386

Biochtmwa et Biophystca Acta, 1073(1991)386-393 © 1991ElsevierSciencePublishersB,V.0304-416S/91/$G3.$O ADONIS 0304~1659100109U

The identification of a structurally important cysteine residue in the glycerol dehydrogenase from Bacillus stearothermophilus P. S p e n c e r 1, M . D . S c a w e n 2, T . A t k i n s o n 2 a n d M . G . G o r e 1 Protein Sequencing Unit, Department of Bi~ +wmistry,Institute of Biomolecular Sciences, Universityo/Southampton, Southampton and 2 Biotechnology Division, PHLS Centrefor Applied Mic~biology Research, Porlon, Salisbury (U.K.)

(Received10 September, 3990) Key words: Glyceroldehydrogenase; Metallo-ergyme;Cysteine; Amino acid sequence Evidence is presented to demonstrate that the Zn 2+ metallo-enzyme glycerol dehydrogenaso from the thermophile Bacillus stearothetTnophilm has one eysteine residue per subunlt which is only available for reaction with thiol reagents in the metal-depleted form of the enzyme. Modification of the metal-depleted enzyme by methyl methauethiosulphouete prevents the reactivation of the enzyme by Znz+ ions and induces dissociation of the oligomer into subanits. The rate of reaction of the cysteine residue with the thiol reagent D T N B is limited by a factor other than reagent concentration and it is proposed that the reagent only reaets with the eys.*eine residue In dissociated monomers. The enzyme has been labelled at the single cysteiue residue by radlwactive iedol2*3Hlacefic acid. Two radinlahelled peptides have been isolated and sequenced; one peptide is ~ component of the other. Spectroscopic evidence suggests that the cysteiue residue is not involved in ligatiort of the essential metal ion. Chemical modification studies using the reagent diethylpyrocarhenate have suggeated that two hlstidines are involved in the ligation of the metal.

Interest in glycerol dehydrngenases (GDH) has arisen in recent years because of their role in clinical diagnostics where they can be used to estimate glycerol derived from of serum triglycerides. G D H enzymes are soluble and pyridiue noclcotide dependent with the exception of the membrane bound G D H from Gluconobacter indu,ctrius which requires pyrroloquinine quinon¢ as a co/actor [1]. The NAD-speeific glycerol dehydrogenase from the thermophile Bacillus stearothermophilus is a type I G D H catalyzing the interconversion of glycerol and dihydroxyacetone (EC 1.1.1.6) [2]. Like the enzyme from Bacillus megaterium [3], it requires divalent metal ions for catalytic activity and binds one •quivalent of Zn 2+ per subunit, although other metal ions such as Cd 2+, Co2+ and Mn :+ will substitute for Zn ~÷ [2]. Previous studies have shown that the metal ions can be removed by chelating agents such as EDTA [2] to yield a catalytically inactive species which can be completely reactivated by the addition of Zn 2+ ions. Fluorescence studies using N A D H as a probe have shown that the metal-depleted GDH from B. stearothermophilus still Correspondence:M.G. Gore, Departmen of B ochemistry,Schoo of

Biochemicaland PhysiologicalSciel~ces,Universityof Southampton, Eassett Crescent East, Southampton 509 3TU, U.K.

binds one equivalent of N A D H per subunit (Mr 42000) with high affinity but that this species is unable to bind the substrate glycerol [2] thus implicating a role for the metal ion in catalysis. No changes in the circular diehroic or fluorescence spectra of the enzyme is noted on removal or replacement of the metal ion suggesting that the secondary and tertiary structure of the protein is litdc affected by the process [4]. In other metallo-enzymes, the metal ion is often co-ordinated by a combination of amino acid residues such as cysteine, histidine and glutamate 15], depending upon the role of the metal ion in catalysis or structural stabilisation [6]. Amino acid analysis and chemical cleavage experiments using the reagent 2-nitro-5-thiocyanobecLzoate [2,7] have suggested that there is only one equivalent of cysteine per subunit of M r 42000 in this enzyme. In the present paper we demonstrate that only one equivalent of indo[2-3H]-acetic acid can be incorporated per subuuit of this enzyme and that modification of the single cysteine residue by methyl methauethiosuiphonate resuits not only in the loss of the ZD2+ binding site but also in the dissociation of the tetramer into subunits. Materials and Methods Bacterial cells (NCIB 11400) were obtained from the Centre tor Applied Microbiology and Research, PHLS, Porton, Salisbury, U.K. lodo[2-3H]acetic acid was oh-

tained from The Radiochemical Centre, Amersham, U.K. Diethylpyrocarbonate (DEP) and TPCK-treated trypsin was obtained from Sigma, London, Chelex 100 from Bio-Rad, Warlord, U.K. and all other chemicals were obtained from BDH, Poole, U.K. Preparation of enzyme. The bacterial cells were grown as described in [8] and the enzyme was purified as described in [2]. Assay. Glycerol dehydrogcnase activity was assayed by following the increase in absorbance at 34O nm at 30°C using a Perkin Elmer Iarabda 3 spectrophotometer. Reaction mixtures of 1 ml contained 0.975 ml of 200 m M triethanolamine-HCl (pH 8.5). comaining 100 m M glycerol, 25 gl of 15 m M N A D and 5 - 1 0 /LI of enzyme solution. 1 unit of activity was taken as the amount of enzyme needed to reduce 1 gmol of N A D per rain. Protein concentrations were deteanined by the method of Lowry et al. [9]. Preparation of the metal-depleted GDH. At all stages precautions were taken to ensure that there was no or minimal contamination by trace elements [10]. Purified enzyme was dialysed against four times 5 litres of 50 m M potassium phosphate buffer containing 10 m M EDTA. A f'mal dialysis against two changes of buffer p H 6.0 (or distilled water if the protein solution was to be lyophilysed) was carried out in order in the p r ~ n c e of Chelex-100 to remove the EDTA. The dlalysate was then used for kinetic studies or lyophilysed for chemical modification.

Determination of the metal content of the protein. A Perkin-Elmer 280 Atomic Absorption spectrophotometer was calibrated by sampling solutions of known concentration of the metal ion under investigation. The concentration of metal ions in the buffer or in the solution of protein were measured and calculated from the appropriate standard curve.

Reaction of the enzyme with methyl methanethlosulphonate. The reagent (1 raM) was added to a solution of 4 # M enzyme ( M r 42000) in 250 m M triethanolamine-HCl buffer (pH 7.4) at 21°C.

ReactWn of the enzyme with 5,5"-dithiobis(2-nitrobenzoic acid). A stock solution of the reagent (10 raM) was made in 250 mM triethanolamine-HCl buffer (pH 7.4) and aliquots (50-fold molar excess) of this were added to samples of metal-depleted enzyme and the absorption monitored at 412 nra in a Perkin Elmer lambda 3 spectrophotometer thermostated at 25°C. Reaetian of GDH with iodo[2-SH]acetie acid. 10 mg of metal-depleted (240 nmol) was dissolved in 1 - 2 mi of 6 M guanldinium hydrochloride in 0,5 M Tris-HCl CoH 8.5) also containing 1 m M EDTA. 1200 nmo! of fl-mercaptcethanol (which represents a 5-fold molar ratio to cysteine residues) were added, the solution was incubated under nitrogen for approx. 30 rain at 37°C. Iodo[2- 3H]acetic acid (143-10 6 d p m per/tmol) pre-dissolved in the above buffer was added to give a final

concentration of 5 mM. The p H was monitored a n d kept at 8.5 and the reaction was incubated for 2 h a t 37~C. The sample was then dialysed exhaustively against distilled water until no more radioactivity was detected in the dialysis media. The sample was then frozen and lyophilysed.

Proteolytic cleavage of [2-3H]~wboxymethyl-enzyme. 10 mg of lyophilysed, carboxymethy]ated enzyme was dissolved/suspended in 1 ml of 200 m M N a H C O 3 (pH 8.5). TPCK-treated trypsin was then added (1% w / w G D H ) and the digestion left for 5 - 6 h at 37°C. Insoluhie material at the end of the digestion was removed by centrifugation at 11 000 rpm in an Eppendorf mierofuge and the supernatant taken for purification of the peptides labelled by the icdo[2-~Hlacetic acid.

Purification of the peptides by reverse phase HPLC. 100-200/LI aliquots of the tryptic digest wese applied to a reverse-phase OS column (Rainin) 250 × 4 nun equilibrated in 0.570 T F A / w a t e r (v/v). A Gilson gradient HPLC system was used to produce a gradient using 0.5,% T F A / w a t e r ( v / v ) and 0.17o TFA/asetonitrile (v/v); for details see the legend to Fig. 2. Pepfides eluted from the column were detected at 235 nrm collected and an aliquot taken for radioactivity measurements. Solutions of peptides containing radioactivity were reduced in volume to 450 g l under vacuum in a Ouivap vacuum centrifuge and then applied to a Rainin reverse-phase O D S column in 5 0 - 1 0 0 / l l aliquots using the same solvent system as above. Details are given in the legend to Fig. 2.

Observation of the dissociation of the tetramer by gel permeation studies~ A Gilson HPLC system was used with a T S K G3000 SW column (600 x 7.5 ram) equilibrated in 50 m M triethanolamine-HCl buffer containing 1 m M EDTA. Samples were applied in 100-200/~1 aliquots at a flow rate of 2.0 ml per rain. Measurement of radieaetivity. The radioactivity was determined by adding 0.1-0.3 ml of aqueous samples to 5 ml of Labseint scintillation fluid obtained from Lablogic, Sheffield, U.K. and counted in a Philips 4700 liquid scintillation counter. Protein sequencing. 200 pmol amounts of the peptides were loaded onto the glass fibre disc (pre-treated with Biobrene) of an Applied Biosystems Instruments (ABI) 477A Pulsed-Liquid sequencer with an on-line ABI 120A P T H detector. The P T H derivative released at each turn was split 1 : 6 and the smaller fraetion analysed by the ABI 120A HPLC to identify the amino acid and the larger fraction taken for detection of radioactivity. The sequence analysis was repeated and the auilino thiazolinone derivatives of the amino acids taken direct for radioactivity measurements. Reaction of GDH with diethylpyrocarbonate. Diethylpyrocarbonate was added m a final concentration of 1 m M to a solution of G D H (13 /~M) dissolved in 100 m M sodium acetate buffer (pH 6.0). Histidine modifica-

tion was followed spectrophotometrically at 240 nm (E240,m=3600) as described in [11]. Alternatively, aliquots from the incubation were taken for assay in the assay system described above. The modification reaction was terminated by the addition of histidine (to 25 mM). Reversal of the modification by diethylpyrocarbonate was effected by the addition of hydroxylamine to a final concentration up to 1 M which by itself has no effect on the activity of the enzyme.

In the native, metallo.form of the enzyme the ¢ysteine residue is unreactive to the thiol ceagents iodoacctie acid, indoacetarnide, methyl methanethiosulphonate and 5,5'-ditkiobis(2-nitrobenzoic acid), (DTNB). However, preliminary experiments showed that D T N B would react with the metabdepleted enzyme. Therefore in order to chemically modify and identify all of the cysteine residues in the protein, samples of metal-depleted enzyme were reacted with iodo[2-3H]acetic acid under reductive denaturing conditions (see Materials and Methods). Reacted samples of protein were dialysed exhaustively against four times 5 litres of 50 m M potassium phosphate buffer (pH 7.4). The radioactivity incorporated was determined by dissolving a small aliquot of the reaction mixture of known protein concentration in an equal vohime of 2% S D S and counting the sample in a water miscible scintillant (see Materials and Methods). In each of three separate experiments approx. 1 mol equivalent (0.92 + 0.15) of iodo[2-3H]acetic acid was incorporated per me1 of polypeptide chain. Incubation of the metal-depleted G D H with solutions of I m M methyl methanethiosulphonate produces a time-dependent decrease in the activity regained by the enzyme on addition to an assay containing 20 ,aM Zn 2÷ ions (Fig. 1). This modification by the reagent can be reversed (see below) by the addition of excess fl-mercaptoethanol. Stoiehiometry measurements using atomic absorption spectrophotometry were made to determine the amount of Zn z+ able to be bound by metal-depleted enzyme or the same i n c u b a t e d with m e t h y l methanethiosulphonate for various times. Samples of unmodified or modified enzyme were mixed with a 25-fold molar excess of ZnC12 (100 p.M), left to eqnil~bcate for 1 rain and then applied to a HPLC gel permeation column (see Materials and Methods) where separation of the enzyme bound Z n 2+ ions and free Zn 2+ ions was achieved. The eluted protein was de. tected at 28O nm, collected, and the metal content measured by atomic absorption spectrophotometry. The experiments showed that there is a decrease in the amount of recta] bound to the protein following reaction with methyl methancthiosniphonate from 1.05 5: 0.15 reel equivalent of Zn 2 + per mol enzyme, Mr 42 000,

o

~"

Mh.

"o

Fig. 1. [a) The correlation between the degree of dissociatiOnof the GDH tetramer following thiomethylation by methyl methanethiosalphunate and the logs of activity able to be regemerated from metal-depleted enzyme incubaled with Zn2+. A solution of metal-depleted enzyme {4 ;zM in 250 mM triethanolamine-HCI buffer (pH 7.4) containing t mM EDTA) was incubated with (QI or without I o) l mM methyl methanethi~ulphonate at 2I°C. Abquots were taken at various times and the activity of the native enzyme regenerated by the presence of Zn2+ ions measured (see Materials and Methods). The change in the quaternary s;ructure of the metal*depleted enzyme incubated with (×) or without ( , I methyl methanethiosulphonate was measured by gel permeation studies [see Fig. tb and Materials and Methods}.(bI The eludon profile from a TSK G3000 SW column t7.~×600 ram) of metal-depleted e ~ y ~ incubated for O l - - l , 35 ( . . . . . ) or S0 ( - - - - - - ) nlln with l mM methyl methanethi~ sulphonate. Areas unmder thert e peaks were determined and taken to represent the amount of protein in each peal The buffer was 50 mM t rielhanalamine-HCI (pH 7.4) and the flow rate was 2 ml per min. at time 0 to less than 0.1 reel of the metal per reel of enzyme subunit when modification had reached the point where no activity could be regenerated by the addition of Z n 2+ ions. A prolonged period of incubation of methyl methanethiosulphonate modified enzyme

with Zn 2+ ions (up to 30 rain) prior to removal of the unbound Zn 2+ ions did not lead to an increase in enzyme-bound metal detected. Parallel experiments us'ng HPLC gel permeation chromatography detected a change in the M r of the protein after modification by the methyl methanmhiosulphonate. Whereas the native enzyme and fhe metal depleted enzyme appear to be stable as the tmramer ( M e four times 42000) in 50 m M potassium phosphate buffer (pH 7.4), the methyl methanethiosulphonate modified enzyme is nearly totally dissociated into single subunits ( M r 42000) and the majority of the protein applied to the column elutes after the peak containing unreacted native enzyme. In Fig. l a the change in the oligometic nature of the enzyme is demonstrated during progressive modification by the methyl methanmhiosulphonate. Protein samples taken from the peak containing high M r species (Fig l b , peak 1) can be reactivated to full activity on addition to an assay containing Zn ~+ ions. However, samples taken from the second eluting peak give no activity in such an assay unless pre-treated by excess ~8-mercaptoethanol. The amount of activity regenerated [rom this treatment is, however, variable and depends upon the length of time before the regeneration is attempted, suggesting that this monomeric form of the enzyme is not stable. in order to identify the eystaine residue apparently located at or near to the metal binding siie [23H]carboxymethylated enzyme (0.88 mol label per mol enzyme snbunit) was subjected to digestion by trypsin and the radioactively labelled peptides isolated. The water soluble peptides from the tryptic digestion contained 95~b of the incon~orated radioactivity and were separated by reverse-phase HPLC on O S or O D S columns in a 0.5% T F A in water/0.1% T F A in acetonitrile solvent system (v/v). Prefiminary separation of peptides was achieved using the system described in Fig. 2a which yielded about 32 individual peaks, two of which containing radioactivity. Peak PL containing approx. 30% of the radioactivity appfied to the column ehited in 35% acetonitrile. The other peak (P2), containing 27o of the radioactivity applied to the column, eluted later in the gradient in 50~ acetonitrile. N o other radioactive peaks eluted from the column and the total radioactivity recovered was about 33~o of that applied to the column. P1 was collected from the OS column and was resolved on a reverse-phase O D S column into three major peaks Ppl, Pp2, Pp3 (Fig. 2b) which einted sequentially at 35~ acetonitrile on an isocratic part of the gradient. Peak Pp3 contained all of the radioactivity recovered from the cohinm in an overall yield of approx. 27f$. The final recovery of peptide Pp3 after chromatography and associated procedures was about 8 5 of the starting amount of the radiolabelled protein (18 mnol). P2 was treated similarly except for the concentration

°o " ," ,; ,o Tim* (~,nl Fig- 2. The purification of the radioactive tryptlc ix'prides from carboxymethylated glycerol dehydrogenase- (al The elutlon profile of the peptides in the tlypfic digest from a R~rdn reverse-phase OS column (250×4 ram). The solvent system was wat~ (4-0.5~ TFA v/v) and acetonitfile (+0.1~ TFA v/v) in a linear gradiem from 0-70~ acetonitrile over 28 non at 1 nO per loin. Peaks PI and P2 contained radioacuvity (see Table 11. (b) The re~olution uf peptides from peak P[ (Fig. 2a) using a Ralnin ODS reverse-phase column 1250×4 nun). The solvent system was as above with a gradient of 0-35~0 acetonltrile over 17 nOn followed by a further 8 mill at 35% acetonafile. The flow rate was 1 ml per rain and peak Pp3 was only radioactive peak eluted (Table 1). (c) The resolution of pepfides c~tained in the peak P'2 on a Rainin reverse-phase ODS coluran using a solvent syctean as above. The gradient was 0-50~ acelonilfile over 17 rain followedby a further 8 rain at 50¢~~tonitrile. The flow rate was I rnl per nOn and peak Pp4 was the only radioactively lehelied peak to einte (see Table II. range of acetonitrile used for the elution (see Fig. 2c) and was resolved into six peaks, the fourth of which (Pp4) contained all of the radioactivity eluting from the column which was approx. 50% of that applied. The final yield of this peptidc was about 1.0~ of the total

radioactivity incorporated into the protein. Both peaks were then submitted to automated Edman degradation using an ABI 477A pulsed liquid sequencer. About 125 pmol of peptide Pp4 were used per run and the sequence of the peptide and the amount of each residue recovered (pmol) was T ( l l 0 ) M(61) A(58) G(105) G(98) I(55) P(23) T(34) 1(27) A(31) A(26) E(20) A(26) 1(20) A(21) E(15) K(16) C ( l l ) E(10) Q(5) T ( < 1) I.(1.5) F(1.5) K( < 1). Ho significant level of radioactivity was found in the PT'H ~e~vative of the amino acid identified as carboxymethyl cysteme or in any of the other P T H derivatives. This is kossibly due to exchange of the tritium under hot acid conditions or due to a combination of this with the effects of small quantities of peptide, the location of the presumed radiolabelled residue [18] and the effects of an average repetitive yield around 91%. 11 pmol of carboxymethyl cysteine would be expected to contain 1500 d p m of 3I-I if no exLhange takes place. Approx. 100 pmol of peptide Pp3 was sequenced and was shown to be derived from Pp4 by a successful tryptic cleavage after residue 17 and therefore consists of the last seven amino acid residues of Pp4, i.e., CEQTLFK. In this particular peptide the S-carboxymethyl-cysteine is the first residue and radioactivity could be recovered in reasonable yield. The metal-depleted enzyme can bind metal ions other than Zn 2+, e.g., cobalt, to yield a catalytically active species [2]. Other workers (Ref. 12 and references cited therein) have shown that the presence of a cobalt ion ligated to a cysteine residue usually gives rise to fight absorption in the wavelength range 250-500 n m (E400 > 500 per participating cysteine residue) due to the presence of charge-transfer bands. In the cobalt reactivated G D H only very weak absorption occurs in the wavelength range 350-750 nm with a maximum at 550 nm ( E ~ 0 = 100). This is not typical of charge transfer bands between cobalt and cysteine residues but more similar to those found when cobalt is ligated via histidine residues as in the case of carbonic anhydrase [11]. This suggests that the sole cystcine residue in G D H is not directly involved in metal binding; however, it is protected from chemical modification hy the presence of the metal ion. An alternative chemical modification of the enzyme by the chromophoric 5,5'-dithiobis(2-nitrobenzoie acid) (DTNB) was used to compare the rates of modification of the cysteine residue, the loss of activity able to be regenerated by the addition of Zn 2÷ ions and the dissociation of the tetramer. Solutions of 5 ~ M metal-depleted enzyme were incubated with D T N B (0.25-8 raM, for conditions see legend to Fig. 3) and samples were taken at various times for analysis of the activity regenerated on addition to an assay containing Z n 2+ ions (see Materials and Methods), the extent of modification of the cysteine residue (absorbanee at 412 rim), and the

loc

ec

6c

/o

Mm

too M.n Fig, 3. The correlation between the perce~lag¢of cysteni¢ modified by DTNB with the loss of metal reaetivaaon of the enzyme by Znz÷ ions and the dissociation of the tetramer. DTNB was added to a rnial concentration of I mM Io a solution of 5 ~M metal-depleted GDH in 250 mM tricthan01amine buffer (pH 7AI containing 1 mM EDTA. Modification of the cysteine residue was monitored by the increase at 412 nm (o) detecting the release of the thlonitrohenzoate ion ~ d samples were also laken for assay at various times in buffer cOnlaining 20 pM Zn:+ ions (o I. Aliquots were also taken to determine the oligomeric status of the protein at vafio~ stag~s of modification by the reagent by passage down a TSK G3000 SW HPLC gel permeation column (7.5×600 ram) equilibrated in the same buffer (@). Inset: the semilogarithmic plot of the data obtained in the experiments from Fig. 3. One single exponential with a rate of 0.O2 rain 1describ~ all curves. 2O

40

~

ao

extent of dissociation of the tetramer measured by HPLC gel permeation as above. Fig. 3 shows that the rates of change of all threo properties were identical indicating that either all three occur at the same rate, i.e., that the modification of the single cysteine residue causes loss of metal binding and induces dissociation of the tetramer, or that one process is perhaps limiting the rate of the other two processes. Metal-depleted enzyme modified by this reagent (see Materials and Methods) was subjected to gel permeation chromatography as above and both protein peaks (monomer and tetramef~ were collected. Each sample was treated with excess ~mercaptoethanol to release any bound thionitrobenzoate which was detected spectrophotometricany as the thioditrobcnzoate ion at 412 nm. Only the peak representing the monomeric form of the enzyme was found to release thionitrobenzoate showing that either the modified tetramer does not exist or does not persist after the reaction. It was also noted that if the concentration of the D T N B was increased from 0.25 to 8 m M no change in the rate of reaction with the enzyme was seen (0,02 alia -1 under these conditions), i.e., the rate was limited by something other

391 TABLE l The r~ooer2, ..f the trit!um label from iodo[2- SHlaceti,. acid ,luring the preparmion a,;o'purificJtion of the carboxymethylawd peptldes 223 nmol of the carboxymethylaledprotein containing 32× 106 dpm of 3H weresubjected to digestionby trypsin (1%.w/w) (see Materials and Methods). The table deSelSbes the recovery of radioacti~ily at each slage of the peptide purification prOCedure. Step After labelling

nMol 223

Dpm× 104 32

After centrifugation

214

30.6

%yield 100 96

Rainin RP OS ¢olunm Peak P1 Peak I"2

67 4.S

9.6 0.6a

30 2

Raiain RP OS colulral Peak Pp3 Peak Pp4

IS 2.23

2.56 0.32

S 1

than reagent concentration, possibly by a change in structure of the enzyme (e.g., dissociation). Chemical modification of histidine residues in the protein by the reagent diethylpyrocarbonate is possible whether the native enzyme or the metal-depleted enzyme is used. However, the number of histidine residues modified in each case is different. Using the absorption change at 240 mn which occurs on carboethoxylatinn of histidine [ll] it was found that four residues per subtmit in the native enzyme are modified by this reagent although only 10~o of the activity of the enzyme is lost. In the metal-depleted enzyme approximately six residues are modified, all at the same pseudo first order rate of 0.313 mix -1 (Fig. 4a). However, the loss of activity able to be regenerated by addition of g n 2+ ions proceeds at approximately twice this rate, i.e., 0.66 rnin - l (see inset to Fig. 4a). This treatment of metal-depleted enzyme results in the total loss of catalytic activity. Thus, the reaction of the extra two histidine residues results in the

loss of almost all (90%) of the activity normally recovered on addition of metal ions. Analysis of the rate of modification by the method of Tsou [13]. described by Fig. 4b. confirms that inactivation proceeds at twice the rate of modification and therefore that modification of either of extra two residues exposed when the metal ion has been removed is sufficient to prevent all of the reactivation normally observed on addition of metal ions. No significant dissociation of the tetramer occurs after modificatlo: of the enzyme by diethylpyrocarbonate. Diethylpyrocarbonate-modified metal-depleted enzyme cannot be reactivated by the addition of Zn 2+ ions unless the enzyme has been previously treated with 1 M hydroxylamine. When incubated with this reagent, the activity of the diethylpyrocarbonate modified enzyme in the presence of Zn2+ (20/~M) increases to 85% of that of unmodified enzyme over a period of 60 rain; the half time for this reaction being 8 Pain at 20°C. A final level of reactivation equivalent to 95% of the original activi',y is achieved after a father 60 mix. Observation of the absorbanea at 240 nm shows that such treatment almost completely removes the carboethoxylate group (98%) and thus regenerates native histidine residues. Discussion Labelling experiments using radioactive iodo[2JH]aeetic acid have shown that there is only one cysteine residue per polypeptidc rain of the G D H from B.stearothermophilus. In the studies described in this paper the sequence of the peptide containing the cystehie residue has been determined ana shows that the methionine precedes the cysteine residue in the peptide sequence by 16 residues, equivalent to a difference in M, of approx. 1700. This is in agreement with previous chemical cleavage studies [21 using the reagents 2-nitro-

TABLEII The sequen~ of 1he ~pade Pp3 1060 praot 052000 dpm) and 6O3pmol (8670Odpm} of pepride Pp3 were sequenced as described in the text and the PTH and A'VZderivatives, reSl~Ctlvely,werecon~t~ "the i,~aal y~lds quoted are based upon either estimation of the pmol of the PTH derivativescollected, or for the ATZ derivatives, on the radioactivitycollected, PepridePp3 Re,due Amino acid

1 C

2 E

3 Q

618

39O

original

64889 43

28056 18

10871 6.6

dpm in Aq'Z

64686

14427

6653

ongh~al

74

15

omol dpm in PTH

Initial yield624 (PTH). 744 (ATZ)

6O7

7.2

4

5

6

7

T

L

F

K

554

678

458

233

8

!![ !:

o

...-t"

. . . . .

oL

e--,

,

~

no

m,.

Io

~

FrOCt,on ufimod~fled

h,stldine~

Fig.4. (a) Co,elation b e t ~ n the modification of histidine residues by diethylpyr~arbonate and the loss of activityable to be regained on additinn of Zn2+ ions. A solution of 13 ~M metal-depleted enzyme was incubated with 1 raM DEP in 100/aM sodium acetate buffer pH 6.0 at 200C.5/ri aiiquots were taken at various times and enzymeactivity(o) determined by a assay in buffer containing 20 I~M Zn2+ ions. Hislidinemodification (o) was monitored spectrophotometricallyand the number of residues reacted was calculated from the absorption at 240 nm (E = 3.6x 10J). Inset: the inset shows senfihigarithml¢ analyses of the data from Fig. 4a. The loss of activity (