of 0.1 M HC1 and dried to remove possible traces of free acetate. Hydrazine (0.2 ml) was added and the sample was heated under vacuum at 100°C for 17 h, after which the hydrazine was removed under reduced pressure. The product was dansylated in 0.3 ml of 0.2 M sodium citrate buffer (pH 3.0) using a 10-fold excess of dansyl chloride at 37°C for 24 h. After drying, the sample was taken up in 0.3 ml H 2 0 and dansylated prod- ucts were extracted with 3 x 0.3 ml chloroform. The products were examined by thin-layer chro- matography on polyamide plates using the conven- tional solvent systems for identification of dansyl amino acids. A sample of 1-acetyl-2-dansyl hy- drazine, prepared by dansylation of acetylhydra- zine, was used as a standard.

Results and Discussion

Reactivity of sulphydryl groups The number of sulphydryl groups of cytosolic

aspartate aminotransferase from horse heart titra- table in the presence of SDS was 4.8, in good agreement with the number of cysteic acid residues (5) found after hydrolysis of the protein in the presence of dimethylsulphoxide and also with the value determined for the homotopic enzyme from pig heart by structural analysis [15]. However, the reactivity of sulphydryl groups in the native equine enzyme under various conditions is markedly dif-

ferent from that of the isoenzyme from pig heart in that two groups can be titrated with 5,5'-di- thiobis(2-nitrobenzoic acid) in the resting enzyme (i.e., in the absence of substrates) with no impair- ment of the catalytic activity but a phenomenon characteristic of the working pig isoenzyme; that is, the 'syncatalytic ' increase of reactivity toward the reagent of a third sulphydryl group when titration is performed in the presence of a sub- strate pair [16] is not detectable. The search for a possible 'syncatalytic ' sulphydryl group in the cy- tosolic equine isoenzyme was extended by mea- surement of its reactivity toward N-ethylmaleimide in the presence and absence of substrates. For the homotopic isoenzyme from pig, an increased 'syn- catalytic' reactivity toward this reagent is observ- able with concomitant loss of enzyme activity. With the equine enzyme a higher concentration of reagent was used, following the report by Petrilli et al. [8] for the bovine cytosolic enzyme. Progress of the reaction could be conveniently monitored by measuring enzyme inactivation and the results ob- tained are shown in Fig. 1. In the same figure, results obtained under identical conditions with a sample of porcine cytosolic isoenzyme and also data taken from the results of Petrilli et al. [8] obtained with the bovine enzyme, are reported for comparison. A second-order inactivation rate con- stant of 0.4 M -~. min 1 was calculated for the equine cytosolic isoenzyme, one order of magni- tude lower than that (4.0 M-~ -rain 1) found for

lOO

N 5O C.?

F- 25 _ •

~ °

\ I I

0 20 40 60 TIME[mini

Fig. 1. Inactivation of cytosolic aspartate aminotransferase by reaction with 42.6 mM N-ethylmaleimide. Horse isoenzyme (e); pig isoenzyme (m). Dashed line: data taken from Ref. 8 for the bovine isoenzyme.

53

the porcine enzyme [16]; we have found a g [ n a c t =

2.0 M -1 . min -1 under slightly different experi- mental conditions and with a porcine enzyme pre- paration kept for some weeks as an ammonium sulphate suspension. Inactivation rates with N-eth- ylmaleimide of 0.25 M - 1. m in - 1 and 0.02 M - 1. min-1 have been determined for the homotopic isoenzymes from bovine [8] and chicken heart [7], respectively.

Thus, our results with the cytosolic isoenzyme from horse heart confirm the idea that 'syncata- lytic' reactivity of a specific sulphydryl group, which in the porcine enzyme has been identified as cysteine-390, is a phenomenon present in all the cytosolic isoenzymes so far analyzed, but ex- pressed to a widely variable extent [8]. On the other hand, mitochondrial isoenzymes of aspartate aminotransferase of various origins, including horse heart, possess a sulphydryl group (cysteine- 166) which is also 'syncatalyticaUy' modifiable but with no impairment of enzyme activity, and whose behaviour remains constant in all the examples so far examined [1,2,7,8]. This strict conservation of the properties of the single titratable cysteine re- sidue is consistent with its postulated involvement in uptake of the isoenzyme into mitochondria [2].

A mino-acid sequence analysis Five cysteine-containing peptides were isolated

from the chymotryptic digest of cytosolic aspartate aminotransferase from horse heart with relative ease, taking advantage of the radioactive iodoace- tate employed for protein carboxymethylation; the sequences of these peptides are shown in Fig. 2, aligned along the sequence of the homotopic pig heart isoenzyme. The five cysteine residues, includ- ing cysteine-390, were found in positions identical to those of the corresponding pig heart isoenzyme; evidently, the observed different extents of 'syn- catalytic' reactivity in the cytosolic enzymes is attributable to variations in the environment pro- vided by more distant parts of the polypeptide chain and not to the immediately adjacent re- sidues.

Additional chymotryptic peptides were isolated and analyzed; on the basis of homology considera- tions they could easily localized along the se- quence of the corresponding porcine isoenzyme, with the exception of a peptide obtained in rela-

54

c-AAT-p A F P S V F A EV PQA Q pV L V FK2LOI A D F R E D c-AAT-e AC-T S "-- I - V I ~ T ) +--

~0 60 P D P R K V N L G V G A R T D D C Q P W V L P V V R K V E Q R I A N

NS S LNHEYLP I LG LA EFBOTcA SR LALC DD SPA LQE

- - - - ~ ~ ~ ~- S-- ) + ~ ~

IvOOG 120 K R GVQSLGGTCALRI CAEFLARWY NGTN NKDTP

) • ) e N--

140 160 VYVSSPTWENHNGVF TTAGFKD IRSYRYWDTEKRG

~o 2oo LDLQGFLSI) LKN EFS I F V L H A C A H N P T G T D P T P

EQWKQIASVMKRRFLFp22FOFDsAYQGFASGNLEKDA - - ) ( m ( ~ )( D R - -

240 260 WA I RYFVSEGFE LFCAQS F S K N F G L Y N E R V G N L TV

S 218OL 300 V A K E P D R V L S Q H Q K I V R V T W S N P P A Q G A R I V.A D4 ) ( I - - - t (

V% 3~o RTLSDPELFHEW N V K T M A D R I L S M R S E L R A R L E

i%( G - - - ' , ( )( ) ( - - - ' - ~'~, ~4 -

A LK TPGTWN H I TDQ I GM3~OS F TGL N PKQV EY L I NQK

D ~ - - -~ ( ) e V - -

H I 3 ~ O L L p S G R I N M C G L Z r K N L D y4vUOA r S I H E A V T K I Q

Fig. 2. The partial primary structure of cytosolic aspartate aminotransferase from horse heart (c-AAT-e) compared with that from pig heart (c-AAT-p). The complete sequence of the isoenzyme from pig heart is given [15]. Arrows beneath this structure show the parts of the isoenzyme from horse heart that have been sequenced with amino acid substitutions inserted into the arrows. Full arrows denote peptides for which both amino acid analysis and complete sequence analysis by the dansyl Edman method were performed. The partially dashed arrow denotes a peptide for which the N-terminal section was sequenced and the remainder of the structure assigned from the amino acid composition and comparison with the sequence of the same region in the isoenzyme from pig heart.

tively high yield (19%), which did not show any N-terminal residue after reaction with dansyl chloride and which was resistant to Edman de- gradation. The latter peptide was placed at the N-terminus of the molecule on the basis of experi- ments and considerations reported below.

Sequence data from all of these peptides are also shown in Fig. 2; 361 residues have been identified (87.6% of the structure) and there are 19 differences between the equine and porcine isoen- zymes in the regions identified. This amounts to a difference of 5.3% between the two isoenzymes as compared with the difference of 3.7% found be- tween the mitochondrial isoenzymes from the same two species [1]. These degrees of sequence identity are similar to that found for cytochrome c from

the same two sources (2.9%), but very much less than for the a-chains of haemoglobin (13.5%). Hence, both isoenzymes of aspartate aminotrans- ferase should be considered to be conservative proteins. Assuming that the regions for which structural information is available are representa- tive of the whole, then sequence data from the horse isoenzymes would indicate a faster rate of change for the cytosolic compared with the mitochondrial forms, in agreement with the results of immunochemical studies [4,5] but in conflict with the predictions from comparisons of amino- acid compositions of isoenzymes obtained from a variety of sources which suggest a similar rate of change for the cytosolic and mitochondrial isoen- zymes [3]. However, the sequence data should be viewed with some caution. One very striking fea- ture of the cytosolic isoenzyme from horse is the low degree of homology of the first five amino acid residues when compared with the homotopic isoenzyme from pig. Three differences are ob- served in these five residues, which is a much greater degree of variability than is found between any other pair of cytosolic mammalian isoenzymes for which information is currently available [17]. This variability of the N-terminal tract obviously contributes substantially to the total sequence dif- ference between the pig and horse isoenzymes; indeed, if these residues are ignored then the dif- ference between the two cytosolic isoenzymes de- creases to 4.4%, a value very similar to that for the mitochondrial forms. It is likely, therefore, that the prediction from immunochemical analysis of a rate of evolution of mammalian cytosolic isoenzymes twice that of the mitochondrial forms [4] is an overestimate in the general case. This is under- standable, since immunochemical methods detect largely differences on the surface of mature pro- teins, which may not always reflect the extent of differences in internal regions. In this context, it is important to note that the complete amino-acid sequences of the isoenzymes from chicken are now available [18,19]. Comparison of these sequences with those of the homotopic isoenzymes from pig shows 14% difference and 17% difference for the mitochondrial and cytosolic pairs, respectively [19]. Hence, these results also suggest a very similar rate of evolution of structure for mitochondrial and cytosolic aspartate aminotransferases.

Isolation and sequence analysis of an N-terminal blocked peptide

The amino-acid composition of the peptide iso- lated from the chymotryptic digest with an unreac- tive N-terminal residue was: Thr (1.1); Ser (2.1); Pro (1.0); lie (1.0); Phe (0.9). Treatment with carboxypeptidase Y released Phe (1.0), lie (0.9) and Ser (0.2) (mol per mol of peptide). After treatment with 25% (v/v) trifluoroacetic acid, the peptide became amenable to dansyl-Edman de- gradation and the following sequence was estab- lished: Thr-Ser-Pro-Ser-Ile-Phe.

These results suggested placement of this peptide with a blocked amino-terminal group at the N-terminus of polypeptide chain; it should be recalled that the cytosolic isoenzyme from horse proved to be resistant to several attempts to de- termine the N-terminal sequence on the intact protein both by manual and automated methods [17]. However, some uncertainty in this positioning remained, since it was based upon a rather low degree of homology to the pig enzyme (three out of six residues were different in marked contrast to the situation with other mammalian isoenzymes [17]). Similarly, although the cytosolic aspartate aminotransferase from chicken heart was found to have an acetylated N-terminal residue [18], this feature is not found in the phylogenetically more related cytosolic isoenzymes from pig, rat, ox or human, all of which have an unblocked alanine residue at the amino end [17].

To provide stronger evidence for the proposed N-terminal sequence, we proceeded to isolate a longer blocked peptide from the original protein. This was done by cleavage with trypsin (a lysine residue was expected at position 19) followed by removal of the terminal residues with carboxy- peptidase to yield a peptide with no positive charge at low pH (see Materials and Methods). Following chromatography on SP Sephadex and further purification, a peptide was obtained with the amino-acid composition Thr (1.0), Ser (1.6), Glu (3.3), Pro (2.7), Ala (1.1), Val (2.8), lie (1.0) and Phe (0.9); the yield was 40%.

After deblocking by mild acid hydrolysis, the sequence Thr-Ser-Pro-Ser-Ile-Phe-Val-Glx-Val- Pro-Glx-Ala-Glx-Pro-Val was determined for this peptide, which could unambiguously be placed at the N-terminus of the protein (Fig. 2).

55

The experiments reported above produced an unambiguous N-terminal sequence, but did not identify the blocking group. This was done by isolation of the blocked peptide from a digest of the native protein with the protease form S. aureus followed by hydrazinolysis and dansylation. Thin- layer chromatography of the products on poly- amide thin layers allowed identification of 1- acetyl-2-dansylhydrazine by comparison with a standard produced by dansylation of acetylhydra- zine. These results clearly established the N-termi- nal blocking group as acetyl. In confirmation of this, a sample (20 nmol) of the blocked peptide from the chymotryptic digest (see above) was analysed using mass spectrometry by Professor H.R. Morris; the sequence obtained was AcThr- Ser-Pro-Ser-Ile-Phe (results not given in detail).

The presence of a blocked N-terminus in the cytosolic aspartate aminotransferase from horse heart was unexpected, given that this feature ap- pears to be absent from the enzyme from other mammalian sources [17]. The possibility was con- sidered that the isoenzymes from other species were partially acetylated and that this partial ace- tylation had gone undetected. It should be recalled in this context that aspartate aminotransferase as usually isolated is a mixture of electrophoretically distinct subforms [20]. A mixture of such subforms isolated from pig heart was subjected to the proce- dures described above. Although the amount of material used was 3-times that employed for isola- tion of the blocked peptide from the horse heart isoenzyme, no trace of a blocked peptide could be detected. Conversely, no trace of an unblocked N-terminal peptide was found in the chymotryptic digest of the isoenzyme from horse. These observa- tions suggest that acetylation of cytosolic aspartate aminotransferase is either complete or absent, de- pending on the species, and is a feature of the amino-acid sequence and specificity of the acetyla- tion system operating in vivo.

JOrnvall [21] has reported on the occurrence of particular amino acids both at the N-terminus and in the first ten positions of acetylated proteins compared with the distributions in these positions in proteins in general, and has noted significant differences. Results available for aspartate aminotransferase are complementary. Alanine is most frequently found to be acetylated, but in the

56

case of cytosolic aspartate aminotransferase of the five isoenzymes with terminal alanine only one (that from chicken) is acetylated [17]. The two isoenzymes that are acetylated, although differing in the terminal residue, share a common feature in that they are the only two cytosolic aspartate aminotransferases with isoleucine at position 5 (according to the numbering system of the isoen- zyme from pig heart); isoleucine is over-repre- sented in the N-terminal region of acetylated pro- teins [21].

No specific functional significance can be ascribed to acetylation of the cytosolic aspartate aminotransferase from horse heart. The isoenzyme shares with that from chicken a reduced 'syncata- lytic' reactivity of a sulphydryl group compared with the form from pig heart, but so does the non-acetylated isoenzyme from bovine heart [8]. Three-dimensional structures of cytosolic [22] and mitochondrial [23] isoenzymes show that in both dimeric proteins the N-terminal segment of one subunit makes important contacts with the other. Acetylation could weaken these contacts and hence reduce the stability of the dimer with important consequences for catalytic activity, since the monomer is catalytically inactive [24].

Finally a possible function in topogenesis should be considered. Studies in vitro have shown that some at least of the features which dictate the specific intracellular localisation of the isoenzymes are present in the native proteins [25]. In the case of cytosolic aspartate aminotransferase, N-termi- nal acetylation may be one such feature in some species. It may be noted in this context that also in the case of malate dehydrogenase from pig heart the cytosolic isoenzyme is acetylated [26].

Acknowledgement

This work was supported in part by a grant from the Ministero Pubblica Istruzione.

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