Mutant aspartate aminotransferase (K258H) without pyridoxal-5′-phosphate-binding lysine residue : Structural and catalytic properties

Eur. J. Biochem. 211, 475-484 (1993) 0 FEBS 1993

Mutant aspar tate aminotransferase (K258H) without pyridoxal-5’-phosphate-binding lysine residue Structural and catalytic properties

Martin ZIAK’, Joachim JAGER’, Vladimir N. MALASHKEVICH’, Heinz GEHRING’, Rolf JAUSSI’, Johan N. JANSONIUS2 and Philipp CHRISTEN’

Biochemisches Institut der Universitat Ziirich, Switzerland Abteilung Strukturbiologie, Biozentrum der Universitat Basel, Switzerland

(Received September 17November 12, 1992) - EJB 92 1318

If the pyridoxal-phosphate-binding lysine residue 258 of aspartate aminotransferase is exchanged for a histidine residue, the enzyme retains partial catalytic competence [Ziak, M., Jaussi, R., Geh- ring, H. and Christen, P. (1990) Eur. J. Biochem. 187, 329-3331. The three-dimensional structures of the mutant enzymes of both chicken mitochondria and Escherichia coli were determined at high resolution. The folding patterns of the polypeptide chains proved to be identical to those of the wild-type enzymes, small conformational differences being restricted to parts of the active site. If aspartate or glutamate was added to the pyridoxal form of the mutant enzyme [A,- 392nm and 330 nm (weak); negative CD at 420 nm, positive CD at 370 nm and 330 nm], the external aldimine (A,,, = 430 nm; negative CD at 360 nm and 430 nm) transiently accumulated. Upon addition of 2- oxoglutarate to the pyridoxamine form (Arna 330 nm, positive CD), a putative ketimine intermediate could be detected ; however, with oxalacetate, an equilibrium between external aldimine and the pyridoxal form, which was strongly in favour of the former, was established within seconds. The transamination cycle with glutamate and oxalacetate proceeds only three orders of magnitude more slowly than the overall reaction of the wild-type enzyme. The specific activity of the mutant enzyme is 0.1 U/mg at 25°C and constant from pH 6.0 to 8.5. Reconstitution of the mutant apoenzyme with [4‘-3H]pyridoxamine 5’-phosphate resulted in rapid release of 3H with a first-order rate constant k’ = 5 X lop4 s-’ similar to that of the wild-type enzyme.

Apparently, in aspartate aminotransferase, histidine can to some extent substitute for the active- site lysine residue. The imidazole ring of H258, however, seems too distant from Ca and C4’ to act efficiently as proton donor/acceptor in the aldimine - ketimine tautomerization, suggesting that the prototropic shift might be mediated by an intervening water molecule. Transimination of the internal to the external aldimine apparently can be replaced by de novo formation of the latter, and by its hydrolysis in the reverse direction.

In all pyridoxal-5’-P-dependent enzymes, the cofactor is covalently bound to the &-amino group of an active-site lysine residue. In aspartate aminotransferase (AspAT; for a treatise on aminotransferases, see [I]), this &-amino group is thought to serve as proton acceptor and donor in the tautomerization of the aldimine to the ketimine intermediate [2]. Replacement of the active-site lysine residue (K258) by alanine [3, 41 or arginine [5] had resulted in virtually inactive enzymes. As previously reported, we have replaced K258 of

Correspondence to P. Christen, Biochemisches Institut der Uni- versitiit Ziirich, Winterthurerstrasse 190, CH-8057 Zurich, Switzer- land

Fax: +41 1 363 79 47. Abbreviations. AspAT, aspartate aminotransferase ; AspAT

(K258H), mutant from Lys2.58 replaced by His; PPxy, 5‘-phosphopyridoxyl.

chicken mitochondrial AspAT by a histidine residue [AspAT (K258H)I; we have found that the pyridoxal form of AspAT (K258H) plus aspartate and the pyridoxamine form plus 2- oxoglutarate undergo the corresponding half-reactions of transamination within seconds or minutes, respectively [6].

In this study, AspAT (K258H) was found to catalyze the overall transamination cycle with glutamate and oxalacetate as substrates at a rate only three orders of magnitude slower than the wild-type enzyme. Stable crystals suitable for X-ray analysis were obtained from the pyridoxamine form of the Escherichia coli mutant enzyme as well as from the N-(5’- phosphopyridoxy1)-L-aspartate (PPxy-Asp) complex of the mitochondrial mutant enzyme. The crystals served to deter- mine the three-dimensional structures of both mutant enzymes at resolutions of 0.24 and 0.23 nm, respectively. Inter- mediates and rate-limiting steps were identified by absorp-

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tion and CD spectroscopic examination of the transamination half-reactions with substrates and substrate analogs. The data are discussed in the light of the three-dimensional structures.

MATERIALS AND METHODS Chemicals

L-Aspartic acid, 2-oxoglutaric acid, L-glutamic acid, tri- fluoroacetic acid were purchased from Fluka; pyridoxal-5’- phosphoric acid and pyridoxamine 5’-phosphoric acid hydro- chloride were from Merck; 2-methyl DL-aspartic acid, L-cysteine sulfinic acid, Hepes and Mes were obtained from Sigma, PPxy-Asp and 5’-phosphopyridoxyl glutamate (PPxy-Glu) were synthesized according to [7] ; [4’-3H]pyridoxamine-5‘-P was prepared as described previously [8].

Bacterial strains and plasmids The E. coli strain BL21 (DE3) pLysS [9, 101 was kindly

provided by Dr W. Studier and the AspAT-deficient E. coli strain MG204 [ l l ] by Dr I. Fotheringham. The pGemex vector was purchased from Promega and pBluescript from Stra- tagene.

Plasmid construction for expression of the mutant protein

The full-length cDNA encoding chicken mitochondria1 AspAT (K258H) was isolated from the previously used expression construct pOTS/mAspAT (K258H) [6] by digestion of the DNA with NdeI and XbaI (from Boehringer). Partial digestion of pGemex DNA with NdeI and blunting of the staggered ends followed by ligation provided the vector with a unique NdeI site at the initiator methionine of gene 10. The NdeI-XbaI DNA fragment containing the mutation was subcloned into the expression vector pGemex, the DNA of which had been digested with the same restriction nucleases. After selection on ampicillin plus chloramphenicol plates, plasmids from transformants of BL21 (DE3) pLysS were an- alyzed by restriction endonuclease mapping. For expression, the cultures were induced at A,, = 0.6 with 0.5 mM isopro- pyl P-D-thiogalactoside (from Bachem) for 4 h at 37°C.

Purification of chicken mitochondrial AspAT (K258H) Purification of the mutant enzyme was performed as de-

scribed previously [6]. Enzyme prepared in this way was in the pyridoxamine form as indicated by its absorption spectrum. The concentration of purified AspAT (K258H) was determined on the basis of an absorption coefficient of the subunit tZ8” = 70000 M-’cm-’ [12].

Assays of aminotransferase activities Enzymic activity was measured in a coupled assay with

malate dehydrogenase and 20 mM aspartate plus 20 mM 2- oxoglutarate as substrates [13]. The specific activity of the wild-type enzyme was 240 Wmg. In an alternative assay, the reaction of glutamate and oxalacetate was followed by measurement of the decrease in absorption of oxalacetate ( E ~ ~ ~ = 499 M-’ cm-’ [14]). The assay mixture contained 80 mh4 glutamate plus 1 mM oxalacetate in 50 mM Mes pH 6.0 or 50 mM Hepes pH 7.5. The specific activity of the wild-type enzyme was 280 U/mg at both pH values.

Spectrophotometric measurements Absorption spectra were measured with a 8450A UVNIS

diode-array spectrophotometer from Hewlett-Packard. Kine- tic measurements were performed with the same instrument equipped with a HP85 computer using software from Hewl- ett-Packard. CD spectra were recorded with a spectropolari- meter model 5-500 from Jasco. For all optical measurements, a l-cm cuvette was used. CD spectra were recorded at least four times (scan speed 20 nm/min, sensitivity 10 mdeg, time constant 1 s).

Mutagenesis of AspAT of E. coli and purification of the mutant enzyme

The vector M13mp18 containing the aspC gene was a gift from Dr J. F. Kirsch [3]. The aspC gene was subcloned into the pBluescript vector. Using single-stranded phagemid DNA, the codon AAA for lysine 258 was replaced with the histidine codon CAC by oligonucleotide-directed mutagenesis [15]. Three out of five clones tested by nucleotide sequencing according to the method of Sanger [16] were positive. The mutant protein was expressed in E. coli MG 204 [ll] and purified as described [3]. The protein appeared as a single band on SDSPAGE with the correct molecular mass.

Preparation of apo enzyme and PPxy-Asp and PPxy-Glu enzyme complex

The apo form of the mutant enzyme was prepared by extensive dialysis of the pyridoxamine form (obtained by ad- ding 2 mM cysteine sulfinate to the holoenzyme) against 0.5 M sodium phosphate pH 5.2 at 4°C [17]. Invariably, the procedure was only partially successful ; even after extensive dialysis for several days, a small amount of cofactor absorbing at 330 nm remained bound to the enzyme (Fig. 6). For the preparation of the complex with PPxy-Asp or PPxy-Glu, the apo form was incubated with 1 mM phosphopyridoxyl amino acid in 50 mM Hepes pH 7.5 for 2 h at room temperature in the dark. Excess phosphopyridoxyl amino acid was removed by gel filtration.

Determination of the rate of 3H release from [4’-3H]pyridoxamine-5’-P upon binding to AspAT

[4’-3H]Pyridoxamine-5’-P was added to apo AspAT as described previously 181. For following the detritiation of [3H]pyridoxamine-5’-P, samples of the reconstituted enzyme (50-100 pl) were quenched with 100 pl0.5 M HC1 and immediately frozen in liquid nitrogen for the determination of sublimable radioactivity. In all samples, the sum of sublimable and residual radioactivity corresponded to the total radioactivity of [3H]pyridoxamine-5’-P added initially.

Crystallization of AspAT (K258H) E. coli AspAT. The pyridoxamine form of E. coli AspAT

(K258H) was crystallized in a monoclinic form by vapor dif- fusion techniques essentially following the protocols of Smith et al. [18], using ammonium sulfate, and of Jager et al. [19], using poly(ethyleneglyco1) as precipitant. Single crystals appeared within two to three days and grew to sizes suitable for X-ray diffraction experiments with maximum dimensions of 1.5 X 0.5 X 0.5 mm in two to three weeks. Re-

477

flections corresponding to a resolution of 0.22 nm were observed on a FAST area detector diffractometer. The crystals belonged to space group P2, with unit cell dimensions of a = 8.65 nm, b = 7.99 nm, c = 8.98 nm and y = 119.1". They were isomorphous with the crystals of the wild-type enzyme [20].

Mitochondrial AspAT. Orthorhombic crystals of the PPxy-Asp complex of mitochondrial AspAT (K258H) were grown by the hanging drop method from poly(ethyleneg1y- col) solutions as described previously for the wild-type enzyme [21]. The crystals were of space group C222, and isomorphous with the corresponding wild-type crystals.

X-ray crystallographic analyses of AspAT (K258H)

E. coli AspAT, pyridoxamine form. The structure of Asp- AT (K258H) of E. coli was solved by difference Fourier techniques, taking initial calculated phases from a refined E. coli wild-type AspAT model (with an R-factor of 0.186 at 0.25-nm resolution [22]), and omitting K258, the cofactor and a sulfate ion from the structure factor calculation. The model, which currently includes 286 solvent molecules, has been refined to an R-factor of 0.205 at 0.24-nm resolution. The overall coordinate error of the mutant enzyme structure is 0.04 nm as determined by the method of Luzzati [23].

Mitochondrial AspAT. The structure of a complex of As- PAT (K258H) with PPxy-Asp was solved by molecular replacement methods using as a starting model the refined structure of a complex of the wild-type enzyme with 2- methyl aspartate [24]. Crystallographic refinement yielded a model with good geometry and an R-factor of 0.159 in the resolution range of 1.00-0.23 nm. From a Luzzati plot [23], the overall coordinate error was determined to be less than 0.03 nm.

RESULTS

The new expression system (pGemexlE. coli BL21 pLysS [9, 101) gave a tenfold higher yield of chicken mitochondrial AspAT (K258H) than the previously used pOTSIE. coli AR120 [6]. The mutant protein was purified to homogeneity and showed the same mobility on SDSPAGE as the wild- type enzyme. The absorption spectra of the pyridoxal and pyridoxamine form, as well as the single turnover rate con- stants for the two half-reactions with the substrates aspartate and 2-oxoglutarate, were virtually the same in mitochondrial and E. coli AspAT (K258H). The following experiments were all performed with mitochondrial AspAT (K258H) ex- cept when explicitly stated that the mutant E. coli enzyme was used.

Crystallographic analyses of AspAT (K258H)

E. coli AspAT (K258H), pyridoxamine form, is an isolo- gous a, dimer with dimensions of about 9.6 X 8.0 X 6.0 nm. The secondary, tertiary and quaternary structures are identical to those of wild-type E. coli AspAT [22] and very similar to those of the mitochondrial enzyme [24-261.

The peak size and shape of a solvent feature in a difference Fourier electron density map indicates that a sulfate ion is bound at the active site of the pyridoxamine form of E.

8 Y225 f Y225

Fig.1. Partial active-site structure of E. coli AspAT (K258H) (pyridoxamine form). A sulfate ion of the crystallization medium (ammonium sulfate was used as precipitant) is bound in the active site between R386 and R292*. It is directly hydrogen-bonded (dashed line) to the latter and the, presumably charged, cofactor amino group. The anion may influence the cofactor conformation that can be characterized by the torsion angles x (C3-C4-C4'-N) of - 97" and 4 (C4-CS-C5'-0) of 70". Thus, the amino group is in front, i.e. on the substrate side, of the pyridine ring and the 5'- phosphate ester oxygen behind it. H258 forms a hydrogen bond with the peptide oxygen of G38. It makes van der Wads interactions with Y225 and with Y263 (not shown). Its side-chain conformation is characterized by the torsion angles xi = - 76" and xz = 83". Note that the amino acid residues around the cofactor have the same conformation as in the wild-type enzyme [18, 20, 221, demonstrating that the mutation has no structural consequences. PMP = pyridoxamine-5'-P.

coli AspAT (K258H) (Fig. 1). It occupies a site in the vicinity of R292* (of the adjacent subunit), where the distal carboxylate group of the substrate would bind. In the half-open conformation of the pyridoxal form of the wild-type enzyme the sulfate ion is located near the guanidinium group of R386 [18, 20, 221. Apparently, the positive charge on the amino group of pyridoxamine-5'-P attracts the sulfate anion towards the cofactor. The electron density of H258 is very well de- fined as indicated by a low average temperature factor of only 0.11 nm2 in both subunits. The relative immobility of H258 can be explained by intimate Van der Waals contacts with the aromatic rings of neighboring Y225 and Y263 (not shown), and by a hydrogen bond between NE2 of the imidazole group and the carbonyl oxygen atom of G38. The dis- tance between Ns2 and C4' of the cofactor is 0.42nm. As compared to the pyridoxamine form of the wild-type mitochondrial enzyme [26], the pyridine ring plane of the cofactor is tilted by an additional 10" towards the substrate binding site above W140.

Mitochondrial AspAT (K258H), complex with PPxy-Asp The structure of the complex of the mutant apo enzyme

with PPxy-Asp (Fig. 2) shows only minor deviations from the structure of the 2-methyl aspartate complex of the wild- type enzyme [24]. The C4'-N4' single bond is rotated out of the pyridine ring plane towards the indole ring of W140 by about 35'. The tilt of the pyridine ring is the same as in the 2-methyl aspartate complex of the wild-type enzyme. The side chain of H258 can be fitted to the electron density in two alternative ways differing by a 180" rotation around the CP-Cy bond. In neither case can hydrogen bonds be made to neighboring residues. Upon performing sterically allowed rotations around CP and Cy, the closest distances between the NE2 atom of H258 and the Ca or C4' atoms in the coenzyme- substrate adduct are 0.42 nm and 0.37 nm, respectively. Esti- mates of the corresponding distances from N, of K258 in the

478

Fig. 2. Partial active-site structure of apo mitochondria1 AspAT (K258H) reconstituted with the coenzyme-substrate analog PPxy-Asp. This complex crystallized in the closed form, demonstrating that H258 does not prevent this conformation. The structure mimics covalent catalytic intermediates with aspartate as substrate. The cofactor conformation is characterized by the torsion angles x (C3-C4-C4'-N) of - 35" and fp (C4-CS-C5'-0) of 40". The substrate moiety makes ion pairs and hydrogen bonds of optimal geometry with R386 (a-carboxylate) and R292* @-carboxylate). N194 is hydrogen-bonded to R386 and both the a-carbxylate and the ionized 3-hydroxy group of the cofactor. The side chain of H258 has a conformation slightly different from that in the pyridoxamine form of the mutant E. coli enzyme. The side-chain torsion angles are ,yl = - 42" and x2 = + 80". This reorientation is presumably caused by the approach of G38 as part of the small domain upon formation of the closed structure. PPLAsp, PPxy-Asp.

wild-type mitochondria1 enzyme were obtained from its 2- methyl aspartate derivative. After deletion of the 2-methyl group of the inhibitor from the structure, the closest distances were 0.27 nm and 0.29 nm, respectively.

Spectroscopic properties of the coenzyme

The noncovalent mode of binding of pyridoxal-5'-P to the mutant enzyme is reflected by significant differences to the wild-type enzyme in both absorption and CD spectra. The spectroscopic properties of the internal aldimine of the wild-type enzyme depend on pH [27, 281; its protonated and deprotonated form showed a positive CD signal corresponding to the absorption bands at 430 nm and 360 nm, respectively (Fig. 3). The pyridoxal form of the mutant enzyme showed a broad absorption maximum centered at 392 nm, which could be dissected by log(norma1 distribution) analysis into three species absorbing at 370 nm, 395 nm and 420 nm, and a second band at 330 nm (Fig. 3, inset). In the CD spectrum, a positive CD signal around 370 nm and a negative one around 420 nm were found; no CD signal around 395 nm corresponding to the major absorption band in the log(norma1 distribution) analysis was observed. In contrast to the wild- type enzyme, no changes in absorption and CD spectra were observed between pH 5.0 and 8.5 (Fig. 4A). In the wild-type enzyme, a small negative CD signal at 295 nm, which is maximal at acidic pH, was detected (Fig. 3). This signal is most likely due to the coenzyme [29] and not to an ionized tyrosine residue as proposed previously [30]. The CD spectrum of the mutant enzyme was independent of pH in this wavelength region also.

The pyridoxamine form of both the mutant and the wild- type enzyme showed an absorption maximum and a CD extremum at 330 nm (Fig. 5). The CD signal of the mutant but not of the wild-type enzyme increased with pH, titration giv- ing a pK value of 7.8 (Fig. 4B). Addition of 50 mM maleate abolished the pH-dependent increase in the CD signal. The pyridoxine form of AspAT (K258H) did not show the pH-

0

Wavelength (nm)

0

4

0

-4

300 400 500

Wavelength (nm)

Fig. 3. Absorption (A) and CD spectra (B) of the pyridoxal form of the mutant and the wild-type enzyme. The pyridoxal form of the mutant enzyme in 50 mM Hepes pH 7.5 (spectrum 1) was obtained after addition of 2.5 mM 2-oxoglutarate to the pyridoxamine form. Excess substrate was removed by gel filtration (Sephadex G- 25). The log(norma1 distribution) analysis (Jandel Scientific Co.) of the absorption band at 392 nm is shown in the inset, measured values (O), calculated spectrum (-). Wild-type enzyme in 50mM Hepes pH 7.5 (spectrum 2); wild-type enzyme in 50 mM sodium acetate pH 5.0 (spectrum 3). The enzyme concentration was 14 pM in each case.

dependent increase of the CD signal at 330 nm (Fig. 4B). The CD signal of the mutant pyridoxamine form at 280 nm was stronger than that of the wild-type enzyme which pos- sessed a negative CD band at 295 nm (Fig. 5).

Both the mutant and wild-type apo enzyme exhibited in the CD spectrum the same single positive signal in the aromatic region. Both apo enzymes could readily be reconstituted with pyridoxamine-5'-P or pyridoxal-5'-P. Titration of the apo form of the mutant enzyme with pyridoxal-5'-P monitored by measurement of CD at 412 nm (see Fig. 3) showed that it can be reconstituted with 1 mol pyridoxal-5'-P/mol subunit. The absorption and CD spectrum of mutant mitochondrial AspAT reconstituted with either pyridoxal-5'-P or pyridoxamine-5'4' were identical with the spectra of the original mutant holoenzyme (Figs 3 and 5).

Transamination intermediates

After addition of glutamate to the pyridoxal form of the mutant enzyme the absorption band at 392 nm disappeared

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5 6 7 8 9

PH Fig. 4. pH-dependence of the molar ellipticity [O] of the pyridoxal form (A) and the pyridoxamine form (B) of both AspAT (K258H) and the wild-type enzyme. (A) Pyridoxal form of AspAT (K258H) at 370 nm (A) and at 420 nm (A); pyridoxal form of wild- type enzyme at 430 nm (O), and at 360 nm (a). For the pH titra- tions, the enzyme was dialyzed against 50mh4 sodium acetate pH5.0. The indicated pHvalues were attained by the addition of solid Tris base. The titration curves correspond to a pK value of 5.7. (B) qrridoxamine form of the wild-type enzyme (e), and of AspAT (K258H) unliganded (A) and after addition of 50 mh4 maleate (A) measured at 330 nm; pyridoxine form of AspAT (K258H) (V) measured at 333 nm. The pyridoxine form was obtained by reduction of enzyme-bound pyridoxal-5'-P with sodium borohydride (for conditions, see legend of Fig. 6). The conversion of the pyridoxal to the pyridoxine form was followed spectrophotometrically.

and a transient species with A,, 430 nm, most probably representing the external aldimine, was produced. This intermediate was rapidly converted to a species with A,, 330 nm. After gel filtration, all the enzyme was in the pyridoxamine form. Formation of the external aldimine has also been observed after addition of aspartate to the pyridoxal form of AspAT (K258H) [6]. The rates of formation of the external aldimine and its absorption spectra were virtually the same for both substrates.

In the reverse half-reaction, the pyridoxamine form of the mutant enzyme was converted to the pyridoxal form upon addition of 2-oxoglutarate. No transient species could be detected by absorption spectroscopy [6]. However, if the reaction was followed by CD spectroscopy, the positive signal centered at 330 nm shifted to 333 nm on addition of the substrate (Fig. 6). This change was accompanied by a decrease in CD at 280 nm and 295 nm. Subsequently, a positive signal at 370 nm and a negative signal around 420 nm appeared and the minimum at 295 nm disappeared. After 2 h, the conversion was complete, the CD spectrum having become identical to that of the pyridoxal form (cf. Fig. 3). The initial shift of the CD signal to 333 nm might reflect the transient appem- ance of the ketimine andlor the carbinolamine intermediate. Attempts to trap the ketimine intermediate by reduction with sodium borohydride failed (for conditions, see legend of Fig. 6). No shift of the CD signal of the pyridoxamine form was observed on addition of the competitive inhibitors maleate (25mM) and succinate (25mM) (not shown). Appar- ently, the initial shift of the CD signal to 333 nm does not reflect formation of the Michaelis complex.

In contrast to the reaction with 2-oxoglutarate, addition of oxalacetate to the pyridoxamine form initiated the rapid

300 400 500

Wavelength (nm)

Fig.5. Absorption (A) and CD spectra (B) of the pyridoxamine form of the the mutant and the wild-type enzyme. Mutant enzyme in 50 mM Mes pH 6.0 (spectrum 1) and in 50 mM Hepes pH 9.1 (spectrum 2); wild-type enzyme in 50 mM Hepes pH 7.5 (spectrum 3). The enzyme concentration was 14 pM in each case.

- 1 1

I 300 400 500

Wavelength (nm)

Fig.6. Reaction of the pyridoxamine form of AspAT (K258H) with 2-oxoglutarate followed by CD spectroscopy. Spectrum 1 is that of the pyridoxamine form of the mutant enzyme (14 FM) in 50 mM Hepes pH 7.5 at 25 "C. Immediately after addition of 1 mh4 2-oxoglutarate, the formation of a catalytic intermediate was detected (spectrum 2; for explanations, see text). In an attempt to reduce the imine bond of the putative ketimine intermediate, 5 p1 sodium borohydride (200 mh4 in 0.1 M NaOH) was added to l ml of the reaction mixture. Trichloroacetic acid (7% by vol.) was then added and the precipitated enzyme removed by centrifugation. Analysis of the supernatant on a C, , reverse-phase HPLC column failed to detect any PPxy-Glu.

480

a,

0 m n

02

E M al 73 v

dl I 0

x 6

r--

Q u

300 400 500

Wavelength (nm)

Fig.7. Absorption (A) and CD spectra (B) of the external aId- imine formed with 2-methyl aspartate in AspAT (K258H) (spectrum 1) and wild-type enzyme (spectrum 2). Spectra were recorded immediately after addition of 1 mM 2-methyl aspartate to the pyridoxal form of AspAT (K258H) (spectrum 1 ; higher concentrations of 2-methyl aspartate did not change the spectrum) or 10mM 2-methyl aspartate to the pyridoxal form of the wild-type enzyme (spectrum 2). The enzyme concentration was in each case 14 pM in 50 mM Hepes pH 7.5 at 25 "C.

formation (ti,* < 3 s) of a stable species absorbing at 430 nm. The absorption spectrum (not shown), which closely re- sembled that formed with the substrate analog 2-methyl aspartate (see below), identified this intermediate as the external aldimine.

Addition of 1 mh4 2-methyl aspartate to the pyridoxal form of the mutant enzyme resulted in a decrease in absorbance at 392 nm and an increase in absorbance at 430 nm (Fig. 7). In the wild-type enzyme, this 430-nm band rep- resents the protonated external aldimine [31, 321. In contrast to the wild-type enzyme, the mutant enzyme showed two negative CD signals at 360 nm and 430 nm which did not change in the pH range 5.0-8.5. The absorption spectrum of the external aldimine of the wild-type enzyme, though different from that of the mutant enzyme, is also insensitive to pH changes [31]. Addition of 5 mM erythro-3-hydroxyaspartate to the pyridoxal form of mitochondiral AspAT (K258H) decreased the absorption at 392 nm under formation of the external aldimine (data not shown). This intermediate disappeared and the pyridoxamine form was produced. In contrast to the wild-type enzyme, no detectable band at 490 nm representing the quinonoid intermediate [33, 341 was observed during this reaction. The pyridoxal form of the mutant enzyme showed no change in its absorption

and CD spectra after addition of the aromatic amino acids phenylalanine (20 mM) or tyrosine (20 mh4).

In the wild-type enzyme, the noncovalent binding of the dicarboxylic acid inhibitors succinate (25 mM) or maleate (25 mM) raises the pK value of the internal aldimine, resulting in an increased absorbance at 430 nm due to the protonated aldimine [35], an effect that is even more pronounced in the crystalline enzyme [36, 371. Addition of dicarboxylic acids had no effect on the absorption and CD spectrum of the mutant enzyme.

Kinetic properties The mutant enzyme had shown no measurable activity

in the standard aminotransferase assay with aspartate and 2- oxoglutarate as substrates. However, upon addition of aspartate, the pyridoxal form had been converted to the pyridoxamine form and addition of 2-oxoglutarate had initiated the reverse reaction. Both half-reactions followed pseudo-first- order saturation kinetics [6]. The rates of both the pyridoxal- 5'-P to pyridoxamine-5'-P half-reaction [6] and the reverse reaction proved pH-independent in the pH range 6.0- 8.5. The reaction with glutamate as substrate obeyed pseudo-first- order saturation kinetics and was of the same magnitude as that with aspartate (Table 1). An exception was the reaction of oxalacetate with the pyridoxamine form in which the external aldimine was rapidly formed within the mixing time and then remained at constant concentration. The question arose whether the accumulation of an apparently stable external aldimine intermediate was due to an equilibrium between the external aldimine and the pyridoxal form or whether the hydrolysis step did not take place. In an overall assay with oxalacetate and glutamate as substrates, a specific activity of 0.1 U/mg was measured both at pH 6.0 and 7.5. This activity is only three orders of magnitude lower than that of the wild- type enzyme (240 U/mg) and in the same range as the rate of the reaction with aspartate or glutamate under single-turnover conditions. From this experiment we conclude that in the mutant enzyme the external aldimine is indeed hy- drolyzed to the pyridoxal form. In order to confirm the exis- tence of an equilibrium between the external aldimine and the pyridoxal form, the reaction mixture was subjected to gel filtration. About equal amounts of the pyridoxal and pyridoxamine form of the enzyme were obtained (not shown). Simi- larly, addition of sodium borohydride to the reaction mixture yielded the pyridoxine and pyridoxamine form of the enzyme in a ratio of 1 : 2 (for conditions and analytical procedure, see legend of Fig. 6). No phosphopyridoxyl amino acid was obtained. As in the wild-type enzyme (Sterk and Gehring, unpublished results), the coenzyme-substrate imines cannot be reduced by sodium borohydride. Both the gel filtration and the reduction experiment confirm that the 430-nm absorbance observed after addition of oxalacetate to the pyridoxamine form is due to the external aldimine in equilibrium with the pyridoxal form; this equilibrium, however, is strongly in favor of the external aldimine. Consonant with this conclusion, spectrophotometric titration of the pyridoxal form of AspAT (K258H) with 2-methyl aspartate gave a K & value of only 22 pM.

The values of k,,, and K , for dicarboxylic substrates in both half-reactions were determined under single-turnover conditions (Table 1). The K, values for aspartate and 2-0x0- glutarate were similar to those of the wild-type enzyme, whereas the K, value for glutamate was somewhat decreased.

48 1

Table 1. Kinetic parameters for transamination half-reactions of AspAT (K258H). Values for AspAT (K258H) were determined in 50 mM Mes pH 6.0 at 25 “C by Lineweaver-Burk analysis of the rate of the increase in A,,, in the case of the amino acid substrates and in A,,, for 2-oxoglutarate. The values for aspartate and 2-oxoglutarate are taken from [6]. For the wild-type enzyme, kcar values for full transamination cycles, determined with radioactively labeled aspartate or glutamate plus oxalacetate or 2-oxoglutarate, respectively, were taken from [38]; K , values, determined by Lineweaver-Burk analysis, were taken from [39]. Similar k,, values for aspartate and 2- oxoglutarate were found for the E. coli mutant enzyme under saturation conditions: kobs for aspartate 6.3 X lo-’ s-‘ and k,,, for 2-oxoglutarate 3.3 X s-’. On addition of oxalacetate, the mutant enzyme forms the external aldimine intermediate at a rate too rapid to be accurately measured by conventional means (tin < 3 s). An equilibrium between the external aldimine and the pyridoxal form is established (see text).

Substrate AspAT (K258H) Wild-type enzyme

k,, Km kcalfKm kcat K m kc.,lKm

S-’ mM M-1 s-1 S-’ mM M-I s-l

Aspartate 7.0 x 0.2 350 225 0.5 4.5 x lo5 2.3 X 104 Glutamate 2.0 x lo-, 1.7 12 250 11 1.5 x 107 Oxalacetate 225 0.015

2-Oxoglutarate 4.0 x 10-4 4.3 0.093 250 1.5 1.7 X lo5

0 40 80

Time (min)

Fig. 8. Release of 3H from [3H]pyridoxamine-5’-P following binding to apo AspAT (K258H). [3H]Pyridoxamine-5’-P (specific radioactivity, 1.7 X lo6 cpm pmol-’) was added in equimolar concentration (40 pM) to apo AspAT (K258H) in 50 mM Hepes pH 7.5 at 25°C. The binding of the coenzyme to the mutant enzyme was followed by measurement of CD (a). The CD spectrum of reconstituted (K258H) enzyme was identical with the spectrum of the original mutant holoenzyme (Fig. 3 B), confirming that [3H]pyridoxamine-5’4’ was bound to the active site of the mutant enzyme. Samples of 100 pl were withdrawn at the indicated times for determination of sublimable radioactivity (A).

Labilization of C4’ p r o 4 hydrogen of enzyme-bound pyridoxamine-5’-P

The key event in the transamination cycle is the tautomerization of the external aldimine to the ketimine intermediate. It appears to be a stepwise process catalyzed by a single acidhase group. The transfer of a proton from Ca of the substrate glutamate to the p r o 3 position at C4’ of pyridoxamine-5’-P had been demonstrated in the case of wild-type mitochondrial AspAT [40]. Reconstitution of wild- type apo AspAT with [4’-3H]pyridoxamine-S’-P has been shown to result in a stereospecific exchange of pro-S ”H with solvent water [8]. Upon reconstitution of the mutant enzyme, a time-dependent release of 3H into the solvent was observed (Fig. 8). The release of 3H followed first-order kinetics and its rate (k’ = S X s-I) was the same as that observed for the wild-type enzyme [8].

Ligand-induced and syncatalytic conformational changes In the reaction of the mutant enzyme with 5,s’-di-

thiobis(2-nitrobenzoate), the single accessible cysteine resi-

Table 2. Rate of modification of Cys166 with 5,5’-dithiobis(Z- nitrobenzoate) in the wild-type enzyme and AspAT (K258H). The substrate concentrations correspond to saturation conditions. The assays were performed in 50 mM Hepes pH 7.5; the enzyme concentration was 14 pM. The concentration of 5,5‘-dithiobis(2- nitrobenzoate) was 25 mM. The absolute value of the second-order rate constant was 69 M-’ min-’.

Conditions Relative rate of modification

wild-type AspAT AspAT (K258H)

Pyridoxal enzyme No substrate Oxalacetate (55 mM) 2-Oxoglutarate (250 mM) Succinate (1 50 mM) Maleate (25 mM) 2-Methylaspartate (35 mM) erythro-3-Hydroxy-

aspartate (20 mM) PPxy- Asp PPx y -Glu

1-00 2.7 2.8 3.7 3.6 5.6

4.6 8.4 8.4

1 .o 1.5 1.3 2.0 2.1 5.2

1.3 5.9 5.7

Pyridoxamine enzyme No substrate 0.9 0.8 Aspartate (55 mM) 1.4 1 .o Glutamate (55 mM) 1.3 1 .o Oxalacetate (1.5 mM) 5.1 Maleate (25 mM) 2.3 1.6

due, C166, is modified. The reactivity of this residue toward sulfhydryl reagents has been shown to be an indicator of the conformational state of the enzyme [12, 211. The rate of modification of C166 was similar for mutant and wild-type enzyme in their unliganded forms (Table 2). However, in the mutant enzyme, the conformational responses to noncovalent binding of nonproductive substrates (2-oxoglutarate or oxalacetate for the pyridoxal enzyme and glutamate or aspartate for the pyridoxamine enzyme) or competitive inhibitors and to the formation of covalent enzyme-substrate intermediates were consistently less pronounced than in the wild-type enzyme (Table 2). In the mutant enzyme, the rate of modification of the aldimine intermediate formed with 2-methyl aspartate is virtually the same as that of the aldimine inter-

482

mediate formed on addition of oxalacetate to the pyridoxamine form.

DISCUSSION In a preceding study, the quite similar CD spectra in the

210-250-nm range of the mutant and the wild-type enzyme had shown that the (K258H) mutation does not bring about major changes in the spatial structure. The unaltered Kk values for aspartate and 2-oxoglutarate had indicated that the substrate binding site remains intact [6]. The X-ray crystallographic analysis now showed indeed that the replacement of the active-site lysine residue by histidine resulted only in changes necessary to accommodate the imidazole ring. Thus, the functional differences between mutant and wild-type enzyme can be interpreted as direct consequences of the substi- tution of K258 by histidine.

Coenzyme binding and orientation The mutant enzyme in solution still tightly binds pyri-

doxd-5’-P and pyridoxamine-5’-P; neither form of the coenzyme is removed by gel filtration at pH 7.5 [61. Due to the absence of the Schiff base, not only pyridoxamine-5’-P but also pyridoxal-5‘-P is held exclusively by noncovalent interactions. In the wild-type enzyme, both pyridoxal-5’-P and pyridoxamine-5’-P are somewhat strained, the first by the covalent linkage to K258 and the second by a hydrogen bond between the amino group of pyridoxamine-5’-P and the E- amino group of K258 [26]. The more relaxed orientations of the coenzyme in AspAT (K258H) must be due to the absence of these interactions. The reduced molar ellipticity of both coenzyme forms in the mutant enzyme must also be due to their altered orientation.

In contrast to the wild-type enzyme, the absorption spectrum of pyridoxal-5’-P in the mutant enzyme proved to be independent of pH (Fig. 4A). Similarly, reductive methyl- ation of the active-site lysine residue has been reported to render the absorption spectrum of pyridoxal-5’-P insensitive to changes in pH [41]. The CD spectrum of pyridoxal-5’-P in the mutant enzyme showed two signals of opposite signs. Neither extremum in the CD spectrum (Fig. 3B) coincides with Am- in the absorption spectrum (Fig. 3A). A possible explanation for this finding is furnished by log(norma1 distribution) analysis of the spectrum (Fig. 3A inset) which indicates that the absorption band centered at 392nm may be deconvoluted into three absorption bands possibly due to different ionic forms of pyridoxal-5’-P. Two of these bands ex- actly correspond with the two extrema.

The pyridoxal form of the mutant enzyme does not show the CD minimum at 295 nm found in the wild-type enzyme. The mutant and the wild-type apoenzyme also lack this pH- sensitive signal. Apparently, the signal of the wild-type pyridoxal form at 295 nm is due to the protonated Schiff base between pyridoxal-5’-P and K258 [29].

The mutant enzyme in its pyridoxamine form showed the same absorption maximum at 330nm as the wild-type enzyme (Fig. 5A). However, the CD signal at this wavelength increased with pH in the mutant enzyme while it was independent of pH in the wild-type enzyme (Fig. 4B). Possible candidates for this reporter group at the active site with a pK of 7.8 are the imidazole group of H258 and the 4’-amino group of pyridoxamine-5’-P. Titration of the pyridoxine form of the mutant enzyme does not show any effect on the CD

signal at 330nm, suggesting that it was the 4‘-amino group of pyridoxamine-5‘-P that was titrated. It seems difficult to reconcile the titration of an ionizable group at the active site with invariable catalytic activity in the pHrange 6.0-8.5. Indeed, upon addition of maleate, which shifts the conformational equilibrium toward the closed form that is assumed by the enzyme during catalysis, the intensity of the signal no longer changed with pH. The spectrum of the external aldimine of the wild-type enzyme with 2-methyl aspartate is also pH-independent, indicating that in both cases either the active site is isolated from the solvent, as shown by X-ray crystallographic analysis of the wild-type enzyme [24], or that the pK value of the titrated group is shifted to consider- ably higher pH values (> 9.5).

Transamination intermediates Addition of 2-oxoglutarate to the pyridoxamine form of

the mutant enzyme (Scheme 1) immediately shifted the maximum of the CD spectrum from 330nm to 333nm (Fig. 6) . The signal at 280 nm was decreased and a new negative signal at 295 nm was induced. During the pro- gression of the enzymic reaction this signal disappeared (data not shown) and the spectrum became identical to that of the pyridoxal form. The transient species might represent the ketimine intermediate. In cryoenzymological studies at - 60 “C, an intermediate with essentially the same properties could be trapped after addition of 2-oxoglutarate to the wild-type pyridoxamine form [29]. In both instances, i.e. wild-type enzyme at -60°C [42] and mutant enzyme at 25°C (see legend to Fig. 6), it was not possible to reduce this intermediate with sodium borohydride. At the stage of the ketimine intermediate, the enzyme is assumed to be in the closed conformation which may not allow access for borohydride to the active site. In the catalytically less active K258A mutant, the reduction of the ketimine, formed after addition of 2-oxoglutarate to the pyridoxamine form, yielded 51% N-phosphopyridoxyl-glutamate [4]. The ketimine-specific negative CD signal at 295 nm of the mutant enzyme might reflect a syncatalytic reorientation of the cofactor or an active-site tyrosine residue, most likely Y225 [29]. There is ample experimental evidence, including X-ray crystallographic analysis [2, 241 and linear dichroic measurements [37, 43, 441, for changes in the tilt angle of the coenzyme during the catalytic process.

Upon incubation of the pyridoxal form of mitochondria1 AspAT (K258H) with erythro-3-hydroxyaspartate, the external aldimine and not, as in the wild-type enzyme [33, 341, the quinonoid intermediate (A,,, = 490 nm) accumulated. Apparently, in the mutant enzyme, formation of the quinonoid intermediate with erythro-3-hydroxyaspartate occurs more slowly than in the wild-type enzyme as indicated by the accumulation of the external aldimine intermediate; on the other hand, reprotonation of the quinonoid intermediate to the ketimine intermediate is faster in the mutant enzyme, as indicated by the eight times more rapid formation of the pyridoxamine form (not shown).

Kinetic properties and reaction mechanism of AspAT (K258H)

Remarkably, the overall transamination cycle of AspAT (K258H) with glutamate and oxalacetate as substrates is only three orders of magnitude slower than that of the wild-type enzyme. Substituting the active-site lysine residue by alanine has been reported to reduce the catalytic activity at least lo6-

483

Pyridoxal form External aldlmine

Scheme 1.

R$ CO;

& - Tauto-,

merization CH3 H

fold [45]. The replacement of the active-site lysine residue by arginine decreased the catalytic activity 103-fold for the forward and 107-fold for the back reaction. The rate of reaction with 2-oxoglutarate approached the value of the nonen- zymic reaction [5, 461. However, replacement of the active- site Lys145 of bacterial D-amino acid transaminase by Gln reduced the activity by only four orders of magnitude [47].

The half-reactions of AspAT (K258H) with aspartate, glutamate and oxalacetate are only three orders of magnitude slower than the corresponding reactions of the wild-type enzyme. However, the half-reaction with 2-oxoglutarate is slower by six orders of magnitude. The reaction of the pyridoxamine form with oxalacetate produces the external aldimine in equilibrium with the free pyridoxal form. In the mutant enzyme, this equilibrium is greatly in favor of the external aldimine, K & for 2-methylaspartate being only 22 pM as compared to 2 mh4 in the wild-type enzyme (unpublished data). In AspAT (K258H), the transimination step from external aldimine to internal aldimine of the wild-type enzyme is replaced by a hydrolysis step, similar to the hydrolysis of the ketimine intermediate, which in the pyridoxal- 5‘-P to pyridoxamine-5’-P reaction yields pyridoxamine-5’-P and the 0x0 acid product (Scheme 1). However, the hydrolysis step proves fast enough for the overall transamination cycle to proceed at a rate that is only three orders of magnitude slower than that of the wild-type enzyme.

The accumulation of the external aldimine intermediate in the pyridoxal-5’-P to pyridoxamine-5’-P reaction upon addition of amino acid substrates and of the ketimine intermediate in the reverse half-reaction with 2-oxoglutarate indicates that for these substrates the deprotonation step of the aldimine- ketimine tautomerization (Scheme 1) is rate-limiting. These data are consistent with those of the wild-type enzyme in which the deprotonation steps are also rate-limiting [29, 481. In the pyridoxamine-5’-P to pyridoxal-5‘-P reaction with oxalacetate, the rate-limiting step could not be identified. The deprotonation of the ketimine formed with the C, substrate 2-oxoglutarate is two to three orders of magnitude slower than the deprotonation of the ketimine with the C, substrate oxalacetate and the deprotonation of both external aldimines with aspartate and glutamate. This striking difference might relate to the nonproductive binding of 2-oxoglutarate as observed for the pyridoxamine form of AspAT (K258H) of E. coli [22].

The PPxy-Asp derivative of mitochondria1 AspAT (K258H) demonstrates that substrates bind in the same way as in the wild-type enzyme, with the labile Ca-H bond in the external aldimine normal to the cofactor pyridine ring plane (Fig. 2). However, in contrast to the &-amino group of K258 in the wild-type enzyme, the imidazole ring of H258 in the crystal structure appears to be too far away from Ca and C4’

Ketimlne Pyridoxamine form

of the coenzyme to be able to act directly as a general acid base catalyst. Possibly, this function is performed by a water molecule situated between H258 and the coenzyme-substrate adduct. There is enough space for a water molecule in this area. Alternatively, the conformational motility of the enzyme might allow H258 to approach occasionally the Ca and C4’ proton sufficiently for direct proton transfer. The partially preserved ability of AspAT (K258H) for protonation and deprotonation of the coenzyme substrate adduct is also substantiated by the undiminished rate of ’H release from [4’-3H]pyridoxamine 5’-P. In contrast, the K258A enzyme failed to release 3H [491.

The equilibrium between the open and closed conformation of mitochondria1 AspAT can be monitored by the reaction of C166 with 5,5’-dithiobis(2-nitrobenzoate) which is faster by a factor of nearly ten in the closed structure of the enzyme [12]. Table 2 shows that the reactivity of C166 is reduced by a factor of 1.5-2 in the liganded forms of the mutant enzyme in comparison to the corresponding functional states of the wild-type enzyme. This difference indicates a slight destabilization of the closed structure in the K258H mutant, perhaps resulting from tighter packing in the region of the H258 side chain, an effect that may be assumed to be more pronounced in the closed than in the open structure. The absence of a significant effect of erythro-3- hydroxyaspartate in the mutant enzyme is explained by the failure to accumulate the quinonoid intermediate.

CONCLUSION Both the partially preserved aminotransferase activity and

the fully preserved C4’ pro-S hydrogen labilizing activity indicate that a suitable polar residue such as histidine, perhaps in conjunction with a water molecule, can to a remark- able extent replace Lys258. AspAT (K258H) is the first catalytically active pyridoxal-5’-P-dependent enzyme without a lysine residue at the active site. The ubiquitous active-site lysine residue in pyridoxal-5’-P-dependent enzymes might thus represent a consequence of the chemical reactivity of the cofactor rather than a mechanistic necessity. On the one hand, covalent binding of the coenzyme, i.e. the internal aldimine linkage, appears not to be essential for the enzymic transamination reaction (Tobler, H. P., Gehring, H. and Christen, P., unpublished results); on the other hand, the function of a proton acceptor/donor in the aldimine-ketimine tautomerization could conceivably be served also by a histidine residue.

We thank B. Fol and U. Sauder for expert technical assistance. This work was supported in part by Swiss National Science Foun- dation Grants (31-27975.89 to P. Christen, 31-25713.88 to J. N. Jan-

484

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2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15. 16.

17.

18.

19.

20.

21.

22. 23. 24.

sonius). R. Jaussi was a recipient of a stipend from the Cloetta Foun- dation, Zurich.

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Documents

Mutant aspartate aminotransferase (K258H) without pyridoxal-5′-phosphate-binding lysine residue : Structural and catalytic properties