Identification of vicinal thiols of phosphoenolpyruvate carboxykinase

THEJOURNAL OF BIOLOGICAL CHEMISTRY 8 1993 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 268. No.3, Issue of January 25,pp. 1628-1636,1993 Printed in U.S.A.

Identification of Vicinal Thiols of Phosphoenolpyruvate Carboxykinase (GTP)* (Received for publication, July 15, 1992)

Cristina T. Lewis$, Jerome M. Seyers, Robert G. Cassells, and GeraldM. CarlsonB From the Departmentof Biochemistry, College of Medicine, The Uniuersity of Tennessee, Memphis, Tennessee38163

Phosphoenolpyruvate carboxykinase (PEPCK) from from oxaloacetate in a reversible reaction thatinvolves phosthe cytosol of rat liver has13 cysteines, a t least one of phoryl transfer anddecarboxylation. which (CysZs8)is known to be very reactive and critical (Reaction 1) for catalytic activity (Lewis, C. T., Seyer, J. M., and Oxaloacetate + MgGTP(1TP) e Carlson, G . M. (1989) J. Biol.Chem. 264, 27-33). phosphoenolpyruvate + MgGDP(1DP) + GO, Previous results provided evidence for the existence of a t least 1 pair of vicinal cysteines within or near the Mammalian PEPCKdisplays aspecific substrate requirement active site of PEPCK (Lewis, C. T.,Haley, B. E., and for guanosine or inosine nucleotides (Miller et al., 1968). Although the gene coding for the cytosolic carboxykinase has Carlson, G . M. (1989) Biochemistry 28,9248-9255). An intramolecular cystine disulfideis induced to form been cloned and the aminoacid sequence of the enzyme has been determined (Beale et al., 1985),the molecular basis for upontreatment of PEPCKwithequimolar5,5’-dithiobis(2-nitrobenzoate)(Nbsz) or upon irradiation of thesubstrate specificity remains unclear. Furthermore,in the enzyme in the presenceof the photoaffinity probe spite of the important role that PEPCK plays in central 8-azidoGTP. In each case, modification is accompanied metabolism, very little isknown about the architectureof the by a substantial loss in catalytic activity, and subenzyme’s active site. strates protect against inactivation andmodification. PEPCK that hasbeen isolated from a variety of sources is We now report the identification of these modified sensitive tosulfhydryl reagents, and thesulfhydryl chemistry thiols by differential alkylationof cysteines and half- of the enzyme has been studied in a number of laboratories. cystines with radioactiveiodoacetate, followed by iso- Low concentrations of exogenous thiols stabilize or optimize lation and sequencing of the modified tryptic peptides. enzyme activity (Ballard and Hanson, 1969;Utter et al., 1954; The results indicate that thedisulfide formed by equi- Hebda and Nowak, 1982),whereas disulfides promote loss of molar Nbsz lieswithin a 15-residueregion of the activity (Ballard andHopgood, 1976;Brinkworth et al., 1981). PEPCKsequence that includes Cys3”,Cys407, and Cys413.In addition, Cys407and/or Cys413also appear to The cytosolic enzyme from rat liver has 13 cysteines, all in participate in formationof the disulfide induced by8- the reduced state, among its 622 amino acid residues (Mr = azidoGTP. These thiols lie very near a consensus se- 69,289)’(Beale et al., 1985;Colombo et al., 1978).By incubation of the enzyme with equimolar levels of a hydrophobic quence that has been suggested to represent the binding maleimide, we recentlyidentifieda very reactivecysteine site for the guanine ring of GTP. residue of PEPCK (CysZm) that is rapidly and exclusively modified, with nearly completeloss of enzymaticactivity (Lewis et aL, 198913). Thiscriticalthiol lies between two Phosphoenolpyruvate carboxykinase (EC 4.1.1.32),here- consensus sequences for a nucleotide-binding site, and nuafter abbreviated as PEPCK,’ catalyzes the first committed cleotide substrates provide almost complete protection against with step of gluconeogenesis by producing phosphoenolpyruvate inactivation andmodification. These data are consistent the hypothesis that CysZm resideswithin or near theenzyme’s * This work was supported in part by Research Grant DMB 85- active site. 20311 from the National Science Foundation(to G.M.C.) and by Previous studies have suggested that several cysteines of research funds from the Veterans Administration (to J.M.S.). The PEPCK exist asvicinal thiol pairs (note thatvicinal is used costs of publication of thisarticle were defrayed in part by the herein to denote spatially proximal residues as opposed to payment of page charges. Thisarticlemusttherefore be hereby sequence) (Carlson et al., marked “aduertisement” in accordance with 18 U.S.C. Section 1734 residues that are adjacent in the 1978). When the homogeneous enzyme is treated with equisolely to indicate this fact. $ Recipient of the Doggett Predoctoral Fellowship fromthe College molar 5,5’-dithiobis(2-nitrobenzoate)(Nbs’), there is a very of Graduate HealthSciences, The University of Tennessee, Memphis. rapid and nearly complete loss of catalytic activity, accomPresent address: Depts. of Chemistry and Molecular Biology, The panied by the release of not 1 but 2 mol of thionitrobenzoate Scripps Research Institute, 10666 N. Torrey Pines Rd., La Jolla, CA (Nbs-)/mol of enzyme. These and other results suggest that 92037. $5 Present address: Veterans Administration Medical Center, 1030 Nbsp attacks a critical cysteine residue causing release of 1 mol of Nbs- and loss of enzymatic activity ( s t e p 1 of Scheme Jefferson Ave., Memphis, T N 38104. 1To whom correspondence should be addressed Dept.of Biochem1). Subsequently, a nearby cysteine residue reacts with the istry, University of Tennessee, 800Madison Ave., Memphis, T N mixed function disulfide to form an intramolecular cystine 38163. Tel.: 901-577-8302; Fax: 901-528-7360. The abbreviations used are: PEPCK, phosphoenolpyruvate car2 T h e residue numbering system usedherein is basedupon 622 boxykinase (GTP);PEP,phosphoenolpyruvate;HPLC, high performance liquid chromatography; Nbsz, 5,5’-dithiobis(2-nitroben- amino acids and includes the amino-terminal methionine as residue is or modified zoate); Nbs-, thionitrobenzoate; 8-N3GTP, 8-azidoGTP; DACM, N - 1. It is notclear whether the NHn-terminal Met cleaved beenconfirmed by amino(7-dimethylamino-4-methylcoumariny1)maleimide;PTH, phenyl- in vivo because itspresencehasnot terminal sequencing (Beale et al., 1985). thiohydantoin.

1628

1629

Vicinal Thiols of PEPCK

of PEPCK was determined spectrophotometrically using a molar extinction coefficient of 1.15 X IO6, and the PEPCK-catalyzed rates of phosphoenolpyruvate or oxaloacetate formation were determined spectrophotometrically at 25 "C as previously described (Colombo et al., 1978; Lewis et al., 1989b). Disulfide Formation by Nbs2-The reaction was initiated by the addition of Nbs2 to enzyme (4-10 p~ in buffer A) and was allowed to continue until the absorbance at 412 nm reached a plateau. The moles of thiol modified were calculated using an extinction coefficient at 412 nm of 14,150M" cm" for thionitrobenzoate (Riddles et al., 1983). Unless indicated otherwise, the molarity of sulfhydryl reagents is compared to enzyme concentration rather than to the molarity of cysteine residues. Aliquots of the reaction mixture were taken during the reaction to measure enzyme inactivation. Disulfide Formation by 8-N3GTP-PEPCK (5 p M ) was irradiated for 45 s with ultraviolet light in the presence of 12.4 pM 8-NaGTP in buffer A as previously described (Lewis et al., 1989a). After irradiation, aliquots of the reaction mixture were taken to measure enzyme inactivation, relative to enzyme that was irradiated in the absence of 8-N3GTP. Double Labeling with Radioactive lodoa~etate"[~H]- and ["Cliodoacetate were supplied by Du Pont-New England Nuclear. Immediately after modification of PEPCK byNbsz or 8-N3GTP, the reaction mixtures were rapidly mixed with a 22000-fold molar excess (to total thiols) of [3H]iodoacetate (1-1.5 Ci/mol), followed by the SCHEME 1 addition of solid urea to 4 M. Samples were incubated in the dark at 37 "C for 1 h, prior to precipitation of the modified enzyme by 10% disulfide,withrelease of the 2nd mol of Nbs- (step 2 of perchloric acid. After centrifugation, the supernatant was carefully Scheme 1).For convenience, we have designated the cystine removed and the pellet was resuspended and washed in 1.5 ml of 1 M disulfide that is formed by the reaction of PEPCK with Tris + 0.5 ml of 10 mM NH,HC03 (pH 8), 2 M urea. Precipitation of equimolar Nbsz (in the absence of any substrates) as "Cystine- the sample with 10% perchloric acid and washing of the pellet was 1." Evidence that the thiols involved in cystine bridge for- repeated three times; the final pellet was suspended in a solution that mation are initially proximal and are not juxtaposed due to contained 0.5 M Tris, 50 mM NH4HC03 (pH7.5), 8 M urea, and 1mM dithiothreitol. The sample was incubated under nitrogen at 37 "C in a n inhibitor-induced conformational change was gained by the dark for 2 h. The thiols constituting the reduced disulfide were using reagents relatively specific for vicinal thiols: low con- then alkylated with a 2-fold molar excess (to total thiols) of["C] centrations ofCd" and arsenite-dimercaptopropanol.The iodoacetate (0.2-0.4 Ci/mol) and incubated at 37 "C in the dark for 4 enzyme also appears to have at least two additional pairs of h. The carboxymethylated enzyme was then subjected to gel filtration in the presence of 10 mM NH4HC0, (pH7.5), 2 M urea. Aliquots were vicinal thiolsbecause titration with additional Nbsz results in t h e formation of at least two more cystine bridges. Thus, taken to measure 3H and "C incorporation, and protein concentration titration of PEPCK witha 3-fold molar excessof Nbsp results was measured using the Bio-Rad protein assay reagent; unmodified PEPCK in the presence of an equivalent concentration of urea was in the release of 6 mol of Nbs-/mol of enzyme, whether the used as theprotein standard. 3-foldmolarexcess of Nbse is added inthree equimolar Samples were counted in Cytoscint scintillant using the Full Specincrements or a t one time (Carlsonet al., 1978). trum Analysis program of aPackardTri-Carb 2000 scintillation In independent studies, we have shown that modification counter, which automatically corrects for counting efficiency of single of PEPCK by the photoaffinityprobe 8-N3GTP results in the or dual-labeled samples. After generation of quench curves using loss of 2 mol of thiol/mol of inactivated enzyme. 8-N3GTP sealed, quenched radioactive standards, control experiments demonfulfills all of the criteria for a n affinity label of PEPCK, strated that 3H, 14C,and 3H 14Csamples were accurately counted within 3% of predicted disintegrationslminute. suggesting that binding and modification occur specifically Proteolysis, HPLC, and Sequence Analysis-Modified enzyme was within the active site. A detailed analysisof the photoaffinity digested with TPCK-treated trypsin (2-4%)for 36 h, lyophilized, and labeling event revealed that 8-azidoGTP inactivates PEPCK redissolved in 0.1% trifluoroacetic acid. The tryptic peptides were bycausing oxidation of 2 cysteine residues, presumably purified by reversed-phase HPLC using a Waters system equipped throughformation of anintramolecularcystine disulfide with a Delta-pak C-18 column (300 A, 5 X 300 mm). Peptides were bridge. Thus, these resultsprovide evidence for the existence eluted in 0.1% trifluoroacetic acid at a flow rate of 1 ml/min using a multiphasic linear gradient of 0.1% trifluoroacetic acid in acetonitrile: ofa pair of proximalcysteine residues within the GTP0-5% from 10 to 20 min, 5 2 5 % from 20 to 150 min, 25-35% from binding site (Lewis et al., 1989a). 150 to 230 min, and 35580% from 230 to 265 min. Fractions of 2 min We now report the identification of vicinal thiol pairs of were collected, and aliquots of each fraction were counted to deterPEPCK using a strategy that involves specific formation of mine total 3H and "C radioactivity. Radioactive peaks were pooled intramolecular disulfides, followed by differential alkylation and repurified by reversed-phase HPLC using an appropriate linear gradient of 0.1% trifluoroacetic acid in acetonitrile. Radioactive peaks of cysteines with radioactive iodoacetate and isolation and sequencing of modified tryptic peptides. The identificationof were again pooled, lyophilized, and subjected to sequence analysis using an automatedpulse liquid sequencer (AppliedBiosystems model these cysteinesbegins t o define the GTP-binding domain and 477). One sample of a labeled tryptic peptide was also submitted to t o outline the framework of the enzyme's active site. the Harvard Microchem Facility for amino acid analysis. A portion of this sample was hydrolyzed in 6 N HC1 for 24 h and derivatized EXPERIMENTALPROCEDURES with phenylisothiocyanate using a 420A Derivatizer fromApplied Enzyme Preparation-PEPCK from rat liver cytosol was purified Biosystems. The resulting phenylthiocarbamyl amino acids were anat o homogeneity and toa high, constant specific activity (18-23 pmol lyzed by reversed-phase HPLC (Applied Biosystems model 130A). of oxaloacetate formed/min/mg at 25 "C) as previously described (Colombo et al., 1978; Lewis et al., 1989a). Immediately prior to each RESULTS experiment, exogenous thiols were removed by gel filtration in the Protection by Substrates againstModification by Nbs2presence of buffer A: 10 mM N-tris(hydroxymethyl)-methyl-2-aminoethanesulfonic acid (pH 7.2), 5% glycerol, and 0.5 mM EDTA. All Previousresultsdemonstratedthatphosphoenolpyruvate, buffers were degassedand saturated with nitrogen. The concentration IDP, and ITP could protect againstdisulfide bridge formation

+


1630

that was induced by treatment of PEPCK with equimolar Nbst (Carlson et al., 1978). To evaluate protection by G T P under the experimental conditionsemployed herein, we have assessed the ability of substrates to protect againstmodification of enzyme thiols by equimolar Nbst (Fig. 1). Inthe absence of substrates, the reaction of equimolar Nbsp with PEPCK resulted in rapid inactivation (not shown) accompanied by the loss of 2 mol of cysteine/mol of enzyme. The presence of phosphoenolpyruvate prevented formationof the intramolecular disulfide, but did not prevent modification of 1 cysteine residue. In contrast, nucleotides dramatically inhibitedtherate of thiol modification by equimolar Nbsp. Unlike M$+, free Mn2+ is an activating divalent cation for PEPCK; inclusion of micromolar levels of Mn2+inthe PEPCK assays stimulates the rate of phosphoenolpyruvate production and is nearly obligatory for oxaloacetate production (Colombo et al., 1981; Colombo and Lardy, 1981). We observed that protection afforded by GTP in the presence of excess Mn2+exceeded that provided by the nucleotide alone. Double Labeling of Modified PEPCK-Faithful trapping of original disulfides and prevention of disulfide exchange is of critical importance in any attempt toidentify disulfide bonds inproteins. Of the two general strategies developed for quenching disulfide exchange, alkylation or acidification of protein thiols, alkylation has the advantage of .being completely irreversible (Creighton, 1984). The strategy we have developed to identify the vicinal thiol pairs of PEPCK is outlined in Scheme 1. The disulfide bond is formed in the native enzyme, either by reaction withequimolar Nbsp as shown, or with 8-N3GTP (Lewis et al., 1989a). After completion of disulfide formation, the mixture is rapidly quenched with at leasta 2000-fold molar excess (to total thiols)of [3H] iodoacetate in the presenceof a denaturant (step 3 of Scheme 1).Denaturant is always added after excess [3H]iodoacetate t o avoid promotion of disulfide exchangefacilitated by protein unfolding (Creighton, 1984). The alkylation is terminated by precipitation of the modified enzymewithperchloricacid, and excess [3H]iodoacetate is removed by repeated cycles of precipitationandresuspension of theprotein pellet. The resolubilized protein is then treated with dithiothreitol at alkaline pH to reduce any disulfide bonds, and subsequent alkylation with a slight excess of ['4C]iodoacetate traps the

newly formed cysteines (step 4 of Scheme 1). This protocol dictates that any thiol not participating in a disulfide bond be 3H-labeled, whereas vicinal cysteines that are induced by Nbsz or 8-NaGTP to form an intramolecular disulfide would be 14C-labeled.Furthermore, anycarboxymethylcysteine bearing both a 3H and a I4C label would reflect the occurrence of either disulfide exchange or nonspecific disulfide formation; this double-labeling technique thus permits a measurement of the extent to which these phenomena occur. Control experiments using enzyme that had beenmodified by equimolar Nbsp showed that only four to five thiols were rapidly alkylated by excess iodoacetate; however, the addition of 4 M urea was sufficient to cause rapid alkylationof all remaining thiols (data not shown). The resultsof the reactionof PEPCK with equimolar Nbs, are shown in Table I; as usual, the modification was accompanied by a nearly complete loss of activity, with release of 1.95 mol of thionitrobenzoate/mol of enzyme. As predicted, 2.0 mol of ['4C]iodoacetate were incorporated/mol of enzyme, whereas 13 mol of [3H]iodoacetatewere incorporated/mol of which would PEPCK. Alkylation of residues other than thiols, result in theincorporation of more than 11 mol of [3H] iodoacetate/mol of enzyme, is not unexpected inasmuch as a largeexcess of [3H]iodoacetate was used under denaturing conditions. Indeed, as discussed below, we obtained evidence for alkylation of a histidine residue by [3H]iodoacetate. Double-labeling of PEPCK that hadbeen modified by a 2fold molar excess of Nbsz also occurred as predicted (Table I). Treatmentof the enzyme with 2 molar equivalents of Nbsp resultedinthe nearlycompleteloss of catalyticactivity, accompanied by the release of 3.75 mol of Nbs-/mol of enzyme. Again, the moles of ['4C]iodoacetate incorporated/mole of enzyme were nearly identical to the moles of thiol modified as measuredspectrophotometrically. Theappropriate decrease in the number of cysteines modified by [3H]iodoacetate is also consistent with the formationof two cystine disulfides. Irradiation of PEPCK in the presence of 12 P M 8-N3GTP resultedin 70% inactivation, relative to enzyme that was irradiated in the absence of the photoprobe (Table I). These data are consistent with published results, in which we reported a similar loss in catalytic activity thatwas not accompanied by significant covalent incorporation of radioactive 8N3GTP (Lewis et al., 1989a). Our conclusion that photoinactivation of PEPCK by 8-N3GTP is caused by the formation 2.0 of an intramolecular cystine disulfide bridgeis consistent with theextent of incorporation of [3H]-and [14C]iodoacetate shown in Table I. Eleven thiols were modified by [3H]iodoacetate, whereas 1.9 mol of [14C]iodoacetate were incorporated/ mol of enzyme, Because onlyoxidized thiols would be labeled by [14C]iodoacetate, these results imply that approximately two thiols were induced t o form a cystine disulfide in the 8N3GTP-modifiedenzyme. Although somewhat greater than2 mol of cysteine were modified per mole of inactivated enzyme, the explanationfor this resultbecame apparent upon analysis of the labeled tryptic peptides. Reversed-phase HPLC of Tryptic Digests ofModified PEPCK-A typical reversed-phase HPLC peptide map of a tryptic digest of modified PEPCK is shown in Fig. 2 A . For I each experiment, aliquots of each fraction were counted to 0 5 25 12 00 15 determine the total 3H and 14Ccontent using the full spectrum Time (min) isotope analysis programdescribed under "Experimental ProFIG. 1. Thiol titration with equimolar Nbsz. PEPCK (5.0p M ) cedures." The elution profile of radioactive tryptic peptides was incubated with equimolar Nbsp in buffer A in the absence of derived from PEPCK that had been modified by equimolar substrates (0),or in the presence of 5 mM PEP (A),1 mM GTP (H), Nbsz displayed a number of radioactive peaks that arelabeled or 2 mM MnClz + 1 mM GTP (0).T h e absorbance at 412 nm was monitored with time andwas measured against a blank that contained alphabetically inorder of elution (Fig. 2B). Most of the radioactive peaks contained predominantly or exclusively tritonly Nbss and buffer A.

1


1631

TABLE I Extent of inactivation and double-labeling of PEPCK PEPCK was modified by 8-N3GTP or by 1 or 2 mol equivalents of Nbs?. At the termination of each reaction, the PEPCK-catalyzed rate of oxaloacetate formationwas assayed, and themodified enzyme was labeledwith [3H]- and [14C]iodoacetateas described under “Experimental as the mean ? S.E. for duplicate or triplicate analyses of representative experiments. Procedures.” The results are expressed PEPCK modified by eauimolar Nbs,

9; inactivation

(Mol of [3H]iodoacetate)/(molof PEPCK) (Mol of [“C]iodoacetate)/(mol of PEPCK) (Mol of Nbs- released)/(mol of PEPCK)

+

PEPCK modified by 2 mol equivalents of Nbsp

PEPCK modified by 8-N,GTP

96.2 0.3 13.0 f 0.4

94.2 70.2f 0.4 8.7 11.0k 0.2

k 1.2

* 0.2

3.7 f 0.1 1.9

f 0.1

2.0

1.95 f 0.01

f 0.4

3.75 f 0.04

ium label. In contrast, most of the 14C label was confined to isolated peptides, in that the yields of contaminating P T H of labeling by derivatives were typically negligible. The peptidepurified the two peaks designatedC and H. The pattern ~ ~ ~ ) an Arg-Pro sequence, [‘4C]iodoacetate was very reproducible, including some exper- from peak C ( G l ~ ~ ” - A r gcontains iments in which unlabeled iodoacetate was substituted for which is usually resistant to tryptichydrolysis. Although the [’HHIiodoacetate. It should be emphasized that the label de- peptides obtained from peaks E (LysZ9-Gly4’)and F (Lys71rived from [“C]iodoacetate was not distributed throughout each begin with a lysine residue, this unusual pattern the carboxymethylated peptides, indicating that widespread of tryptic cleavage is explained by the fact thateach of these disulfideexchange didnot occur underourexperimental peptides is immediately preceded by a lysine or an arginine conditions. Peaks C and H clearly contained the majority of residue within the amino acid sequence. Each experimental the 14C label, suggesting that the half-cystines making up sequence matches exactly that predicted from the sequence Cystine-1 are found within these labeled peptides; however, of PEPCK if the unidentifiedresidues X and are substithese two peaks also contained 3H label as well. Cleavage of tuted by cysteines and histidines, respectively (Beale et al., PEPCK by trypsin isexpected to yield 10 cysteine-containing 1985). These results allowed us to unequivocally identify 11 peptides,three of which shouldcontain 2 cysteineseach carboxymethylated cysteines within the PEPCK sequence, as (Beale etal., 1985). indicated in Table I1 and Fig. 2. No P T H derivatives were detected in those cycles desigThe elution profile of radioactive tryptic peptides derived from PEPCK that had been modified by a 2-fold molar excess nated with a dash (-). In each case, a histidine residue was of Nbsp (Fig. 2 C ) displayed a similar pattern of radioactive predicted based on the amino acidsequence, but we were peptides. Again, peaks C and H bore the majority of the I4C unable to determine from the elution profile of the PTH label, and in fact, the ratio of 14C/3H radioactivity in these derivatives whetherthesehistidine residues were carboxytwo peaks was increased in Fig. 2C as compared to Fig. 2B methylated or were unmodified. Although we anticipated that (Table 11). In addition, the amount of14C radioactivity was measurement of the total radioactivity recovered from each D, and toa lesser extent, sequencing cycle would clarify the extent of [3H]- or [“C] increased in the peaks designated B, F. In contrast to Fig. 2B, the elution profile of radioactive carboxymethylation of each aminoacid, we found instead that tryptic peptides displayed in Fig. 2C exhibited a prominent most of the 3H and 14Cwas retained on the cartridge seal and peak of radioactivity (labeled I) that was not retained by the sample filter onto which the peptides were applied for sereversed-phase HPLC column. Peak I was also apparent in quencing.Althoughsomeradioactivity was released at the the elution profile of radioactive tryptic peptidesderived from appropriate cycles for each peptide, the total countswere not PEPCK that had been modified by 8-N3GTP (Fig. 20). The sufficiently high to permita statistical analysisof the ratioof majority of the I4C label in Fig. 2 0 coeluted with peaks I and labeling for a given amino acid, particularlywithinthose H. tryptic peptides that containedmore than 1 cysteine, or that Thus, peaks C, H, and I were targeted as containing pep- contained histidines aswell as cysteines. tides with half-cystine residues; these peaks, in addition to Sequence analysis of the peptide derived from the tritiummost of the other radioactive peaks, were subjected to further labeled peak 1-1that was purified by anion-exchange HPLC purification. PeaksA through H were each pooled, lyophilized, (Fig. 3) yielded the following sequence. and purified by reversed-phase HPLC using an appropriate Ser-Glu-Ala-Thr-Ala-Ala-Ala-Glu- -Lys (Sequence 1) expanded linear gradient of 0.1% trifluoroacetic acid in acetonitrile (data not shown). Peak I, in contrast, was purified This sequence matches that of the tryptic peptide Ser4‘j2by first desalting the lyophilized fractions over a column of L Y S ~of ~ ’PEPCK if the blank at cycle 9 is replaced by a Bio-Gel P-2 equilibrated in 10 mM ammonium acetate (pH histidine residue (position 470, Beale et al., 1985). It thus 7.5), followed by anion-exchange HPLC (PL-SAX 1000 A, 8 appears that the tritium label derived frompeak I in Fig. 2, C pm, 50 X 4.6 mm, PolymerLaboratories) using a linear and D was caused bythe carboxymethylationof His470in this gradient of 10-500 mM ammonium acetate (pH7.5). As shown acidic tryptic peptide. The I4C-labeledpeak 1-2 that was in Fig. 3, the anion-exchange HPLCcolumn resolved peak I purified by anion-exchange HPLC (Fig. 3) yielded no detectinto two distinct radioactive peaks, one containing predomi- able sequence in one case, and in another preparation yielded nantly 3H label (peak I-1 ) and the other containing predom- exclusively the following sequence. inantly label (peak 1-2).Each radioactive peak was pooled, Ile-Glu-Gly-Glu-Asp-Ser-Ala (Sequence 2) lyophilized, and subjected to sequence analysis. Sequence Analyses of the Purified Labeled Peptides-The Because of diminishing yields, no P T H derivatives were desequence of each of the purified peptides labeled in Fig. 2 was tected beyond the seventh cycle of sequencing. This sequence determined as described under “Experimental Procedures” overlapswith that of thetrypticpeptide IleS44-Lys551of (Table 11). The sequence analyses confirmed the purityof the PEPCK (Beale et al., 1985). We considered the possibility ‘I-”

1632

Vicinal Thiols of PEPCK 1.o

A

A210 0.5

B

2500 FIG. 2. Reversed-phase HPLC of the tryptic digests of modified PEPCK. The tryptic digest of modified, double-labeled PEPCK was analyzed by reversed-phase HPLC as described under“ExperimentalProcedures.”Peptides were detected by their absorbance at 220 nm; fractions of 2 min were collected and aliquots were counted to decontent. termine 3H (0) and ‘*C).( Panel A , a representative elution profile of modified, double-labeled enzyme. Panel B , tryptic peptide mapof PEPCK modified by equimolar Nbsp anddoublelabeled. Panel C , tryptic peptide map of PEPCK modified by 2 molar equivalents of Nbs2 and double-labeled. Panel D, tryptic peptide map of PEPCK modified by 8-N3GTP anddouble-labeled.

2000

1500 1000 500

0

C

400

300

I

20 0 100

0 3000 2500 2000

H

c y s 38 c y s 46

D

Cy6 245

E

Cys 192 Cy6 198

P sr,::

G

:: .*

c y s 407 Cyr 413

Y

1500 1000 500 0

D-

Fraction

120

1633


TABLEI1 Sequence analysis of modified peptides Samples of each of the labeled peptides from Fig. 2B were sequenced as described under "Experimental Procedures." The ratio of I4C to "H radioactivity is shown for each radioactive HPLC peak purified from a tryptic digest of PEPCK that had been double-labeled after incubation with equimolar,or a 2- or 3-fold molar excess of Nbss. [Nhs,]/[PEPCK] Peak

Amino acid sequence

3

1

3'

Carhoxymethylcysteine

pmol "Cfpmol 3H

F P G X O X F A L R E W R P Q A"" Y L A A A K F V E G Y I K Y D N X X L H S V F X T P A A W a

M K D P F N W G S E

E E P X

0.24 0.18 0.70

0.22 0.43 1.5

0.11 0.42 2.0

Cys'33 cys245 cy299

0.26 0.13

0.60 0.21

0.81 0.30

0.17 0.11 1.1

0.50 0.29 1.1

0.70 0.38 1.7

Cys2M Cys38 Cy@ cy25 Cys'98Cys'92, Cys407 CYS413

N S R P A I L X Q

S Q X A P X S P

A L D L L P E

X G K X Q P E G...b T D P R P L K I I D P G...b

PTH derivatives that could not be detected oridentified are indicated by a dash (-) Sequencing was discontinued after 20 cycles.

or an X .

TABLE 111 Amino acid analysis of the "C-labeled tryptic peptideI-2 A sample of the purified tryptic peptide was subjected to amino acid analysis by the Harvard Microchem Facility as described under "Experimental Procedures." 1.0 1-1

Fraction

1.3

Amino acid

Pmol found

Asx Glx Ser GlY His Arg Thr Ala Pro TYr Val Met PECys" Ile Leu Phe LYs

89 149 119 136 4 15 28 112 0 0 0 35 0 87 0 32 43

Residues found

1.7 1.5 0.0 0.2 0.3 1.3 0.0 0.0 0.0 0.4 0.0 1.0 0.0 0.3 0.5

FIG. 3. Ion-exchange HPLC of peak I. After reversed-phase HPLC, peak I was pooled and desalted prior to anion-exchange HPLC using a linear gradient of 10-500 mM ammonium acetate (pH 7.5). Aliquots of each fraction were counted to determine 3H (0)and I4C (D)content. Detection of a significant corresponding peak in absorbance at 220 nm was difficult due to a large background subtraction from the ammonium acetate gradient. a Detected as S-(pyridylethy1)cysteine.

Residues predicted

1 2 1 1 0 0 0 1 0 0 0 0 0 1 0 0 1

that this sequenced tryptic peptide simply represented an contains a histidine, which if carboxymethylated, may account acidic, contaminating peptide that was also not retained by for a significant fraction of the 3H label in this peptide; thus, the reversed-phase HPLC column and that coincidentally the ratio of 14C to 3H label in Cys3" may be higher than the comigrated with the 14Cradioactivity on the anion-exchange value listed for peak C in Table 11. Identification of the halfHPLC column; however, amino acid analysis of a different cystinesthat compose the second andthird disulfides is sample of this purified peptide derived from another prepadifficult because several peptides display an increase in the ration of modified PEPCK was in excellent agreement with ratio of 14C/3Hradioactivity. For example, the peptides conthisassignmentand showed nosignificantcontaminating taining C Y S ~Cys245, ~, and Cyszss showed an increased incoramino acids (Table 111).We are therefore confident that Ile544poration of [14C]iodoacetate when PEPCK was treated with Lys551represents the14C-labeledpeptide of peak 1-2, although 2 equivalents of Nbs,. These results suggest that some disulthe question remains as towhich residue was labeled by ["C] iodoacetate and why it was not significantly labeled by [3H] fide exchange may have occurred between formation of the disulfides and quenching by [3H]iodoacetate. Nevertheless, iodoacetate. The identification of the labeled peptides provides a more based on theincrease in [14C]iodoacetate incorporation, Cys7', and CysZMare obvious candidates for half-cystines of clear picture of the thiols that participate in intramolecular disulfide bond formation. The sumof the data indicates that the second disulfide formed by 2 equivalents of Nbs2. SimiCys407or Cys413may participate in the disulfide formed the thiols that are oxidized by equimolar Nbsz are C Y S ~ ~larly, , with the 3rdmolar equivalent of Nbsz. Cys407 and/or Cys413. Theincorporation of nearlyequal The HPLC tryptic peptide map of enzyme that had been amounts of [3H]- and[14C]iodoacetate into the peptide purified from peak H suggests that only 1 of the cysteines in this modified by 8-N3GTP (Fig. 2 D ) consistently displayed lower peptide participated indisulfide bond formation. It should be 14C/3Hratios than did the Cystine-1 peptide map (Fig. 2 B ) , emphasized that the tryptic peptide containing Cys3= also although the stoichiometry of labeling in both cases indicated

1634


that approximately two thiols were oxidized per enzyme mol- sum of these results is consistent with the hypothesis that the ecule. Nevertheless, because the majority of [14C]iodoacetate thiols that arerapidly modified by equimolar Nbsz lie within was incorporated intopeak H, we propose that the cysteine(s) or near theenzyme's active site. modified by 8-NZGTP include Cys407and/or Cys413.The trypDisulfide formation that is induced by irradiation in the tic peptide Ile644-Lys551 from peak 1-2 of Fig. 3 is apparently presence of 8-N3GTP isalso accompaniedby a lossin catalytic a n unusual artifact of labeling by [14C]iodoacetate, inasmuch activity, and published data established a direct correlation as it was also present in peptide maps derivlep from PEPCK between the loss in activity and themodification of approxithat was modified by 2 and 3 equivalents of Nbsz (Fig. 2C and mately two thiols/mol of inactivated enzyme (Lewis et al., data notshown). Again, alkylation of non-thiol residues could 1989a). Those results also demonstrated that the inactivation caused by ultravioletirradiation alone explain greater incorporationof radioactive iodoacetate than of PEPCKisnot because inactivation was assessed relative to control enzyme was predicted from the number of cysteines modified. The 2 cysteine residuesof PEPCK thatwere not identified, that was irradiated in theabsence of 8-N3GTP. Furthermore, fulfills all of CysZ1' and Cys307,presumably occur within the minor radio- because those studies established that 8-N3GTP active peaksof Fig. 2 containing solely, or predominantly, 3H- the criteria of a specific affinity label for PEPCK, it islikely labeled peptides. Onlythe most prominentlylabeled peptides, that themodified cysteines lie within or near the GTP-binding especially those bearing substantial 14C label, were isolated site. Our characterizationof the sulfhydryl chemistry of PEPCK and sequenced. The absence of significant incorporation of [14C]iodoacetate into many of the radioactive peaks in Fig. 2 and the identification of the ['4C]carboxymethylatedhalfsuggests that several cysteines, including Cys3', Cys4'j,C Y S ' ~ ~ ,cystines of Cystine-1 asCys3", Cys407,and/or Cys413implicate active site residues, and also CYS'~', and CYS'~*,do not participate in disulfide bond for- thesecysteinesaspotential suggest that 2 of these residues exist as vicinal thiol pairs. mation under anyof the experimental conditions tested. The possibility that all3 cysteines are spatiallyproximal, and that Cystine-1is defined by a disulfide bond between any two DISCUSSION of the three thiols seems unlikely based on stereochemical Modification by Nbsz that resultsin the release of 2 mol of constraints (Creighton, 1978). The same thiol(s),Cys407and/ thionitrobenzoate/mol of reagent has been considered an in- or Cys413, were apparentlytargeted by bothNbszand 8dication of dithiol pairs in proteins. Previous data (Carlson N3GTP, a result that is not surprising considering the intiet al., 1978) in support of the hypothesis thatmodification of matecorrelation between inactivationand disulfide bond PEPCK with limiting Nbsz causes cystine bridge formation formation that exists in each case. The 3 cysteines lie relainclude the following. 1) Modification of homogeneous en- tively close together within a hydrophobic region of the enzyme with equimolar Nbsz always resulted in twice as many zyme sequence (Beale et d., 1985). More importantly, these moles of Nbs- released as moles of Nbsz added. 2) Two mol thiols lie very near a consensus sequence thathas been of 4-thiopyridone were also released/mol of 4,4'-dithiodipyrsuggested to represent the binding site for the guanine ring idine added, even if 2 or 3 equivalents of 4,4'-dithiodipyridine portion of the nucleotide: A ~ n ~ ~ - L y s - G l u - (Fig. T r p ~4,~Cook ~ were used. 3) Reagents that are commonly presumed diagnos- et al., 1986). Two of the cysteinesimplicated in disulfide bond tic for avicinal dithiol (arsenite-dimercaptopropanol and formation by the second molar equivalent of Nbsz also lie Cd2+) were potentinhibitors of PEPCK activity. 4) The near consensus sequences for a GTP-binding site. Cys245is 2 addition of sodium dodecyl sulfate and excess Nbsz never resulted in greater than 13 cysteines modified, and this was MPPQLHNGLDF~AKVIQGSLDSL@EVRKFVEGN@ 35 identical to the number of cysteines determined by amino QLCG@YIHI@DGSE@EYGRLLAHMQEEGVIRKLK 70 acid analysis (Colombo et al., 1978) and sequence analysis of KYDNCWLALTDPRDVARIESKTVIITQEQRDTVPI 1 05 the cloned PEPCK gene (Beale et al., 1985). Our current data PKSGQSQLGRWMSEEDFEKAFNARFPGCMKGRX 1 4 0 confirm that treatment of PEPCK with equimolar Nbsz reVIPFSMGPLGSPLAKIGIELTDSPYVVASMRIMTR 175 sults in the rapid release of 2 mol of Nbs-/mol of enzyme. In addition, these new data show that doublelabeling of the MGTSVLEALGDGEFIKCLHSVGCPLPLKKPLVNNW 210 modified enzyme under conditions inwhich each half-cystine ACNPELTLIAHLPDRREIISF-NSLLGK~KC 245 is carboxymethylated by [14C]iodoacetate causes the incorpoFALRIASRLAKEEGWLAEHMLILGITNPEGKKKYL 280 ration of 2 molof ['4C]iodoacetate/mol of enzyme, results AAAFPSACGKTNLAMMNPTLPGWKVECVGDDIAWM 315 that are entirely consistent with the formation of a cystine NLRAINPENGFFGVAPGTSVKTNPNAIKT 350 disulfide bridge, asillustratedinScheme 1. It should be IQKNTIFTNVAETSDGGVYWEGIDEPLAPGVTITS 385 emphasized that the appropriate number of thiols were carW K N K E W R P Q D E E P C A H P N S R F ' C T P A S Q ' C P I I4D2 P0 A W boxymethylated by [14C]iodoacetate and that the total incoror equal poration of iodoacetate was consistently greater than ESPEGVPIEGIIFGGRRPAGVPLVYEALSWQHGVF 4 5 5 t o 13 mol/mol enzyme (Table I). These results, as well as the VGAAMRSEATAAAEHKGKVIMHDPFAMRPFFGYNF 4 90 limited distribution of [14C]iodoacetate, suggest that quenchGKYLAHWLSMAHRPAAKLPKIFHVNWFRKDKNGKF 525 ing by alkylation of free cysteines was complete and attest to LWPGFGENSRVLEWMFGRIEGEDSAKLTPIGYVPK 5 60 the validity of the double-labeling method. EDALNLKGLGDVNVEELFGISKEFWEKEVEEIDKY 595 Inactivation by equimolar Nbsz usually occurs too rapidly L E D Q V N A D L P Y E I E R E L R A L K Q R I S Q M t o allow correlation of the extent of disulfide formation with the loss in activity, but itis clear from Table I that formation FIG. 4. Amino acid sequence of PEPCK from rat liver cyof one disulfide bridge results in the nearly complete loss of tosol. Regions of interest are noted as follows: bold, CysZm;*, bold, catalytic activity. Different substratesfor the enzyme provide Cys3", Cys407,and Cys413;triple underlined, consensus sequence for a many ATP-bindingproteins different degrees of protection against modification by Nbsz; phosphorylbindingsitecommonto (Hannink andDonoghue, 1985);boxed, consensus sequences for phosphosphoenolpyruvate prevents formationof the disulfide, but phoryl binding sites of GTP-binding proteins (Cook et a/., 1986); does not preventmodification of 1cysteine. In contrast, GTP double underlined, consensus sequencefora guanine binding site; provides much greater protection against the rateof disulfide circled, putative phosphoenolpyruvate-binding domain (Cook et al., bridge formation, and MnGTP is even more effective. The 1986); solid underlined, hydrophobic regions (Beale et al., 1985). "


1635

TABLEIV residues removed fromaglycine-richsequence that often Sulfhydryl chemistry of PEPCK representsthebindingsite for thephosphorylportion of The characteristics of PEPCK's 13 cysteines and the probable adenine and guanine nucleotides (Hannink and Donoghue, 1985; Cook et al., 1986), andCys2=, previously identified as a participation of each thiol in disulfide bond formation by 8-N3GTP or by Nbs2 (upon incubation of the enzyme with one, two or three critical, hyperreactive thiol (Lewis et al., 1989b3, lies between molar eouivalents of Nbs,) are listed. this consensus sequence and a second consensus sequence for Description Cysteine residue a phosphoryl binding site that is common to GTP-binding Does not participate in disulfide formation" 38 proteins (Fig. 4,Cook et al., 1986). Does not participate in disulfide formation" 46 As discussed in the Introduction, we have reported that May participate in the 2nd disulfide formed 75 CysZM canbe rapidly and exclusively modified, with nearly by 2 eq of Nbsz complete loss of enzymatic activity,by incubation of PEPCK Does not participate in disulfide formation' 133 with equimolar levels of a hydrophobicmaleimide. Again, Does not participate in disulfide formation" 192 Does not participate in disulfide formation" 198 nucleotide substrates provide almost complete protection No information available 212 against inactivation and modification of this cysteine (Lewis May participate in the 2nd disulfide formed 245 et al., 1989b). Based upon a considerable sum of data, which by 2 eq of Nbs2 is summarized only briefly below, we had previously raised 288 Hyperreactive, pK, = 7, specific modificathe possibility that CysZrnmight in fact be 1 of the cysteines tion causes nearly complete inactivation, modified by equimolar Nbsz in the prest h a t forms the disulfide bond induced by equimolar Nbsz or ence of phosphoenolpyruvateb andmay by 8-N3GTP (Carlsonet al., 1978; Lewis et al., 1989a, 1989b). participate in the 2nd disulfide formed For example, the modification of C Y S ' ~ by the hydrophobic by 2 eq of Nbsp maleimide N-(7-dimethylamino-4-methylcoumarinyl)male307 No information available imide (DACM) and the inactivationof PEPCK by equimolar 399 Participates in Cystine-1 Nbsz or by 8-N3GTP all share common characteristics. In 407/413 One of the two thiols participates in Cystine-1, one or both of the thiols particieach case, the enzyme's three different catalytic activities are pates in thedisulfide formed by 8decreased in parallel, theresidual activity isvery low, and the NSGTP, both oxidize upon treatment relationship between fractionalinactivationandfractional with 3 eo of Nbs9 of substrate modification is 1:l. Inaddition,thepattern Up to formationof three disulfides/molecule of PEPCK. protection against inactivation and modification by equimolar Lewis et al., 1989b; Carlson et al., 1978. Nbsz and DACM is very similar, and the rates of enzyme inactivation are in both cases extremely rapid. Finally, modification of PEPCK with equimolar Nbsz in the presence of dry1 chemistry is presented in Table IV. Our identification of the substrate phosphoenolpyruvate causes modificationof one these critical dithiols begins to define the folding pattern of thiol, but does not result in disulfide bridge formation (Fig. 1, the polypeptide backbone of PEPCK, andalso targets particCarlson et al., 1978). The cysteine modified by Nbs2 in the ular regions of the enzyme as likely binding or catalytic domains. In thisregard, it is noteworthy thatnearly all of the presence of phosphoenolpyruvate was not directly identified but was estimated to lie approximately 44% of the distance oxidized thiols identified in this study lie within the middle et al., 1978); CysZM, at 46% one-third of the PEPCKsequence. Vicinal cysteine pairs that from the amino terminus (Carlson of the distance, is theclosest of all of the 13 cysteines to this are apparently critical for catalysis have been described in a location. In spite of these previous data, our resultsclearly do number of enzymes including the ATP-dependent PEPCK from rabbit not implicate CysZrnas a participant in disulfide bridge for- from yeast (Cardemilet al., 1990), pyruvate kinase muscle (Annamalai and Colman, 1981), and mevalonate 5mation induced by equimolar Nbsz or by 8-N3GTP; rather, Cys2= appears to undergo oxidation in the absence of phos- diphosphate decarboxylase from chicken liver (Alvear et al., phoenolpyruvate only upon reaction with 2 or 3 equivalents 1989), and it has been suggested that vicinal thiols may be a common feature of enzymes having phosphate-binding sites of Nbsz. The cysteines that are apparentlymodified by equi(Rippa et al., 1981). The identity of the critical cysteines of molar Nbs2 in the absence of substrates, Cys3" and Cys407/413, PEPCK will be valuable for the preparation of heavy-atom lie 64, 65%, and 66%, respectively, of the distance from the derivatives of the enzyme for x-ray crystallographic analyses; amino terminus; these thiols are thereforevery unlikely can- determination of the enzyme's three-dimensional structure didates for the cysteine modified by Nbs2 in the presence of will undoubtedly clarify the location of the proximal cysteines phosphoenolpyruvate. In summary, although CysZMis exclu- with respect to the active site and may begin to explain how sively modified by DACM in the absence of substrates and thiol modificationcauses such a dramatic loss in catalytic apparently by equimolar Nbsz in the presence of phosphoen- activity. olpyruvate, the cystine disulfide induced by equimolar Nbsz in the absence of substrates apparently consists of Acknowledgments-We thank Dr. Bill Lane and colleagues of the Cys407and/or Cys413,and does not containCysZR8. One conclu- Harvard MicrochemistryFacility for excellent technical assistancein sion that emerges from our data then, is that different thiols peptide sequencing and aminoacid analysis. of PEPCK can be specifically modified under different experREFERENCES as inclusionof substrates. More Alvear, M., Jabalquinto, A. M., and Cardemil, E. (1989)Eiochim. Biophys. Acta imentalconditions,such 9 9 4 , 7-11 importantly, although different cysteines are modified under A. E., and Colman, R. F. (1981) J Biol. Chem. 256, 10276-10283 different circumstances, each modification event is at least Annamalai, Ballard, F. J., and Hanson, R. W. (1969) J . Eiol. Chem. 244,5625-5630 partially prevented by inclusion of substrates and is charac- Ballard, F. J., and Hopgood, M. F. (1976) Biochem. J. 1 5 4 , 717-724 E. G., Chrapkiewicz, N. B., Scoble,H. A., Metz, R. J., Quick, D. P. terized by a dramatic loss in catalytic activity that correlates Beale, Noble, R. L., Donelson, J. E., Biemann, K., and Granner, D. K. (1985) J.' with the extentof modification. One possible explanation for Biol. Chem. 260,10748-10760 R. I.. Hanson. R. W.. Fullin. F. A,. and Schramm. V. L. (1981),~J . these results is that a cluster of cysteine residues lies within Brinkworth, Biol. Chem. 266,10795-10802 or near the active site of PEPCK or at such a location that Cardemil E., Encinas, M. V., and Jabalquinto, A. M. (1990) Biochim. Biophys. Acta 1040.71-76 their modification prevents or severely impairs catalysis. Carlson, G. M., Colombo, G., and Lardy, H. A. (1978) Biochemistry 1 7 , 5329A brief summary of existing knowledge of PEPCK's sulfhy5338

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Colombo G., and Lardy H. A. (1981) Biochemistry 20,2758-2767 Colombo, G . , Carlson, G. M., and Lardy, H. A. (1978) Biochemistry 17, 53215329 Colombo, G., Carlson, G. M., and Lardy, H. A. (1981) Biochemistry 20, 27492757 Cook, J. S., Weldon, S. L., Garcia-Ruiz, J. P., Hod, Y., and Hanson, R.W. (1986) Proc. Natl. Acad. Sci. U. S. A. 83,7583-7587 Creighton, T. E. (1978) Prog. Biophys. Mol. Biol. 33,231-297 Creighton, T. E. (1984) Methods Enzymol. 107,305-329 Hannink, M.,and Donoghue, D. J. (1985) Proc. N&L Acad. Sci. U. S. A . 82, 7894-7898 Hehda, C. A., and Nowak, T. (1982) J. Biol. Chem. 257,5503-5514

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