Identification of a cysteine residue of glutamate dehydrogenase that binds p-chloromercuribenzoic acid

Vol. 72, No. 4, 1976

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IDENTIFICATION OF A CYSTEINE RESIDUE OF GLUTAMATEDEHYDROGENASE THAT BINDS p-CHLOROMERCURIBENZOICACID

M.P. Cosson, C. Gros, J.C. Talbot Laboratoire d'Enzymologie du C.N.R.S. 91190 Gif-sur-Yvette, France

Received July 29,1976 SUMMARY. A method has been developed which allows the selective replacement ~urials bound to glutamate dehydrogenase by radioactive iodo acetic acid. This lead for p.chloromercuribenzoic acid replacement to the recognition of an unique labelled peptide which is i d e n t i f i e d as peptide 303-329, including cysteine 319. Cysteine 319 is part of the ~E sheet of the coenzyme binding domain not involved in a c t i v i t y and competitively occupied by ADP (10). In view of the great modification of the regulatory properties of ADP in the modified cysteine 319 enzyme , a conformational role is suggested for this sequence alignment.

INTRODUCTION Previous reports have demonstrated the a b i l i t y of one amino acid residue of glutamate dehydrogenase (L-glutamate-NAD(P) oxidoreductase (deaminating) (E.C.I.4.1.3)) to be s p e c i f i c a l l y modified by mercurials (1-4). The resulting labelled enzyme is active but exhibits a slower rate for the prestationnary steps (burst phase) of the glutamate oxidation. The binding s i t e for the nucleotide activator, ADP, is conserved, but i t s regulatory properties are greatly affected : the enzyme with one p-mercuribenzoic acid (pMB) mole bound per subunit is inhibited by ADP whereas the enzyme with one methyl mercuric chloride is only desensitizated to ADP (4). The present experiments were designed to i d e n t i f y the involved amino acid residue of the enzyme sequence. At this residue was only s p e c i f i cally labelled with mercurials which are not usefull in sequence determination, we used an indirect i d e n t i f i c a t i o n

Copyright © 1976 by Academic Press, Inc. All rights o/reproduction in any/orm reserved.

procedure : we replaced as selectively

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as possible, the mercurial derivative bound to the enzyme with iodo (2-14C) acetic acid and i d e n t i f i e d the resulting peptide.

MATERIAL AND METHODS Bovine l i v e r glutamate dehydrogenase (EC 1.4.1.3) was prepared according to Kubo et a l . - ( 5 ) and further purified by G200 chromatography. Protein concentrat~n~as spectrophotometrically measured using a A~9n m value of 0.97 (6). Substrates and buffers were obtained from Sigma Chem. and Merck AG respectively - Methyl mercuric chloride was obtained from K and K, p-chloromercuribenzoic acid from Serlabo and the 14C labelled derivatives from Radiochemical Centre, Amersham. All other products were Pierce Chemical Company reagents except trypsin-TPCK treated which was bought from Worthington. Enzyme modification was performed as previously described (2-3) in 0.05 M phosphate buffer pH 7.3, ~ = 0.2 (Na2S04), 15°C, using an excess of p-chloromercuribenzoic acid (carboxyl-14C) 1.78 ~Ci/umole. The completion of the reaction was measured by the r a d i o a c t i v i t y incorporated in the protein after removing free p-mercuribenzoic acid by extensive d i a l y s i s . The procedure chosen for the peptide recognition was peptide mapping.~e enzyme aliquote(usually 5 mg) was alkylated for 30 mn by IO-~M iodo (2-±~C)acetate (0,5 uCi/umole) in 8M urea pH 8.5, in darkness and at room temperature. Trypsin hydrolysis was performed in 0 . I M ammonium bicarbonate at pH 8.5 and 37°C for 20 hours using an enzyme to substrate r a t i o of 4 % (w/w). Peptide maps were obtained by submitting the hydrolysatespotted on Whatman n ° 3MM sheets - to high voltage electrophoresis (0.2 M pyridinium acetate pH 6.5 44 volts/cm I hr) in one dimension, and descending chromatography (butanol 37.5 % - pyridine 25 % - acetic acid 7.5 % - water 30 %) for 20 hrs in the second dimension. Peptides were located both by ninhydrin staining and by autoradiography. Radioactive spots were either d i r e c t l y measured for r a d i o a c t i v i t y in v i a l s containing s c i n t i l l a t o r (omnif l u o r toluene 4/1000 w/w) or eluted with 0 . I M acetic acid. Peptide p u r i f i cation was obtained by the two steps of two dimensional separation and microelution of the peptide from the paper. N-terminal amino acids were i d e n t i f i e d by reaction with dansyl chloride (7) and amino acid analysis performed on a Technicon Amino Acid Analyzer (8). RESULTS F i r s t l y , evidence for a change in the extent of alkylation of the cysteinyl residues of glutamate dehydrogenase a f t e r treatment with mercurials was looked for. The p-mercuribenzoic acid-enzyme is less alkylated than the native one by iodo (2-14C) acetate and comparison of the two peptide maps shows that a radioactive peptide spot in the control corresponds to a nonradioactive peptide spot in the mercurial enzyme.

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Direct location of the 14C-pMB peptide on f i n g e r - p r i n t s always f a i l e d to, as a r e s u l t of p-MB taking o f f during high voltage e l e c t r o phoresis. Conditions for the selective replacement of p-mercuribenzoic acid by radioactive iodoacetic acid on t h i s residue were then selected. The mercurial enzyme (one mole of 14C-pMB bound per subunit) was alkylated with non radioactive iodoacetic acid in denaturing conditions as described in the Methods. The excess of iodoacetic acid was removed by extensive d i a l y s i s and the amount of 14C-pMB bound to the enzyme was controlled. The assay was then submitted to reducing conditions (10-2M d i t h i o t h r e i t o l

in 8M urea under

nitrogen bubbling f o r four hours). A second a l k y l a t i o n step was carried out with iodo (2-14C) acetic acid and the r e s u l t i n g completely alkylated protein submitted to t r y p t i c hydrolysis. The r e s u l t s of such experiments

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0

o A

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Figure 1 : Peptide map of a t r y p t i c digest of 14C-carboxymethylated glutamate dehydrogenase. Letters r e f e r to the r a d i o a c t i v e l y labelled peptides. (a) electrophoretic and chromatographic m o b i l i t y of orange G, (1) electrophor e t i c m o b i l i t y of xylene cyanol FF.

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are shown in Table I. I t is clear that the f i r s t

alkylation does not dis-

place the bound mercurial, which is completely removed a f t e r d i t h i o t h r e i t o l reduction. The second alkylation step leads to a l i t t l e

greater incorporation

of iodo (2-14C) acetic acid than the quantity of mercurials derivatives displaced but is consistent with the incorporation obtained at a second alkylation step performed on native enzyme. Finger Prints The peptide map of a trypsic hydrolysate is shown in Figure I. There are six major radioactive spots for the control, and an unique radioactive peptide spot (E) for the pMB-treated enzyme. This spot is located in an area of the peptide map which is identical to that where the previously i d e n t i f i e d peptide was found (see above) the r a d i o a c t i v i t y of which f a l l s in pMB-enzyme. Table I shows the extent of carboxymethyl groups incor-

Table 1 : Mercurials replacement by iodo(2-14C) acetate. Repartition of the ral-a-dT6-a-ctivity between the labelled peptides. The values were expressed in mole per subunit after corrections of paper quenching•

pMB treated enzyme 14C-pMB bound a f t e r the f i r s t alkylation step

1.05

14C-pMB bound a f t e r the reduction step

.06

methyl mercure native enzyme treated enzyme

iodo(2-14C)acetic acid incorporated at the second alkylation step

1.9

2.5

.67

14C-carboxymethyl groups incorporated in peptide : A B C D E F

.25 .14 .08 .20 1.06 .16

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.35

.20

• 19

.09

• 13 .34 .93 .56

.02 .16 .10 .12

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porated into each radioactive spot when selective replacement of mercurials by iodo (2-14C) acetic acid is performed. The pMB corresponding peptide is radioactively labelled to a similar extent in the assay and in the control ; the background of r a d i o a c t i v i t y incorporated by the other peptides in the assay is proportionally similar to that incorporated in the control suggesting an i n s u f f i c i e n t alkylation at the f i r s t

step or too drastic reducing

conditions at the second step ; but these experimental conditions were

Table 2 :

Amino acid composition of the peptide labelled during mercurial

replacement.

Amino acid

Radioactive peptide Amount (nmol)

Amount of amino acid/alanine

lysine

7.7

0.94

histidine

3.0

0.37

18.9

2.3

Amino acid composition of peptide 309-329

1

arginine CM-cysteine +~ aspartic acid j

1 2

threonine serine

16.9

2

2

glutamic acid

25.7

3.15

3

proline

7.5

0.92

1

glycine

15.7

1.92

I

alanine

16.3

2

2

valine

7

0,86

I

methionine isoleucine

24.2 (a)

3

4

leucine

13.2 (a)

1.62

2

0.8

I

tyrosine

6.4

phenylalanine (a) only 24 hours hydrolysis performed.

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maintained since they give a b e t t e r proportion of s p e c i f i c to aspecific alkylations. This peptide was p u r i f i e d as described in the Methods and end group determination shows only one dansyl amino acid : valine. The amino acid composition of the peptide is given in Table II and shows a good correspondance with the 309-329 amino acid sequence which is the t r y p t i c peptide expected f o r the cysteinyl residue number 319. The e l e c t r o p h o r e t i c m o b i l i t y of the peptide is - 0.67 which is that of peptide 309-329 (according to Offord - 9).

DISCUSSION The i d e n t i f i c a t i o n of the amino acid residue which binds pMB, depends both on the s t a b i l i t y ficity

of the enzyme-pMB complex and on the speci-

of the replacement method used. Using 14C-pMB allows the control of

bound pMB a f t e r the f i r s t

a l k y l a t i o n step in denaturing conditions ;

we don't believe that pMB is bound to a hypothetical amino acid residue in the native enzyme and displaced to cysteinyl residues during the denat u r a t i o n procedure since the s e l e c t i v i t y of the replacement obtained in the case of pMB modification is conclusive. The same experiments were performed to a t t a i n s p e c i f i c replacement of methyl mercuric chloride and the major l a b e l l i n g of residue occurs in the same peptide confirming previous data obtained on the competitive l a b e l l i n g of the enzyme by these two reactants (3). Amino acid analysis, end group determination and electrophoretic mobilities

show this residue to be cysteine 319.

This residue is included in the sequence of one of the predicted coenzyme-binding domains of glutamate dehydrogenase ( i 0 ) , namely in the BE sheet of domain 3. The alignment of domain I which locates the reactive lysine 126 in the bend sequence immediately following BF is l i k e l y to be the coenzyme

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binding site involved in a c t i v i t y since lysine 126 modification always leads to enzyme inactivation whatever reactants is used. "The domain 2, consisting of the sequence between domain 1 and domain 3, is the candidate for the functions of catalysis and 2-oxoglutarate or L-glutamate binding" (10). Indeed lysine 126 is protected by both NADH and 2-oxoglutarate ( i i ) .

The

domain 3 may be the second coenzyme binding site i d e n t i f i e d on spectrophotometric c r i t e r i a and for which competition between ADP and NADH has been proved (12). Cysteine 319 modification does not interfere with coenzyme binding or ADP binding at this site (4) but does interfere with the associated regulatory consequences on the catalysis : ADP i n h i b i t s the oxidation of L-glutamate by the pMB enzyme and does not affect-the catalysis by the methyl mercurial enzyme. These results

suggest that the i n t e g r i t y

of this sequence alignment is necessary for the expression of the regulatory properties of ADP. That would indicate a c r i t i c

conformational role

for these residues. REFERENCES 1) Bitensky, M.W., Yieldings, K.L. and Tomkins, G.M. (1965) J. Biol. Chem. 240, 663-673. 2 Nishida, M. and Yieldings, K.L. (1970) Arch. Biochem. Biophys. 141, 409-415. 3 Cosson, M.P. and Pantaloni, D. Eur. J. Biochem.,in press, 4 Cosson, M.P. and Pantaloni, D. submitted for publication. 5 Kubo, H., lwatsubo, M., Watari, M., Soyama, T., Kawashura, N., Mitani, A. and Ito, K. (1958) Bull. Soc. Chim. Biol. 40, 431. 6 Olson, J.A. and Anfinsen, C.B. (1952) J. Biol. Chem. 197, 67-79. 7 Gros, C. and Labouesse, B. (1969) Eur. J. Biochem. 7, 463-470. 8 Piez, K.A. and Morris, L. (1960) Anal. Biochem., I, 187-201. 9 Offord, R.E. (1966) Nature, 211, 591-593. I0 Wootton, J.C. (1974) Nature, 252, 542-546. I I ' Talbot, J.C., Gros, C., Cosson, M.P. and Pantaloni, D., submitted for publication. 12) Dessen, P. and Pantaloni, D. (1973) Eur. J. Biochem., 39, 157-169.

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