Cysteinyl peptides of pig heart NADP-dependent isocitrate dehydrogenase that are modified upon inactivation by N-ethylmaleimide.

Pig heart NADP-specific isocitrate dehydrogenase is inactivated by N-ethylmaleimide (NEM) (Colman, R. F., and Chu, R. (1970) J. Biol. Chem. 245, 601-607), and is completely protected against inactivation, but not against the incorporation of NEM, by isocitrate plus Mn2+. We have now treated the enzyme with [3H]NEM in the absence and presence of isocitrate plus Mn2+, digested it with trypsin, and isolated and sequenced the labeled Cys peptides. In the inactive enzyme, two major peptides, SSGGFVWACK and DLAGCIHGLSNVK, and two minor peptides, CATITPDEAR and EPIICK, were labeled at Cys. Upon reaction with [3H]NEM in the presence of isocitrate plus Mn2+, full catalytic activity was retained and only DLAGCIHGLSNVK was labeled; the Cys of this peptide is therefore not essential for catalysis. The modification of SSGGFVWACK appears to be the major cause of inactivation by NEM. The Cys in SSGGFVWACK may have a catalytic role, most likely in the strengthened binding of Mn2+ in the presence of isocitrate. Isocitrate dehydrogenase was carboxymethylated under denaturing conditions with [14C]iodoacetate and digested with trypsin; 6 unique labeled Cys peptides, containing 6 unique Cys residues, were purified and sequenced. Six corresponding peptides were isolated from enzyme treated under denaturing conditions with [3H]NEM. These results eliminate the previous uncertainty regarding the number of Cys residues in the enzyme. A comparison of the sequences of the NH2-terminal 30 residues and the 6 Cys peptides of the pig heart NADP-dependent isocitrate dehydrogenase with the Escherichia coli NADP enzyme provides evidence for great dissimilarity between the two enzymes.

F., and Chu, R. (1970) J. Biol. Chem. 245, 601-607), and is completely protected against inactivation, but not against the incorporation of NEM, by isocitrate plus Mn2+. We have now treated the enzyme with [3H] NEM in the absence and presence of isocitrate plus Mn2+, digested it with trypsin, and isolated and sequenced the labeled Cys peptides. In the inactive enzyme, two major peptides, SSGGFVWACK and DLAGCIHGLSNVK, and two minor peptides, CA-TITPDEAR and EPIICK, were labeled at Cys. Upon reaction with [3H]NEM in the presence of isocitrate plus Mn2+, full catalytic activity was retained and only DLAGCIHGLSNVK was labeled; the Cys of this peptide is therefore not essential for catalysis. The modification of SSGGFVWACK appears to be the major cause of inactivation by NEM. The Cys in SSGGFV WACK may have a catalytic role, most likely in the strengthened binding of Mn2+ in the presence of isocitrate.
Isocitrate dehydrogenase was carboxymethylated under denaturing conditions with [ 14C]iodoacetate and digested with trypsin; 6 unique labeled Cys peptides, containing 6 unique Cys residues, were purified and sequenced. Six corresponding peptides were isolated from enzyme treated under denaturing conditions with [3H]NEM. These results eliminate the previous uncertainty regarding the number of Cys residues in the enzyme. A comparison of the sequences of the NH2terminal 30 residues and the 6 Cys peptides of the pig heart NADP-dependent isocitrate dehydrogenase with the Escherichia coli NADP enzyme provides evidence for great dissimilarity between the two enzymes.
Isocitrate dehydrogenase (isocitrate:NADP+ oxidoreductase (decarboxylating), EC 1.1.1.42) from pig heart mitochondria catalyzes the oxidative decarboxylation of isocitrate to a-ketoglutarate. A divalent metal ion is required for activity (I), the highest activity being obtained with Mn2+ (Z), and it is thought that the metal-isocitrate complex is the true substrate (3, 4). The enzyme is a dimer of identical subunits under many conditions (5, 6) and is not known to be allosterically regulated. Neither the sequence nor the three-dimen-* This work was supported by United States Public Health Service Grant DK39075. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Previous studies (13,14) demonstrated that the enzyme was inactivated by N-ethylmaleimide (NEM)' concomitant with limited reagent incorporation. Isocitrate plus Mn2+ (but not other ligands) protected the enzyme against inactivation, although NEM was still incorporated. Inactive NEM-treated enzyme was shown to bind isocitrate or Mn2+ normally, but it did not display the strengthened binding of Mn2+ in the presence of isocitrate that is characteristic of unmodified enzyme. These results suggested that NEM reacts at the manganous isocitrate site. Although cysteine was shown to be the only type of amino acid modified by NEM in isocitrate dehydrogenase, no attempt was made to identify particular peptides (14).
The present study was undertaken to isolate and ascertain the amino acid sequence of tryptic peptides containing the cysteine residues of pig heart NADP-dependent isocitrate dehydrogenase that are modified when the enzyme reacts with NEM in the presence or in the absence of isocitrate and Mn2+, and hence the cysteine residue(s) that may be involved in the catalytic function of the enzyme. The total number of cysteine residues in the pig heart NADP-dependent isocitrate dehydrogenase has been reported to be 8. 2-8.8 mol/mol subunit (15, 16), although the number of cysteines in isocitrate dehydrogenases from various sources ranges from 1 to 9 mol/mol subunit (4,17,18). We have now determined the total number of cysteine residues in the pig heart NADP-dependent isocitrate dehydrogenase by an independent method the radioactive labeling, isolation, and sequencing, of all of the unique tryptic cysteinyl peptides. In addition, the sequence of the NH2-terminal region of the protein has now been determined.
A preliminary version of this work has been presented (19).

DISCUSSION
It is clear that, under denaturing conditions, 6 unique cysteine residues of pig heart NADP-dependent isocitrate The abbreviations used are: NEM, N-ethylmaleimide; HPLC, high performance liquid chromatography; PTH, phenylthiohydantoin; TPCK, N-tosylphenylalanine chloromethyl ketone.
Portions of this paper (including "Experimental Procedures" and "Results," Figs. 2-5, and Tables 1-111) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.  and digested with trypsin as described under "Experimental Procedures." The tryptic digest was applied to a Vydac C,, column equilibrated with 0.1% trifluoroacetic acid and eluted a t a flow rate of 1 ml/min with Solvent System I. No significant absorbance a t 220 nm or radioactivity was detected after 150 ml. A , absorbance of the eluate a t 220 nm (-).
dehydrogenase reacted with iodoacetate (Table I). However, the extent of the reaction of [14C]iodoacetate with the 6 residues varied, as judged by differences in the areas of the radioactive peaks on HPLC chromatograms (e.g. Fig. 1). The cysteine corresponding to Peaks IVa and IVb was always the least extensively labeled; of the remaining cysteines, that corresponding to Peak I11 was usually the least extensively labeled, while that corresponding to Peak I was usually the most extensively labeled. With a view to obtaining an equal distribution of radioactivity among the labeled cysteinyl peptides, we varied the conditions of denaturation to include the use of an alternative denaturant (urea), the use of higher concentrations of guanidine hydrochloride (7.4 M instead of 5.0 M), the use of dithiothreitol during denaturation, and rapid denaturation of the enzyme before carboxymethylation. Although it was hoped by these methods to render the enzyme more susceptible to complete denaturation and/or to protect the thiol groups from oxidation, none of the resulting tryptic digests displayed a distribution of radioactivity appreciably different from that obtained by the routine method.3

~ ~~
In initial experiments, iodoacetate-labeled isocitrate dehydrogenase was digested for shorter time periods with lower amounts of trypsin (1% for 90 min with a further 1% added after 45 min). HPLC chromatograms of such tryptic digests displayed a diminished Peak In the region of Peptide Ivbii, two proteolytic cleavages occurred that did not reflect the normal specificity of trypsin (Table I); both cleavages were incomplete. One of the cleavages occurred at a His-Ser bond, the other at a Tyr-Ala bond. Although no precedent was found for tryptic hydrolysis on the COOH-terminal side of His, there is precedent for tryptic hydrolysis on the COOH-terminal side of hydrophobic residues, even by trypsin preparations treated with TPCK (25).
Such chymotryptic-like activity is thought to be due to J.trypsin, which arises by autolysis during prolonged incubation I1 and an extra peak eluting a t -117 min. This extra peak was rechromatographed with Solvent System 11. The resulting pure peptide, which yielded the sequence Asn-Ile-Leu-Gly-Gly-Thr-Val-Phe-Arg-Glu-Pro-Ile-Ile-CmCys-Lys, features an internal Arg, and its COOH-terminal6 residues correspond in sequence to Peptide 11. The incomplete cleavage of the Arg-Glu bond under the milder digestion conditions accords with the known diminished rate of tryptic hydrolysis of peptide bonds close to an acidic side chain (25). Longer digestion periods with higher amounts of trypsin (5% for 6 h with a further 5% after 3 h) were therefore routinely used to ensure complete proteolysis. (The generation of Peptides I and VI also involves the cleavage of a peptide bond on the NH2-terminal side of an acidic residue (Cys(Cm) and Asp, respectively), and the generation of Peptides I1 and V involves cleavage a t a Lys preceded in the sequence by a Cys(Cm) residue. Yet all of these cleavages appear to have proceeded to completion even under the milder digestion conditions.)

Cys Peptides
of NADP Isocitrate Dehydrogenase of trypsin and may be present in commercial preparations (26,27). Specifically, the selective tryptic hydrolysis of a single Tyr-Ala bond in a-chymotrypsin has been previously reported (28). It is unlikely that such a cleavage in isocitrate dehydrogenase was catalyzed by contaminating chymotrypsin because the trypsin preparation had been treated with TPCK; furthermore, three other potential chymotryptic cleavage sites (Phe-Gln in Peptide IV and Phe-Val and Trp-Ala in Peptide V) remained intact even under our severe digestion conditions. The use of [3H]NEM as an alternative to [14C]iodoacetate for labeling cysteine residues in isocitrate dehydrogenase yielded 6 major tryptic cysteinyl peptides ( Fig. 5; Tables I1 and 111) that correspond to the 6 ['4C]iodoacetate-labeled cysteinyl peptides ( Fig. 1; Table I). The sequences of these 6 tryptic cysteinyl peptides are summarized in Table IV. NEMlabeled enzyme (Fig. 5) exhibited a single radioactive peak (IV,) corresponding to Peaks IVa and IVb of iodoacetatelabeled enzyme (Fig. l), indicating that the two unexpected tryptic cleavages in the region of Peptide IVbii, which were incomplete in iodoacetate-labeled enzyme, had proceeded to completion in NEM-labeled enzyme.
The labeling of only 6 unique tryptic cysteinyl peptides by two reagents that differ in their mechanism of reaction with thiols casts doubt on the previous quantitative determinations of the number of cysteine residues in the enzyme of 8.2-8.8 mol/mol subunit (15, 16). A possible source of error is the relative molecular mass of the enzyme subunit. The value used by Johanson and Colman (15,16), 58,000, had been determined by sedimentation-equilibrium measurements on the native enzyme (8). However, this value is at the upper end of a range of values determined using different methods, including sedimentation equilibrium under denaturing conditions (52,900) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (48,500) (20). The use of the value of 48,500 instead of 58,000 yields a range of 6.9-7.4 mol/mol subunit from the data of Johanson and Colman (15,16), considerably reducing the discrepancy. of isocitrate and Mn2+ shows that the cysteine residue in Peptide VI is not essential for activity: isocitrate plus Mn2' protected the enzyme against inactivation by NEM but did not protect the cysteine of Peptide VI against modification by NEM. In the absence of isocitrate and Mn2+, NEM inactivated the enzyme and extensively modified not only Peptide VI, but also Peptide V, and partially modified Peptides I and 11. The cysteines in Peptides I, 11, and V are all protected by isocitrate plus Mn2' against modification by NEM. In view of the extent of its reaction with NEM, the cysteine in Peptide V is likely to be the one whose modification causes the enzyme to lose activity. However, our results do not exclude the possibility that the modification of Peptides I or I1 also leads to inactive enzyme; indeed it is possible that all 3 cysteine residues, which protection studies suggest are at or near the substrate site, could react with NEM in a mutually exclusive manner to yield 3 different modified inactive enzymes, of which the predominant one is that modified at Peptide V.
These studies significantly extend those of Colman and Chu (13,14), which yielded evidence for 2 major NEMreactive cysteine residues: in the absence of isocitrate and Mn2+, the modification by NEM of one of these residues (Cys-A) inactivated the enzyme, while the modification of the other (Cys-B) did not affect the activity; and in the presence of isocitrate plus Mn2+, Cys-A was protected against modification, whereas Cys-B was modified, yielding a fully active enzyme. In the earlier studies, Cys-A and Cys-B were not identified. We have now isolated cysteinyl peptides that correspond to these residues: Peptide V presumably contains the critical substrate-protectable Cys-A, and Peptide VI the nonessential Cys-B, which is modified in the absence or presence of the substrate. Cys-A was postulated to be involved in the strengthened binding of manganous ion in the presence of isocitrate (29), so that Peptide V is expected to be close to the manganous isocitrate site.
The incorporation of NEM into native isocitrate dehydrogenase was determined by Colman and Chu (14) to be 2 mol/ mol subunit in the absence or in the presence of isocitrate and Mn2+. The corresponding values of incorporation into the native enzyme determined in the present study were significantly lower but, as expected, they differed depending on whether the reaction was carried out in the absence (0.75 mol of reagent/mol of subunit) or in the presence (0.35 mol of reagent/mol of subunit) of isocitrate and Mn2+. The difference between the earlier and the present values of incorporation may be accounted for by the method of removal of the excess reagent: in the earlier studies, excess reagent was removed under nondenaturing conditions, allowing the possibility of residual noncovalent binding, whereas in the present studies it was removed under denaturing conditions. The difference between the incorporation in the absence of isocitrate and Mn2+, 0.40 mol/mol subunit, presumably corresponds to the incorporation into the protectable cysteine residues, those in Peptides I, 11, and V. This difference, which is close to 0.5 mol/mol subunit, is most easily interpreted as the reaction of NEM at only 1 subunit per dimer; reaction of NEM can occur with any 1 of the 3 protectable cysteine residues, of which that in Peptide V is the most reactive.
The product of the reaction of NEM and Cys is S-(N-ethy1succinimido)cysteine (30). This compound is rather stable in acid; only upon prolonged acid hydrolysis is it converted to ethylamine and S-(1,2-dicarboxyethyl)cysteine (31). Therefore the adduct of a cysteine residue and NEM is expected to be stable during Edman degradation of the peptide in the gasphase sequenator. Accordingly, a PTH-derivative of this adduct was detected in the cycle corresponding to Cys; furthermore, this derivative, which eluted as a double peak between Pro and Met, occurred in the cycle in which the bulk of the recovered radioactivity was detected. Because the radioactiv-

Ser-Ser-Gly-Gly-Phe-Val-Trp-Ala-Cys-Lys
ity of the [3H]NEM resides in the N-ethyl group, the recovery of radioactivity indicates that the adduct had not hydrolyzed. Although S-(N-ethy1succinimido)cysteine could undergo partial hydrolysis to yield S-(l-carboxy-2-N-ethylcarbamoyl)ethylcysteine, the hydrolysis product is thought to recyclize rapidly in acidic solution (31). The reaction of Cys and NEM generates a new asymmetric center, giving rise to a pair of diastereomers (derivatives of a cysteinyl peptide or of PTH-Cys). The diastereomeric adducts of NEM and Cys, which are interconvertible in acidic solution, have been resolved by ion-exchange chromatography (31). Such diastereomeric products could account for the double peaks observed both upon HPLC of NEM-labeled Peptides 11,-IV, and VI, in Solvent System I1 and upon HPLC of the PTH-Cys derivative resulting from Edman degradation. Fast atom bombardment-mass spectrometry of Peptide 11, yielded an M , for the m + 1 parent ion consistent with the expected S-(N-ethy1succinimido)cysteine residue; and the observation of identical M , values for both peaks of the doublet rules out the possibility that one of the peaks corresponds to a partial hydrolysis product containing an S-(l-carboxy-2-N-ethylcarbamoy1)ethylcysteine residue, which can form under mildly alkaline conditions (31). This result agrees with the findings of Colman and Chu (14) who used paper chromatography to identify S-(N-ethy1succinimido)cysteine as the modified residue in NEM-treated isocitrate dehydrogenase.
S-(N-Ethy1succinimido)cysteine is reported to undergo an intramolecular transamidation reaction under very mildly alkaline conditions. This rearrangement, which proceeds to completion at pH 9.0, yields 2-(N-ethylcarbamoylmethyl)-3-keto-1,4-thiazine-5-carboxylic acid (30). Such a rearrangement could occur in the NHz-terminal S-(N-ethylsuccinimido)cysteine residue of Peptide I, during tryptic digestion at pH 8.0; it would block the NHn-terminal amino group of Peptide I, and account for the absence of PTH-derivatives upon application of the peptide to the gas-phase sequenator. We therefore also expect the radioactive fragment generated by thermolytic cleavage of Peptide I, to be refractory to NHzterminal sequencing. The possibility of a blocked NHz terminus is not excluded by fast atom bombardment mass spectrometry of Peptide I,, which yielded an M , for the m + 1 parent ion consistent with both the S-(N-ethylsuccinimido)cysteinyl peptide and its rearrangement product.
NADP-dependent isocitrate dehydrogenase from pig heart mitochondria is considered, on the basis of physical evidence, to consist of a single type of polypeptide chain (20). Our identification in this study of a unique NHn-terminal sequence of 30 residues, combined with our isolation of 6 unique tryptic cysteinyl peptides in approximately equal amounts, provides confirmatory evidence that the enzyme is indeed composed of a single type of subunit. The amino-terminal 30 residues of the pig heart enzyme show striking sequence similarity to the amino-terminal region of the NADP-dependent enzyme from yeast mitochondria (32), 20 out of 29 aligned residues being identical. In contrast, the amino-terminal region of the pig heart enzyme shows no apparent resemblance to the aminoterminal region of the Escherichia coli enzyme (33); nor do the cysteinyl peptides of the pig heart enzyme show obvious similarity to any sequences of the E. coli enzyme.
In this study we have isolated and sequenced all of the tryptic cysteinyl peptides of the pig heart mitochondrial NADP-dependent isocitrate dehydrogenase. The peptides were labeled using either of two thiol reagents, iodoacetate or NEM, that differ in the mechanism of their reaction with thiol groups. With both reagents, 6 unique cysteine residues were identified, yielding a definitive determination of the number of cysteine residues in isocitrate dehydrogenase. NEM reacts rapidly with a cysteine residue in Peptide VI with no effect on activity, so that this cysteine is not essential for catalytic activity. NEM also reacts rapidly with 1 cysteine residue (in Peptide V) and slowly with 2 other cysteines (in Peptides I and 11) concomitant with the inactivation of the enzyme. One or more of these 3 cysteines (which are probably at the substrate site) could be involved in catalysis, most likely by strengthening the binding of manganous ion in the presence of isocitrate.  The enryne was then digested with trypsin (5% u/w) for 6 h at 37'C. with a further 5% (u/w) Of t r y p s i n added after 3 h. and the digest was lyophilized.  observed. Each of these peaks was rechromatographed with Solvent System I I (Fig. 2). Peaks I -111. V. and V I (Fig. 21-C. F, L 6) each yielded a single major radioactive peptide; Peak IVa major and two minor radioactive peptides.
( Fig. 20) yielded a major and a minor radioactive peptide; and Peak IVb (Fig. 2E)  , Im"lrwn, FIG. 2-continued the peptides yielded VTH.CmCyr i n the cycle i n which the bulk of the recovered radioactivity The IldiOaCtiVe peptides were subjected to automted E h n degradation ( Table   I ) . A l l of (80-9OX) was detected. Peptlde 1 has lrg as the C-teminal anino acid.  radiOaCtivitY ( 1 -I l l . V. & VI) previously observed (Fig. 1).
On the chromatogram for The chronatogmm for unnodified mzme (Fig. 3A) displays the five major peaks of unprotected enzyme (Fig. 3s). Peaks V and V I w e S t r i k i n g l y decreased 1c.f. Fig. 3A). indicating that the cysteine residue i n each o f the corresponding peptides had largely reacted with NEM. BY contrast. on the chromatogram for protected e n z m (Fig. I C ) . only Peak V I i s decreased. modification by NEW; whereils Peptide V I reacted with NEM i n the plslence or in the absence of Thus, in the presence of isocitrats plus M$+. Peptide V was essentially fully protected against isocitrate and Mn2+. radioactive peaks. as Well &I several minor mer. Each Of the t W large and the tw rull peat$ was reckroutographed with Solvent System 11. Each yielded 1 single major radioactlv. peptide (data not a h a ) .

I d e n t i f i c a t i o n of
The radiolctive peptides were subjected t o autmated sequence analysis ( Table 11). The peptide eluting at 77 .in mith Solvent System I (Fig. 40). i n three trials. yielded no PTH derivativesIafter application to the gas-phase requenator rugglrting the absence Of a free .-amino group i n t h i s modified peptide.
The peptide elutin; a t 85 .in (Flp. 1 yielded a sequence (Table I1 C  The peptide eluting at 110 .in (Fig. 1 ) i s identical i n sequence (Table I 1  The psptlde eluting at 113 .in (Fig.   48). when rechrmtograpked with Solvent Systm 11. eluted as an 1220 dnublnt coinciding with I broad peak of r a d i o a c t i v i t y t h a t Was S m t i r r resolved i n t o two peaks. Because  for 95% inactive em- (Fig. 4A. B); however in the 27% and 54% inactive o n z m s . the peaks they wore i n 96% inactlve enzyme (Fig. 41. 8). Two other features of the kinetics of peptide labeling by NEW were revealed by the HPLC Chraatogram: (1) the amount of radioactivity i n than peptide V.
Peptide V, increased I S the enzyme l o s t a c t i v i t y ; and (11) Peptide V I was labeled more rapidly Upon reaction of isocitrate dehydrogenase with [%INEM in the absence of isocltrate and In2*, theretom. tw major labeled tryptic peptides. Vn and V I , . and t n less extenrively labeled tryptic pe tides. In and 11, (Fig. 4) Ye therefore used an reacting With lodoacetate. lsedtrate dehydrogenase was treated with [IHINEM under denaturing conditions and the wdlfied enzyme was dlgested with trypsin and ckwmtographed with Solvent System I (Fig. 5). Six large radioactive peaks. as we11 as f o u r r m l l e r ones. were observed.
Each o f these peaks was rechromtogrlphed with Solvent System I1 (data not show). and the s i x large peaks each yielded a single major radioactive peptide. The radioactive peptides were a l l .in. and 110 8in (Fig. 5) are identical i n sequence respectively to Peptldes 11.. Vn. and V I . subjected to autmated sequence analysis. Themajor radioactive peptides eluting at 82 mi", 107 previoYIlY isolated f r a inactlve Nm-treated e n z m (Table 11). The radioactive peptide eluting at 89 mi" (Fig. 5)  The psptlde eluting at 76 .in (Fig. 5) (Table I l (Fig. 5) was therefore named I i n a g r H l n t w i t h th. tentatlw derlgnation pmviously made (Fig. 48). and i t s nonradioa%ire