Specific modification of a single cysteine residue in both bovine liver glutamate dehydrogenase and yeast glyceraldehyde-3-phosphate dehydrogenase Difference in the mode of midification by pyrene maleimide

Biochimica et Biophysica Acta, 707 (1982) 267-272

267

Elsevier Biomedical Press BBA31324

SPECIFIC M O D I F I C A T I O N OF A SINGLE CYSTEINE RESIDUE IN B O T H BOVINE LIVER GLUTAMATE D E H Y D R O G E N A S E AND YEAST G L Y C E R A L D E H Y D E - 3 - P H O S P H A T E DEHYDROGENASE DIFFERENCE IN T H E M O D E OF M O D I F I C A T I O N BY PYRENE MALEIMIDE IHAB RASCHED and STEPHEN BAYNE

Fakulti~t fiir Biologie, Universiti~t Konstanz, Postfach 5560, D-7750 Konstanz (F.R.G.) (Received April 5th, 1982)

Key words: Chemical modification; Pyrene maleimide; Glutamate dehydrogenase," Glyceraldehyde-3-phosphate dehydrogenase; (Bovine liver, Yeast)

Pyrene maleimide is shown to be a 'half of the sites' reagent for glutamate dehydrogenase and for glyceraidehyde-3-phosphate dehydrogenase. The modified residues are identified as cysteine-ll5 for glutamate dehydrogenase and cysteine-149 for glyceraldehyde-3-phosphate dehydrogenase. The two enzymes react differently with pyrene maleimide. Whereas the hydrophobic environment of cysteine-ll5 directs the modification of glutamate dehydrogenase, the high reactivity of cysteine-149 determines the specific modification of glyceraldehyde-3-phosphate dehydrogenase. Glutamate dehydrogenase activity is unaltered by the modification; glyceraldehyde-3-phosphate dehydrogenase activity in inhibited.

Introduction Since the synthesis of pyrene maleimide was first reported [1], this reagent has been successfully used to label thiol groups in proteins [2,3]. The reagent consists of two distinct moieties: a polycyclic four-ring hydrocarbon system and a maleimide ring. The advantage of the reagent is that, after reaction with thiol groups, it fluoresces, indicating that protein modification has occurred. An unfortunate consequence of the pyrene moiety is the low solubility of the reagent in aqueous buffers. An advantage of the hydrophobic moiety, however, is its potential ability to interact with hydrophobic regions in protein structures, thus lending an additional function to the reagent as a minotor for such regions. Bovine liver glutamate dehydrogenase is a hexameric enzyme in which each subunit contains six cysteine residues and no disulphide bridges 0167-4838/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press

[13]. The six cysteine residues of the glutamate dehydrogenase subunit have been shown to be unreactive, under native conditions, to various thiol reagents [4]. This unreactivity could be due to steric hindrance or to hydrophobic protection of the cysteine residues. If the latter is the case, pyrene maleimide might be the reagent of choice to modify these groups, since the hydrophobic moiety of the reagent would enhance the local concentration of the thiol-specific maleimide ring of the reagent. Yeast glyceraldehyde-3-phosphate dehydrogenase is a tetrameric enzyme with only two cysteine residues per subunit and no disulphide bridges. One of these cysteine residues (cysteine149) has been shown to be required for activity and is sensitive to thiol modifying reagents [9]. Pyrene maleimide would therefore be expected to show the usual properties of a thiol modifying reagent with this enzyme.

268 In this report, we show that pyrene maleimide modifies a specific cysteine residue in both enzymes but that the mode of reaction is different. For both enzymes, however, a 'half of the sites' reaction occurs - - a single cysteine residue in three out of the six subunits of glutamate dehydrogenase, and in two out of the four subunits of glyceraldehyde-3-phosphate dehydrogenase is modified.

min for glyceraldehyde-3-phosphate dehydrogenase. In typical experiments, 20 /.tl of 30 ~M pyrene maleimide in acetone were added to 2 ml phosphate buffer containing various amounts of enzyme. As the reaction time for glutamate dehydrogenase is longer than the half-life of the reagent (40 min, results not shown), a second pulse of reagent (20/~1) was added to this enzyme solution after 40 min.

Materials and Methods

Mapping, isolation and identification of the modified tryptic peptides General. Tryptic peptides required for both the

Materials Glutamate dehydrogenase and glyceraldehyde3-phosphate dehydrogenase were obtained from Boehringer Mannheim. Glyceraldehyde-3-phosphate dehydrogenase was further purified as described previously [5]. The enzymes were dialysed at 4°C against 67 mM phosphate buffer, pH 7.6, (hereafter referred to as phosphate buffer) before use. The protein concentration was determined 1% spectrophotometrically at 279 nm using an Aic,n value of 9.7 and a subunit molecular weight of 56000 [13] for glutamate dehydrogenase; and an A1% I c m value of 8.9 and a subunit molecular weight of 36000 [5] for glyceraldehyde-3-phosphate dehydrogenase. Unless otherwise stated, protein concentrations refer to the subunit molecular weight. Since pyrene maleimide has an absorption band overlapping that of the proteins, protein concentrations of modified samples were determined, when required, by the assay of Lowry et al. [7]. Enzyme activities were measured as described previously [5,6]. N-(1-Pyrene)maleimide was synthesised as described previously [ 1]. Iodo[14 C2 ]acetic ~cid was a product of Amersham Buchler (Braunschweig, F.R.G.). TPCK-trypsin was supplied by Worthington (Freehold, U.S.A.). All other chemicals were of analytical grade. Double-distilled water was used throughout.

Methods Fluorescence titrations. Fluorescence titrations were performed at 20°C using a Perkin-Elmer spectrofluorimeter UPF3. The excitation wavelength was 342 nm. The fluorescence increase at 379 nm was followed until a maximum was attained. This generally required between 60 and 70 rain for glutamate dehydrogenase and 8 and 10

mapping and isolation procedures described below were preparared essentially as described previously [8] with the following modifications. For modification and carboxymethylation of glutamate dehydrogenase, pyrene maleimide and iodoacetic acid were used. For the same reactions of glyceraldehyde-3-phosphate dehydrogenase, pyrene maleimide and iodo[laCz]acetic acid were used. Peptide mapping. Tryptic peptides were prepared and mapped essentially as described previously [8] with the above-mentioned alterations. For glutamate dehydrogenase, the maps were examined first for the presence of fluorescent peptides, then stained with ninhydrin. For glyceraldehyde-3-phosphate dehydrogenase, fluorescent peptides were first noted, radioactive peptides positioned by autoradiography and finally ninhydrin staining was performed.

Peptide isolation and identification Glutamate dehydrogenase. To glutamate dehydrogenase (56 mg, 1 /~mol) dissolved in 200 ml phosphate buffer was added an equimolar amount (relative to the cysteine content) of pyrene maleimide dissolved in 5 ml acetone. After 40 min, a further pulse of pyrene maleimide was added and incubation continued at room temperature for 60 min. Filtration over a Sephadex G-25 column (5 × 40 cm) removed excess reagent. The protein-containing fractions were pooled, dialysed against three changes of distilled water (5 litres, containing 5 ml concentrated ammonia) and then freeze-dried. Carboxymethylation and tryptic digestion of the freeze-dried product were performed as described above. To isolate the modified peptide, a solution con-

269

taining 50 mg of the tryptic digestion was applied to a Sephadex G-50F column (3 × 95 cm), equilibrated and eluted with 100 mM ammonium bicarbonate, pH 8.8. Fractions (10 ml) were collected and measured for absorbance (at 280 nm) and for fluorescence (at 379 nm). Appropriate fractions were pooled as depicted in Fig. 3 and freeze-dried. The freeze-dried fractions were analysed by amino acid analysis and by partial sequenching [14] in order to identify the modified peptide. Glyceraldehyde-3-phosphate dehydrogenase. (i) The pyrene maleimide-modified peptide of glyceraldehyde-3-phosphate dehydrogenase was isolated and purified by a combination of high-voltage electrophoresis and descending paper chromatography techniques. To 10 mg (280 nmol) protein (1 m g / m l in phosphate buffer) were added 160 nmol reagent dissolved in 100 ffl acetone. After incubation at room temperature for 60 min, the solution was dialysed against phosphate buffer for 4 h then freeze-dried. The modified peptide was subsequently carboxymethylated and digested with trypsin as described above. The resulting freeze-dried hydrolysate was dissolved in 200 #1 pyridine acetate buffer, pH 6.5, applied as a 10 cm wide strip to Whatman 3 MM paper and electrophoresed at 3000 V for 90 min. Two radioactive bands were isolated and chromatographed prior to a final purification by electrophoresis at pH 1.9. The ensuing radioactive peptides were analysed by amino acid analysis and partially sequenced using the Beckman 890 C sequencer as described previously [ 14]. (ii) Tryptic peptides of pyrene maleimide-modified glyceraldehyde-3-phosphate dehydrogenase were prepared a described above but instead of freeze-drying, the tryptic solution was heated at 95°C for 15 min to inactivate the protease. Separation of the peptides was accomplished using high-performance liquid chromatography. A Waters /~C18 reverse-phase column employing acetonitrile as the organic solvent effected the separation. Conditions were: 20°C; flow, 1 ml/min; buffer A, 80 mM triethylammonium phosphate, pH 2.7; buffer B, 60% acetonitrile/40% buffer A. A linear gradient over 24 min was run from 5-65% buffer B. 100 #1 (about 4 nmol protein) of the tryptic digest were applied. The ab-

sorbance was measured continuously at 220 nm and the eluted peaks were collected manually. Samples of each peak (50 /~1) were analysed for radioactivity and the remainder of the radioactive peaks was dried and analysed for amino acid content.

Amino acid analysis Samples of the modified peptides were dried and hydrolysed with 200/~1 constant boiling HC1 for 24h. The hydrolysed samples were analysed using either the Biotronik amino acid analyser or the Durrum D-500. Results

The titration of pyrene maleimide with increasing amounts of both enzymes is described in Fig. 1. The intersection points extrapolated on to the abscissa indicate that, for the reagent concentration range studied, 0.5 mol reagent was incorporated per mol enzyme polypeptide chain. This implies that three out of the six subunits of glutamate dehydrogenase and two out of the four subunits for glyceraldehyde-3-phosphate dehydrogenase had been modified. This result was also confirmed by incorporation of radioactively 2.5

//l /I •

i

"/Z

1.5

•

/ ---~-°-°

-~

~04 r

/o~/O~O - .....

~

/2/-

~" ,.0

o.,/ 0

--I

/ / ,.~_o_O_O__o__ o

2.0

Z

•

I

I

0.4

/

I

,,

o,t// :o .....

°

0.2

/

.~ /

o

I

06 0.8 I~M Protein Subunit

2 oO:~,.,

I

1.0

I

1.2

I

1.4

Fig. 1. Titration of pyrene maleimide with increasing amounts of protein. Increasing amounts of protein were added to solutions containing various amounts of pyrene maleimide and the resulting fluorescence was measured. A, 0.1 ffM; ©, 0.2 ffM; O, 0.26 #M; IS], 0.3 #M; I , 0.35 #M. Open symbols, glutamate dehydrogenase additions. Filled symbols, glyceraldehyde3-phosphate dehydrogenase additions. The inset indicates the linearity of the 'half of the sites' reaction over the reagent concentration range studied.

270

labelled pyrene maleimide (results not shown). For glutamate dehydrogenase this is the first instance of a chemical modification displaying a 'half of the sites' reactivity. In contrast to previous modifications using iodoacetic acid, the activity of the enzyme is unaltered by pyrene maleimide, suggesting that methionine-169 is not being modified [6]. Determination of the total cysteine residues by Ellman [11] confirmed the loss of 0.5 mol cysteine per tool polypeptide chain. In the case of glyceraldehyde-3-phosphate dehydrogenase, two cysteine residues per four polypeptide chains have been modified, i.e., another example of the 'half of the sites' reactivity of this enzyme [9]. The corresponding rabbit muscle enzyme, on the other hand, shows with the same reagent 'all of the sites' reactivity [10]. In contrast to glutamate dehydrogenase, glyceraldehyde-3phosphate dehydrogenase is inactivated by reaction with pyrene maleimide, suggesting involvement of the active-site cysteine [9]. Ellman determination [11] also indicated the loss of cysteine. Tryptic peptide maps of pyrene maleimidemodified glutamate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase a r e presented in Fig. 2a and b, respectively. Glutamate dehydrogenase contains six cysteine residues per polypeptide chain. Hence if pyrene maleimide had reacted unspecifically, several fluorescent peptides could be expected. However, only one fluorescent area was found (Fig. 2a), indicating the specificity of the modification. The two cysteine residues per polypeptide chain of glyceraldehyde-3-phosphate dehydrogenase are found in the same tryptic peptide [12]. Thus only one radioactive peptide is obtained after carboxymethylation with radioactive iodoacetic acid. After modification with pyrene maleimide and subsequent carboxymethylation, two radioactive peptides, one of which also fluoresced (hatched area, Fig. 2b), can be observed. The isolation of the respective pyrene maleimide-modified peptides was performed as described in Methods. The gel filtration profile for the tryptic digest of the modified glutamate dehydrogenase is shown in Fig. 3. An unusual feature of this chromatography is the fluorescent peptide (TA) which is eluted with a larger volume than the total volume of the column (Vt), suggesting reten-

9

Electrophoresis

IL

_

o

I 0

O0

a)

iE/9~

fi

0

009 ~ O' O 0 ~ °c>

b)

c-

Fx~o,

0 Fig. 2. Two-dimensional tryptic peptide maps of pyrene maleimide-modified (a) glutamate dehydrogenase (b) glyceraldehyde-3-phosphate dehydrogenase. Tryptic digests of both enzymes were subjected to high-voltage electrophoresis at pH 6.5 in the first dimension followed by descending paper chromatography in butan-l-ol/acetic acid/pyridine/water (15:3: 12: 10, v/v) in the second dimension. Peptides were located by ninhydrin staining (open spots); radioactivity (vertical hatching); fluorescence (visualised under an ultraviolet lamp) (horizontal hatching); radioactivity and fluorescence (cross-hatching).

tion of the peptide on the gel. Amino acid and sequence analysis of peptide TA indicated a pure peptide which could be alligned as the tryptic peptide 115-126 in the primary structure of the protein [13]. After one step of the manual Edman degradation, the peptide lost its fluorescence, indicating that cysteine-115 was the modified residue. Two radioactive peptides, one of which also

0

o

0.3 I

0.2

°" 0

°

o =

tO

,~

i

20

i

30

i

I

/*0 50 60 Froction number

.

70

~ 80

~ 1I° '

90

~"

Fig. 3. G e l filtration of a t r y p t i c digest of p y r e n e m a l e i m i d e m o d i f i e d g l u t a m a t e d e h y d r o g e n a s e . A t r y p t i c digest of m o d ified p r o t e i n w a s p r e p a r e d a n d f r a c t i o n a t e d as d e s c r i b e d in M e t h o d s . Vt, c o l u m n v o l u m e ; TA, f r a c t i o n s p o o l e d ( s h a d e d area) which showed both absorbance ((3) and fluorescence

(x).

271

fluoresced, were purified from the same tryptic digest of pyrene maleimide-modified glyceraldehyde-3-phosphate dehydrogenase by classical electrophoretic techniques. Both peptides were shown to have the same amino acid composition, corresponding to the tryptic peptide 142-160 in the primary sequence of the protein [5]. However, automatic liquid-phase sequencing proved inconclusive because the hydrophobic modified peptide was washed out by the benzene extraction step following the cyclization step in the Edman degradation. The two radioactive peptides from the same tryptic digest of modified glyceraldehyde-3-phosphate dehydrogenase could also be separated by the more modern technique of high-performance reverse-phase chromatography. Chromatograms of the tryptic digests of unmodified and modified protein are presented in Fig. 4a and b, respectively. The overall patterns of both chromatograms are similar; minor differences are probably due to

a)

0.5

I00

0.4

80

E= 0.3

6O % IB

<

o

Q2

4O

0.1

2O

Time (rain)

tn

0.5

IL

b)

/-100

o 0.3 ,.~ <

~ ~

- 60 °/o B

0.2

'

0.1

40 20

Time tO

(rain) Dr

Fig. 4. Separation of tryptic peptides of (a) carboxymethylated and (b) pyrene maleimide-modified and carboxymethylated glyceraldehyde-3-phosphate dehydrogenase by reverse-phase high-performance liquid chromatography. Carboxymethylation was performed with iodo[14Cz]acetic acid. Tryptic peptides were prepared and fractionated as described in Methods. Peaks at 17.04 min in (a) and 16.98 min, 23.12 min and 24.36 min in (b) showed radioactivity and were analysed further for their amino acid content.

deamidination of amino acid amides and to small changes in the column characteristics between each run. Unmodified protein gave only one peak in the chromatogram (Fig. 4a; 17.04 min), whereas modified protein showed a smaller radioactive peak eluting at the same time (Fig. 4b; 16.98 min) in addition to other radioactive peaks (23.12 min and 24.36 min) eluting very late in the chromatogram. These latter two peaks are due to the pyrene maleimide-modified peptide, which as it is more hydrophobic will elute later (at a higher acetonitrile concentration) than the unmodified peptide. Amino acid analyses of the radioactive peaks confirmed the results obtained using the classical electrophoresis techniques. The question as to which of the two cysteine residues has been modified can very probably be answered by the loss of enzymatic activity after modification i.e. cysteine-149 [9]. Discussion

Previous investigations [4] have shown that all the six cysteine residues of glutamate dehydrogenase are particularly unreactive towards alkylating reagents such as iodoacetic acid and Nethylmaleimide. The specific reaction of the bulky pyrene maleimide molecule with one cysteine residue (in three out of the six polypeptide chains in the hexamer of glutamate dehydrogenase) can be explained if pyrene maleimide is envisaged as a 'bffunctional' reagent: both moieties of the reagent are functional in the modification of a specific cysteine residue. The reaction order of the modification of glutamate dehydrogenase with pyrene maleimide is less than first order [15], which means that the local concentration of the reagent at the site of reaction is higher than its formal molarity in solution. This is probably due to a hydrophobic interaction of the pyrene ring system with the peptide environment of cysteine-ll5, since it has been previously shown [15] that pyrene succinimide (in which the maleimide ring is saturated and hence cannot react covalently with the thiol group) completely inhibits the covalent reaction of pyrene maleimide with glutamate dehydrogenase. On the other hand, N-ethylmaleimide neither inhibits the reaction nor reacts with any of the thiol residues in

272

the enzyme. Furthermore, the modification is concomitant with a dissociation of the enzyme to its 13 S component (the hexameric species) [15]. In the case of glyceraldehyde-3-phosphate dehydrogenase, the high reactivity of cysteine-149 determines the specificity of the modification. The reaction rate of pyrene maleimide with this enzyme is much faster than with glutamate dehydrogenase under identical conditions. In contrast to glutamate dehydrogenase, pyrene succinimide does not inhibit the reaction of pyrene maleimide, but N-ethylmaleimide does inhibit the modification by reacting with cysteine-149 [9]. Ultracentrifuge studies (results not shown) indicated no gross structural changes. In summary, we have shown that pyrene maleimide modifies a single cysteine residue in half of the subunits in the active enzyme species of both glutamate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase. However, the mode and consequences of modification are different for each enzyme. (a) The modification of glutamate dehydrogenase requires interaction with the pyrene moiety of p y r e n e m a l e i m i d e ; the m o d i f i c a t i o n of glyceraidehyde-3-phosphate dehydrogenase is independent of that moiety. (b) Although structural alteration is observed, no loss of glutamate dehydrogenase activity occurs. (c) Glyceraldehyde-3-phosphate dehydrogenase, however, is inactivated by the modification, but no structural alteration could be detected by analytical ultracentrifugation.

Acknowledgements This work was supported by research grants from the Deutsche Forschungsgemeinschaft

(Sonderforschungsbereich 138 Biologische Grenzfl~ichen und Spezifit~it). Thanks are owed to the following: to M. Nickel and B. Corneliusson for performing the amino acid analyses; to Dr. B. Martin for a thorough introduction to automatic liquid-phase sequencing:, and to Dr. B. Svensson for reading the manuscript and for many helpful suggestions.

References 1 Weltman, J.K., Szaro, R.P., Frackehon, A.R., Jr., Dowben, R.M., Binting, J.R. and Cathou, R.E. (1973) J. Biol. Chem. 248, 3173-3177 2 Wu, C.-N., Yarbrough, L.R. and Wu, F.Y.-H. (1976) Biochemistry 15, 2863-2868 3 Lux, B. and G6rard, D. ( 1981) J. Biol. Chem. 256, 1767-1771 4 Hucho, F., Rasched, I. and Sund, H. (1975) Eur. J. Biochem. 52, 221-230 5 Bayne, S., Martin, B. and Svendsen, I. (1980) Carlsberg Res. Commun. 45, 195-200 6 David, M., Rasched, !. and Sund, H. (1977) Eur. J. Biochem. 74, 379 385 7 Lowry, O.H., Rosebrough, N.J., Farr. A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275 8 Rasched, I., Bohn, A. and Sund, H. (1977) Eur. J. Biochem. 74, 365-377 9 Stallcup, W.B., Mockrin, S.C. and Koshland, D.E. (1972) J. Biol. Chem. 247, 6277-6279 10 Levitski, A. (1974) J. Mol. 90, 451-458 11 Ellman, G. (1959) Arch. Biochem. Biophys. 82, 70 77 12 Jones, G.M.T. and Harris, J.I. (1972) FEBS Lett. 22, 185189 13 Moon, K. and Smith, E.L. (1973) J. Biol. Chem. 248, 3082-3088 14 Johansen, J.T., Overballe-Petersen, C., Martin, B., Hasemann, V. and Svendsen, 1. (1979) Carlsberg Res. Commun. 44, 201-217 15 Rasched, I., Bohn, A., Peetz, D. and Sund, H. (1977) in Pyridine Nucleotide-Dependent Dehydrogenases (Sund, H., ed.), pp. 157-172, Springer Verlag