Stereospecificity of sodium borohydride reduction of pig kidney dopa decarboxylase

ARCHIVESOFBIOCHEMISTRY AND BIOPHYSICS Vol. 251, No. 2, December, pp. 762-766,1986

Stereospecificity of Sodium Borohydride Reduction of Pig Kidney Dopa Decarboxylase’ PAOLA DOMINICI, BRUNELLA TANCINI, AND CARLA BORRI VOLTATTORN12 Institute

of Biological

Chistry,

Faculty

of Pharmacy,

University

of Per&a,

Peru&a,

Italy

Received July 19,1986

Sodium boro[3H]hydride reduction of pig kidney 3,4 dihydroxyphenylalanine decarboxylase followed by complete hydrolysis of the enzyme produced t-[3H]pyridoxyllysine. Degradation of this material to 4’-[3H]pyridoxamine and stereochemical analysis with apoaspartate aminotransferase showed that the re side at C-4’ of the coenzyme is exposed to solvent. In order to determine the face exposed to the solvent in the external SchifYs base, attempts to trap reaction intermediates were made by reduction with sodium boro rH]hydride of the holoenzyme in the presence of various substrates or substrate analogs. In all cases, covalently bound radioactive material was found which was identified as t-l\r-pyridoxyllysine. These results suggest that the internal SchifF’s base is in mobile equilibrium with the external Schiff’s base and that sodium borohydride reduction dis places this equilibrium, resulting in complete reduction of the internal Schiff’s base. 0 1986 Academic

Press, Inc.

Stereochemical studies of sodium borohydride reduction of Schiff’s bases at the active site of different pyridoxal-P enzymes (l-4) provided evidence that the protonation at C-4’ of coenzyme proceeds stereospecifically and with the same absolute stereochemistry. The conservation of stereochemistry around the coenzyme suggests a similar regularity in the geometry of coenzyme binding. This finding, in combination with the observed structural homology of amino acid residues at the active center of pyridoxal-P enzymes with different functions and from different sources, can be interpreted as evidence for the evolution of this entire family of enzymes from a common ancestor. In order to provide additional support to this hypothesis, we decided to extend stereochemistry studies of sodium boro i This work was supported by grants of Minister0 Pubblica Istruzione. *To whom correspondence should be sent at the Istituto di Chimica Biologica, Facoltb di Farmacia, Via de1 Giochetto CP 3’7,WCC.3, Perugia 06100, Italy. 0003-9861/86 33.00 Copyright All rights

0 1996 by Academic Press, Inc. of reproduction in any form reserved.

[3H]hydride reduction to Dopa decarboxylase (EC 4.1.1.28)from pig kidney, another pyridoxal-P-dependent enzyme. Dopa decarboxylase, indeed, an enzyme which promotes decarboxylation of aromatic amino acids, has already been shown to possess a considerable sequence homology with other pyridoxal-P enzymes around the coenzyme binding lysine (5). MATERIALS

AND METHODS

Materials. Dopa decarhoxylase has been purified from pig kidney according to the method of Borri Voltattorni et al (6). Aspartate aminotransferase from porcine heart was obtained from Sigma and converted to the apoenzyme by published procedure (7). Proteaae from Stre-ptom~ces grieeus was purchased from Sigma and used without further purification. Sodium boro mydride (340 mCi/mmol) was obtained from NEN. N-Pyridoxyl-amino acids and t-N-pyridoxyl-L-lysine were prepared according to previously described methods (8, 9). All the commercially available chemicals were of reagent grade or highest purity available. 3Abbreviations used: Dopa, 3.4 dihydroxyphenylalanine; 5HTP, 5-hydroxytryptophan. 762

DOPA

DECARBOXYLASE

Instrumentation Ultraviolet spectra were obtained on a Cary 219 spectrophotometer. HPLC chromatography was carried out on a LKB apparatus consisting of two Model 2150 pumps, a Model 2152 HPLC controller, a UV detector, Model 2151, and a two-pen recorder, Model 2110. Scintillation counting was done in Aquasol (NEN) on a Intertechnique liquid scintillation counter. Identification of e-pyridoxyllysine qfkr reduction of Dopa decarbw$ase holoenzyme. In a typical holoenzyme reduction, the holoDopa decarboxylase (100 mg), dissolved in 0.1 M potassium phosphate buffer at pH 6.8 (3.5 ml), was reduced with a solution of 25 mCi of sodium boro[*H]hydride and 10 mg of sodium boro[‘H]hydride in 1 ml of water. After 2 h, the mixture was dialyzed, heated 5 min in boiling water, cooled, brought to pH 8.5 with concentrated NaOH, and incubated with 10 mg of protease from Streptomyces griseus (24 units/mg) at 37°C for 12 h. The mixture was evaporated to dryness, dissolved in 6 N HCl (3 ml), and hydrolyzed for 12 h at 110°C. The residue, dissolved in distilled water (2 ml), was chromatographed on AG 50 H+ resin (1.5 X 15 cm) with a linear gradient of 2 to 5 N HCl (650 ml each). Thinlayer chromatography against a reference of inactive t-pyridoxyllysine allowed for identification of ej3H] pyridoxyllysine in the eluate. Fractions containing the desidered substance were combined and taken to dryness. Final purification was achieved by HPLC on a Ultrasil CX column (0.46 X 25 cm) using as a mobile phase Tris-HCI 0.1 M buffer, pH 7, + 10% methanol to yield 5.5 pCi of [‘Hlpyridoxyllysine. This product coelutes in the above HPLC system with authentic t-pyridoxyllysine. Reduction of substrates OTanalogs bound Dopa decarboxylase. Testing of different substrates and different pH values for effectiveness and determination of optimal conditions for trapping enzyme-substrate intermediates was performed according to the above procedure at 100X smaller scale. In a typical experiment, substrate or substrate analog, at concentrations at least W-fold higher than their K,,,or KD, was added to an enzymatic solution (1 mg). After 1 to 5 min the mixture was reduced with a solution of 2 mCi of sodium boro[3H]hydride and 0.6 mg of sodium boro[‘H]hydride. After 1 h, the mixture was dialyzed, digested with protease, hydrolyzed in 6 N HCl, and then evaporated to dryness in vuacuo. The residue, dissolved in 0.3 ml of water, was chromatographed on AG 50 H+ (0.6 X 11 cm) with a linear gradient of 50 ml each of 2 and 5 N HCI. The fractions containing radioactivity were combined, evaporated to dryness, and their content was identified by HPLC on a Ultrasil CX column. Stereochemical analysis of [%@yridmyUysim. Oxidative degradation of the [8H]pyridoxamine was performed with calcium hypochlorite by means of a previously published methodology (8), with the fol-

763

REDUCTION

lowing modification: the isolation procedure of [aH]pyridoxamine relied on the use of a cationic-exchange HPLC (Ultrasil CX column, 0.46 X 25 cm at 1 ml min-‘; 50 mM potassium phosphate buffer, pH 3.5, t 5% methanol, isocratic) instead of a conventional cationic chromatography followed by a preparative paper electrophoresis. The stereochemistry of the tritium at C-4’ of the pyridoxamine was determined by incubation with aspartate aminotransferase apoenzyme according to Dunathan et al (10). The apoenzyme (0.576 mg) was incubated with [8H]pyridoxamine and [‘Hlpyridoxamine (0.159 pmol) and 1 mM cY-ketoglutarate in a total volume of 1 ml of 0.1 M Tris-HCl buffer, pH 8, until pyridoxal formation was complete. The reaction was usually complete in 4 h. The incubation mixture was freeze-dried and the water was trapped. A modification of the cited procedure was introduced as follows: the [8H]pyridoxal formed was purified from the residue by reverse-phase HPLC (Viosfer ODS LO.46 X 25 cm, at 1 ml mini; potassium phosphate buffer, 0.1 M, pH 2.5, + 0.1 M NaC104, isocratic), instead of a conventional cationic chromatography followed by a preparative paper electrophoresis. RESULTS

AND

DISCUSSION

Digestion of sodium boro[3H]hydride reduced holoDopa decarboxylase with a protease followed by complete hydrolysis with hydrochloric acid afforded tritiated E-pyroxyllysine. The phosphate was cleaved during hydrolysis. This product was purified and identified by HPLC cochromatography with authentic material (Fig. 1). Oxidative degradation of the [3H]pyridoxyllysine with calcium hypochlorite (8) produced 4’-[3H]pyridoxamine which was isolated and identified by HPLC cochromatography with synthetic pyridoxamine (Fig. 2). Analysis of the tritium distribution between the two heterotopic hydrogens at C-4’ of pyridoxamine took advantage of the known stereospecificity of apoaspartate aminotransferase for removal of the pro-4’S hydrogen (10) and of the notion that the apoenzyme does utilize pyridoxamine as a substrate, rather than binding it tightly as do the natural coenzymes, pyridoxamine-P and pyridoxal-P (11). Thus, incubation of the pyridoxamine samples with apoaspartate aminotransferase under condition of conversion into pyridoxal (presence of substrate amounts of a-ketoglutarate) would be expected to release tritium from the pro-4’S position

764

DOMINICI,

TANCINI,

AND

BORRI

VOLTATTORNI

r

opposite faces are exposed in the internal Schiff’s base and in the complex obtained upon substrate binding. These observations 14 suggest a conformational reorientation of the coenzyme upon substrate binding, which exposes opposite faces in the two SchifF’s bases. Either a rotation around the 10 w C-4/C-4’ axis, leading from a transoid to a ‘0 cisoid conformation, or a rotation around the C-5/C-5’ axis, involving a reorientation ; of the ring plane, can account for the obz served change (12). The only exception ap6 pears to be carbamylated aspartate aminotransferase; the complex of this modified enzyme with L-aspartate has been reported to be reduced predominantly from the re 2 face (3). In pyridoxal-P-dependent enzyme-cata+20 MIN lyzed reactions, the bond to be broken in FIG. 1. HPLC purification of <-N-[3H]pyridoxyl-Lthe substrate is expected to be perpendiclysine produced from acid hydrolysis of reduced ular to the plane of the coniugated ?rsystem holoDopa decarboxylase. (-) Absorbance at 294 nm; of the substrate-pyridoxal-P complex (13). (Cl) cpm; (. -. ) synthetic t-N-pyridoxyk-lysine. In decarboxylases, it is the bond between C, and the carboxyl carbon that must be aligned in this way. The reaction of Dopa into the water of the reaction mixture. So, decarboxylase with tyrosine (14) and trypthe specific radioactivity of the isolated tophan (15) has been reported to proceed pyridoxal would reflect the amount of tri- with retention of configuration; that is, the tium present in the pro-4’R position. Re- incoming proton occupies the same stereoduction of holoDopa decarboxylase oc- chemical position as did the departing carcurred primarily from the re face since 99% of the tritium of the pyridoxamine derived from degradation of pyridoxyllysine was retained in the residue and not in water (Table I). Further, the [3H]pyridoxal isolated and identified by reverse-phase HPLC 0.2 from the residue had the same specific radioactivity as the [3H]pyridoxamine (Table I). These results parallel those obtained ti with the other pyridoxal-P enzymes so far a3 a tested, e.g., aspartate aminotransferase (3), i2 0 alanine aminotransferase (8), tyrosine dez 0.1 carboxylase (2), tryptophanase (l), and tryptophan synthase (4), all of which showed a greater accessibility of the C-4’ Te face in the internal pyridoxal-P-lysine Schiff’s base. Interestingly, it is known that in the above-mentioned enzymes, the pyridoxalOS -30 MINUTES---e--P-substrate or pyridoxal-P product complex is reduced predominantly from the si FIG. 2. HPLC separation of oxidative degradation face at C-4’, thus showing that only one face products of t-N-[8Hlpyridoxyllysine. (-) Absorbance at 294 nm; (0) cpm; ( * -. ) synthetic pyridoxamine. is accessible in each Schiff s base, although

1

DOPA TABLE RADIOACTIVITY

DECARBOXYLASE

I

RELEASED

FROM [~H]PYRIDOXAMINE DURINGTRANSAMINATION WITHCX-EETOGLUTARATE BY APOASPARTATE AMINOTRANSFERASE Yield

[3H]Pyridoxamine” Residue Water [‘H]Pyridoxal*

cm

(%I

12.000 11.900

100 99

300 11.650

2.5 97

a 0.159 rmol. * 0.144 pmol.

boxy1 group. Retention of configuration has been also observed with lysine decarboxylase (16), glutamate decarboxylase (17), and tyrosine decarboxylase (18). These results continue the general pattern of consistency suggested by Dunathan and Voet (19), according to which all pyridoxal-pdependent enzymes are derived from a common ancestor and all reactions are expected to show a consistent stereochemistry. However, it should be noticed that in the case of B. sph~~&ous (20) or wheat germ (21) meso-diaminopimelate decarboxylase the reaction has been shown to operate with inversion of configuration. Where retention of configuration occurs, protonation must take place on the same face of the planar carbanionic intermediate as did decarboxylation; this implies that one face only of the pyridoxal-P-substrate complex is available to solvent. In order to establish which face of the pyridoxal-P-substrate in Dopa decarboxylase is exposed to solvent, the enzyme was reduced in the presence of various substrates or substrate analogs in an attempt to trap reaction intermediates. The compounds chosen for this study were those which had previously been shown to form intermediates that could be detected spectroscopically: Dopa and 5-HTP, both in L and D forms (22), and Dopa methyl ester (23). Different pH conditions (6.8 and 8) were also tested, where these compounds show a different affinity for the enzyme (6), in order to determine optimal conditions for “trapping” enzyme substrate intermediates. We have also attempted the re-

765

REDUCTION

duction with NaB3H4 of holoDopa decarboxylase after 1 h of incubation with 8.6 mM L-tyrosine at pH 9.1; at this pH value, an intermediate enzyme-substrate complex can be detected spectroscopically, whose rate of formation, characterized by an increase of absorbance at 420 nm and a concomitant decrease at 330 nm, is very - 15 min) (data not shown). slow h2 In all cases, we found that sodium boro[3H]hydride reduction in the presence of an essentially saturating concentration of substrate or substrate analog causes a very rapid bleaching of the absorbance at 420 nm; however, the following hydrolysis did not give detectable amounts of pyridoxyl derivative, pyridoxyl substrate, or pyridoxyl product. The major product was pyridoxyllysine, nearly in the same amount as that obtained from reduction of the holoenzyme. These results indicate that the intermediates formed are very resistant to reduction and are in a readily reversible equilibrium with enzyme-bound pyridoxalP. The failure to give appreciable yields of trapped intermediates is an event already observed by reduction of native aspartate aminotransferase enzyme-substrate complex (3), and by reduction of tryptophan synthase in the presence of some substrates or substrate analogs (4). A possible explanation for our data is that the internal Schiff s base is so reactive with sodium borohydride over the external Schiff’s bases that the equilibrium between these species is shifted during reduction to form the internal Schiff’s base, predominantly. REFERENCES 1. VEDERAS, J. C., SCHLEICHER, E., TSAI, M.-D., AND FLOSS, H. G. (1978) J. Biol Chem 253, 5350-

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