ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS

Vol. 267, No. 1, November 15, pp. 38-45,1988

Affinity Labeling of Saccharomyces cerevisiae Phosphoenolpyruvate Carboxykinase with the 2’,3’-Dialdehyde Derivative of ATP’

CLAUDIA SAAVEDRA, SANDRA ARANEDA, AND EMIL10 CARDEMIL’

Departamento de Q&mica, Facultad de Ciencia, Universidad de Santiago de Chile, Gas&z 5659, Santiago 2, Chile

Received March 29,1988, and in revised form July 5,1988

Saccharcwnyces cerevisiae phosphoenolpyruvate carboxykinase [ATP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.491 is completely inactivated by the 2’,3’- dialdehyde derivative of ATP (oATP) in the presence of Mn’+. The dependence of the pseudo-first-order rate constant on reagent concentration indicates the formation of a reversible complex with the enzyme (Kd = 60 f 17 PM) prior to covalent modification. The maximum inactivation rate constant at pH 7.5 and 30°C is 0.200 + 0.045 min-‘. ATP or ADP plus phosphoenolpyruvate effectively protect the enzyme against inactivation. oATP is a competitive inhibitor toward ADP, suggesting that oATP interacts with the enzyme at the substrate binding site. The partially inactivated enzyme shows an unal- tered K, but a decreased Vas compared with native phosphoenolpyruvate carboxyki- nase. Analysis of the inactivation rate at different H+ concentrations allowed estimation of a pK, of 8.1 for the reactive amino acid residue in the enzyme. Complete inactivation of the carboxykinase can be correlated with the incorporation of about one mole of [8- 14C]oATP per mole of enzyme subunit. The results indicate that oATP can be used as an affinity label for yeast phosphoenolpyruvate carboxykinase. o 198s Academic PM, I~C.

Yeast phosphoenolpyruvate carboxyki- nase (PEPCK)3 [ATP:oxaloacetate car- boxy-lyase (transphosphorylating), EC 4.1.1.491 catalyzes the reversible decarbox- ylation of oxaloacetate in the presence of ATP and Mnzf to yield phosphoenolpyru- vate, bicarbonate, and ADP. The enzyme plays an important role in the control of gluconeogenesis and in most animal spe- cies it requires guanosine or inosine nucle- otides (1); the carboxykinases from yeast (2), plants (1, 3), and Trypanasoma cruxi (4) are specific for adenine nucleotides. The molecular masses of most PEPCKs are

’ This work was supported by research grants from DICYT-USACH and FONDECYT-CHILE.

*To whom all correspondence should be addressed. 3 Abbreviations used: PEPCK, phosphoenolpyru-

vate carboxykinase; oATP, 2’,3’-dialdehyde derivative of ATP; PEP, phosphoenolpyruvate; Hepes, N-2-hy- droxyethylpiperazine-N’-2-ethanesulfonic acid.

about 70 kDa (l), whereas the molecular mass of the yeast enzyme is 261 kDa, and it is composed of four 64.3-kDa subunits (5). The presence of quaternary structure has also been reported for PEPCK isolated from C4 plants (3) and from !l’. cruzi (4).

Considerable information exists about the kinetic mechanism and stereochemis- try of several carboxykinases (1, 6-12). In contrast, relatively little work has focused on the characterization of amino acid resi- dues at the enzyme active site. It is clear, however, that most PEPCKs contain es- sential sulfhydryl groups (1, 12) and we have recently provided evidence that there are two functional arginyl residues per en- zyme subunit in yeast PEPCK (13). Also, Silverstein et al. (14) have shown the cova- lent incorporation of N-(iodoacetyl-ami- noethyl)-5-naphthylamino-1-sulfonic acid into hog liver PEPCK, and Jadus et aL (15) have demonstrated the covalent incorpo-

0003-9861188 $3.00 Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

38

AFFINITY LABELING OF YEAST PHOSPHOENOLPYRUVATE CARBOXYKINASE 39

ration of 5’-p-fluorosulfonylbenzoyl-gua- nosine into the rat liver enzyme.

The complete amino acid sequences of the enzymes from rat liver, chicken liver, and Drosophila melanogaster heads have been reported (16-18), and specific se- quences for the nucleotide- and phospho- enolpyruvate-binding sites have been sug- gested (17). Recently, Burlini et al. (19) have identified a phosphorylated form of yeast PEPCK, and they have indicated ser- ine as the phosphorylated amino acid res- idue.

As part of a study aimed at obtaining structural information of the active site of yeast PEPCK, we decided to explore the possibility of introducing a radioactive probe into the nucleotide-binding site of the enzyme, suitable for further isolation and sequencing of active-site peptides. As affinity labeling has the potential to yield more specific chemical modification than is usually achieved with group-specific re- agents (20), we decided to evaluate the use of periodate-oxidized ATP (21) as an affinity label for the nucleotide-binding site of Saccharomyces cerevisiae PEPCK.

The studies we report here present evi- dence that oATP can effectively be used as an affinity label for this enzyme. A prelimi- nary report of these results has been given earlier (22).

EXPERIMENTAL PROCEDURES

Materials. ADP, ATP, PEP, malate dehydrogenase, pyruvate kinase, lactic dehydrogenase, NADH, so- dium cyanoborohydride, and Sephadex G-10 were ob- tained from Sigma (St. Louis, MO). [8-i4C]ATP (1 mCJmmo1) was from Amersham Searle, England; Blue Dextran-Sepharose 4B was a gift of Dr. M. A. Valenzuela (Universidad de Chile).

Enzyme assays. The enzyme assay in the direction of PEP carboxylation was performed as previously described (13), and was employed in all experiments except when oATP was tested as a substrate for the enzyme, where the assay for the reverse reaction was employed (12). In this last assay, the reaction mixture contained 100 mM imidazole-HCl buffer, pH 7.0,1 mM MnClz, 2 mM MgClz, 0.5 mM ATP, 100 mM KCI, 1.5 units of lactic dehydrogenase, 2.7 units of pyruvate kinase, and 0.26 mM NADH. To 0.98 ml of this mixture at 30°C 10 ~1 of freshly prepared 50 mM oxaloacetic acid solution was added and the oxidation rate of

NADH was registered at 340 nm. Then, 10 ~1 of en- zyme solution was added and the oxidation rate of NADH was again registered. When oATP was tested as a substrate, 0.11 mM oATP was included instead of ATP. Under these conditions, no appreciable inacti- vation of the auxiliary enzymes occurred.

Enzyme purz&atim S. cerevisiae phosphoenolpy- ruvate carboxykinase was purified from yeast strain S288C according to Tortora et al. (5) except that two additional chromatographic steps had to be included since the enzyme obtained according to the above-

mentioned procedure retains about 10% contaminat- ing protein, as revealed by polyacrylamide gel elec- trophoresis in the presence of sodium dodecyl sulfate. Homogeneous enzyme could be obtained by adding a cellulose phosphate ehromatographic step (sample adsorbed in 50 mM potassium phosphate buffer, pH 6.0, containing 0.1 mM EDTA, and eluted with a linear gradient of 50 to 350 mM of the same buffer) and a final Blue Dextran-Sepharose 4B step (sample ad- sorbed in 5 mM potassium phosphate buffer, pH 7.0, containing 0.1 mM EDTA, and eluted with 20 mM of the same buffer). The enzyme obtained (specific activ- ity 32-33 units/mg of protein) showed a single band in polyaerylamide eleetrophoresis in the presence of sodium dodecyl sulfate. The carboxykinase concen- tration was estimated spectrophotometrically using an extinction coefficient A:Fm = 12.3 at 280 nm (5).

Synthesis qf2’,3’-dialdehyde ATP. The 2’,3’-dialde- hyde derivative of ATP was prepared by periodate ox- idation as described by Easterbrook-Smith et al. (23) and modified by Lowe and Beechey (21). Only iodate- free fractions (24) collected from the Sephadex G-10 column were pooled. The purity of the product was checked by thin-layer chromatography in polyethy- leneimine-cellulose plates eluted with 0.8 M NH*HCOa (23). The position of the nucleotide was detected by ultraviolet light. The purified material showed only one spot which stayed at the origin. No ATP (Rf = 0.68) was detectable. [8-‘4C]oATP was prepared in the same way, except that [8-14C]ATP was used.

Enzyme modi&caticm with oATP. The enzyme was dialyzed 4 h against 300-400 vol of 50 mM Hepes buffer, pH 7.5, at 4°C before modification. A solution containing the enzyme in the same buffer plus 5 mM sodium cyanoborohydride and 4 mM MnClz, was pre- incubated for 3 min at 3O”C, and then a certain amount of oATP was added to start the reaction. At given intervals, 5- to lo-p1 aliquots were removed and assayed immediately for residual phosphoenolpyru- vate carboxykinase activity. The addition of the mod- ifier, 100-200 times diluted to the assay medium, had no effect on the enzyme activity or on the activity of the auxiliary enzyme, malate dehydrogenase.

Stoichiometry of 2’,318-‘4C]dialdehyde ATP bind- ing. Approximately 0.5 mg of enzyme was incubated with 0.145 mM [8-‘4C]oATP (specific radioactivity 1900

40 SAAVEDRA, ARANEDA, AND CARDEMIL

100 805 60 -

40 -

I I I I I I I I I

5 10 15 20 25 0 40 80 120 Time (min ) l/ [oATPI mbl’

FIG. 1. Inactivation of yeast PEPCK by oATP. (A) The enzyme (0.10 pM) was incubated at 30°C in 50 mM Hepes buffer, pH 7.5, containing 4 mM MnClz and 5 mM NaCNBH, in the absence (0) or presence of 8 pM (m), 17 pM (A), 33 pM (0), 66 pM (Cl), or 265 pM (A) oATP, in a final volume of 0.2 ml. At the indicated times, aliquots of 5-10 ~1 were taken for the activity assay as described under Experimental Procedures. (B) Double-reciprocal plot of the pseudo-first-order inactivation rate con- stants versus oATP concentration. The pseudo-first-order inactivation rate constants (k& were calculated from the data shown in A.

cpm/nmol) as described above. Aliquots were taken at different times to measure enzyme activity and protein-bound radioactivity. The bound radioactivity was measured in 30-pl aliquots of the incubation mix- ture as described before (13). The amount of enzyme present in the experiment was estimated based on a subunit molecular mass of 64.3 kDa (5).

RESULTS

Inactivation qf phosphoenolpyruvate car- boxykinase by 2’,3’-dialdehyde A TP. Incu- bation of the enzyme with oATP resulted in its progressive and total inactivation. This inactivation followed pseudo-first-or- der kinetics with respect to active enzyme, as shown in Fig. 1A. Figure 1B shows the double-reciprocal plot of the pseudo-first- order inactivation rate constants as a function of oATP concentration. The straight line obtained suggests that the re- action follows saturation kinetics (25, 27) as depicted in the equation

Kd 4 E+I =EI+E-I 111

where EI is the reversible complex and

E-I is the covalently modified inactive en- zyme. From the data shown in Fig. lB, val- ues for Kd and Kl of 60 * 17 FM and 0.200 4 0.045 min-‘, respectively, could be ob- tained.

No reversion of the inactivation was ob- served when a sample of enzyme treated with oATP in the absence of cyanoborohy- dride was extensively dialyzed against 100 mM Tris-HCl buffer, pH 8.4.

Since the enzyme is specific for adenine nucleotides (1,2), it could be expected that any alteration in the adenine ring should render oATP a much less effective inacti- vator. To test this point, the 2’,3’-dialde- hyde of 1-N6-etheno-ATP (synthesized from l,N’-etheno-ATP, essentially as de- scribed for the synthesis of oATP under Experimental Procedures) was employed, and we found that it is a much less effective inactivator. When a sample of enzyme was inactivated by 33 yM oATP, only 15% of its activity remained after 30 min of incuba- tion, whereas when a 130 times higher con- centration of the 2’,3’-dialdehyde of l-N6-

AFFINITY LABELING OF YEAST PHOSPHOENOLPYRUVATE CARBOXYKINASE 41

2’ I I I I I I 0 5 10 15 20 25

Time (min)

FIG. 2. Effect of substrates on enzyme inactivation by oATP. The enzyme (0.10 KM) was incubated under the conditions given in Fig. 1A in the absence (0) or presence of 0.265 mM oATP (Cl), 0.265 mM oATP plus 100 mM NaHCO, (A), 0.265 mM oATP plus 1.92 mM ADP (0), 0.265 mM oATP plus 6.8 mM PEP (m), 0.265 mM oATP plus 2.5 mM ATP (A), or 0.265 mM oATP plus 1.92 mM ADP and 6.8 mM PEP (0). At the indi- cated times, aliquots of 10 ~1 were taken for the activ- ity assays. The concentrations of NaHCOa, ADP, PEP, and ATP employed were 20,22,22, and 71 times their reported Km values, respectively [see (1, 2) and the text].

etheno-ATP was employed, the remaining enzyme activity after 30 min of incubation was still 35% (results not shown).

E#ect of substrates on the inactivation process. Pseudo-first-order kinetic data were also observed for the inactivation of S. cerevisiae PEPCK by oATP in the pres- ence of saturating concentrations of sub- strates. Figure 2 shows the effect on inacti- vation of ligands and on combinations of them, at the concentrations indicated. Maximal protection is observed with ATP alone or with PEP plus ADP. These results suggest that oATP binds to the active site of the enzyme.

2’,3’-Dialdehyde ATP as inhibitor of phosphoenolpyruvate carboxykinase. oATP was assayed as a possible substrate of the enzyme, by adding it to an incubation mix- ture (prepared as described under Experi- mental Procedures for the assay in the di- rection of oxaloacetate decarboxylation) in

place of ATP and by employing a sixfold higher amount of enzyme than in the usual assay. No PEPCK activity could be de- tected under these conditions. We then in- vestigated whether oATP behaves as an in- hibitor of the carboxykinase. This experi- ment is technically difficult, since the analog is a strong irreversible inactivator of the enzyme. Therefore, low analog con- centrations and short-time assays had to be used. Figure 3 shows the results ob- tained, which suggest competitive inhibi- tion of oATP against ADP, and from these data a Ki of 0.24 InM could be estimated. This same pattern or inhibition was ob- tained when the inhibitory effect of ATP against ADP was investigated [KiCATPj = 0.26 mM, results not shown]. Under these conditions, the assays were linear with time for 1 to 2 min, so that initial velocities could be obtained.

pH dependence of the inactivation pro- cess. Figure 4A shows the effect of pH on the velocity of inactivation of the carboxy- kinase by oATP. The rate of inactivation increases with pH, within the pH range studied. This increase in reactivity with pH may be due to the ionization of the re- active group on the enzyme to form a more reactive nucleophilic species. If the ioniza- tion of one group only is responsible for the reactivity (and, in turn, for the loss of en- zyme activity), the following mechanism applies:

Kd ‘il E +I =EI+E-I

+

H’

Jr K,

EH+

121

Here, K, is the equilibrium constant for the ionizable group in the enzyme.

From the above mechanism, we derive the relation (25)

k ‘id K [H+l [31 1’ . a

where k is the pseudo-first-order inactiva-

42 SAAVEDRA, ARANEDA, AND CARDEMIL

tion rate constant determined at several concentrations of H+, and I is the concen- tration of oATP employed. Thus, a plot of l/k against [H+] should give a straight line with an extrapolated intercept on the [H+] axis:

abscissa intercept = - (I + rEf,)K, L4] nd

Thus, the value of K,, and hence the pK, of the ionizable group, can be readily calcu- lated by substituting the oATP concentra- tion employed in the experiment (r) and the previously determined value of Kd (60 PM, Fig. 2). From Fig. 4B, a pK, of 8.1 was obtained. Even when the pK, values for the ionization of oATP have not been deter- mined, we do not expect them to differ greatly from those of ATP [pK, values of 3.92 and 6.63 (26)], so the determined pl(, of 8.1 most likely reflects the ionization of the reactive group in the enzyme.

Kinetic properties of the partially inacti- vated enzyme. The enzyme was incubated with 33 PM oATP until 47% activity re- mained, and then the mixture was chro- matographed in a Sephadex G-10 column to eliminate the unreacted oATP. No sig- nificant differences were observed in the

0 0.05 0.10 0.15

[ADPI mM

FIG. 3. Hanes-Woolf plot for the inhibition of yeast PEPCK by oATP, when ADP is the variable sub- strate. Initial velocities were measured by assaying the enzyme (0.002 pM) at different concentrations of ADP (0) as described under Experimental Proce- dures. After the initial velocity was recorded (1-2 min after addition of the last component of the assay), oATP was added at a final concentration of 78 pM, and velocity was again determined (0).

apparent K, values for PEP or ADP when compared with those of the native enzyme. For the modified enzyme the K, values were 0.34 and 0.093 mM for PEP and ADP, respectively, while the corresponding val- ues for the native enzyme were 0.31 and 0.086 mM. This may signify that a partially inactive enzyme preparation consists of a mixture of native and totally inactive en- zyme molecules.

Determination of the number of residues modijied. Yeast PEPCK was incubated with [8-14C]oATP as described under Ex- perimental Procedures, and the correla- tion between the loss of enzyme activity and the number of moles of [8-14C]oATP bound per mole of enzyme subunit [assum- ing a subunit molecular mass of 64.3 kDa (5)] is shown in Fig. 5. It can be seen that there is a direct relation between loss of enzyme activity and incorporation of [8- 14C]oATP, with an extrapolated value of 1.15 mol of [8-14C]oATP bound per mole of enzyme subunit at 0% residual enzyme activity. The nonspecific binding of [8- 14C]oATP is very low, as it was found that when an experiment similar to that de- scribed in Fig. 5 was performed (except that the enzyme inactivation medium con- tained 6.8 mM PEP plus 1.92 mM ADP), the incorporation of [8-14C]oATP after 10 min of reaction was only 0.053 mol/mol of en- zyme subunit, while the enzyme retained 95% of its original activity. For the experi- ment described in Fig. 5, the incorporation of [8-14C]oATP and the enzyme activity were 0.85 mol/mol of subunit and 25%) re- spectively, after 10 min of reaction.

DISCUSSION

The work described in this communica- tion suggests that the 2’,3’-dialdehyde de- rivative of oATP serves as an affinity label- ling reagent for the nucleotide-binding re- gion of S. cerevisiae phosphoenolpyruvate carboxykinase. Inactivation of the yeast enzyme by oATP fulfilled the following generally accepted criteria for an affinity label: (1) the inactivation obeyed satura- tion kinetics, and a double-reciprocal plot of the pseudo-first-order inactivation rate

AFFINITY LABELING OF YEAST PHOSPHOENOLPYRUVATE CARBOXYKINASE 43

0 8 12 16 0 5 10 15 20 25 30

Time (min ) [WI II 108t.I

FIG. 4. Effect of pH on enzyme inactivation by oATP. (A) The enzyme (0.084 KM) was incubated at 30°C with 66 pM oATP in 50 mM Hepes buffer, pH 6.6 (U), 6.9 (0), 7.2 (a), 7.6 (m), 7.9 (0). or 8.2 (A), in a final volume of 0.2 ml. Aliquots of 10 PL were taken at different times for assay of the remaining activity. Spontaneous enzyme inactivation at the different pH values (which ranged from 27% at pH 6.6 to 22% at pH 8.2 after 16 min) has been subtracted in each case. (B) Dependence of the reciprocal of the pseudo-first-order inactivation rate constant on H+ concentration. The pseudo-first-order in- activation rate constant (kobs) of yeast PEPCK was calculated from the data in A.

constants versus concentration of oATP gave a & of 60 k 17 PM; (2) the presence of substrates (especially ATP or PEP plus ADP) effectively prevented inactivation of

1 I

.-

\

I I I I I \ \ ‘\ \

OO I.11

0.4 0.8 1.2

moles oATP /mole subunit

FIG. 5. Relation between enzyme activity and bind- ing of [S-‘*Cl oATP to S. cerevisiae PEPCK. The en- zyme (9.76 @M) was incubated with 0.145 mM [8-

‘“C]oATP at 30°C in 50 mM Hepes buffer, pH 7.5, con- taining 4 mM MnCla plus 5 mM NaCNBHa in a final volume of 0.30 ml. The remaining enzyme activity and the incorporation of radioactivity into the protein was determined as described under Experimental Procedures.

the enzyme by oATP, and oATP appeared to competitively inhibit ADP binding; (3) extrapolation of titration data showed that 1.15 mol of oATP were bound per mole of enzyme subunit at 100% inactivation. In addition, the 2’,3’-dialdehyde of l-N6- etheno ATP was a poor inactivator, as ex- pected for the nucleotide specificity of the enzyme.

Despite having a free triphosphate side chain, oATP is not a substrate of yeast PEPCK. This may suggest that an intact ribose moiety is an essential requirement for the substrate. The reactive amino acid residue may be located in the vicinity of the phosphoryl-binding site of ATP, since the protection of inactivation was most effective by ATP or by the mixture of ADP plus PEP.

It has been reported that periodate-oxi- dized nucleotides react with lysyl residues in proteins by forming simple Schiff bases (23,2’7), conjugated Schiff bases (28), stable dihydroxymorpholine derivatives (21, 29), or mixtures of them (30). Also, ,&elimina- tion of the triphosphate side chain has been reported (31). The interaction of yeast PEPCK with oATP, although not studied in detail in the present communication, ap- pears not to involve a simple Schiff base

44 SAAVEDRA, ARANEDA, AND CARDEMIL

formation, since when the enzyme was in- activated in the absence of cyanoborohy- dride, no reversion of the inactivation was observed on extensive dialysis. In this work, and to avoid the possible problems outlined by Rayford et al. (32), we decided to maintain a 5 mM concentration of NaCNBH3 in all the experiments.

Up to now, periodate-oxidized nucleo- tides have been reported to react only with lysyl residues in proteins, and it is in fact relatively common to find them among the component amino acid residues of the nucleotide-binding region of nucleotide- utilizing enzymes (20, 23, 27, 29, 33). The pK, of 8.1 obtained in this work for the re- active amino acid residue in yeast PEPCK agrees with the expected pK, for a lysyl residue in a low-polarity environment (34). However, even when modified cysteine res- idues have not previously been isolated, we cannot rule out formation of a hemi- thioacetal on reaction of oATP with PEPCK.

Recently, Cook et al. (17), based on com- parisons of the amino acid sequences of cy- tosolic PEPCK with those of other pro- teins that bind guanine nucleotides, sug- gested the presence of lysyl residues in the phosphoryl-binding regions of these car- boxykinases. Also, according to the conclu- sions of Cook et al. (1’7), all of the putative binding regions identified in cytosolic PEPCK from chicken occur in hydrophobic regions, which agrees with the previous findings of Silverstein et al. (14, 35), who had provided evidence of a hydrophobic en- vironment for the active site of mitochon- drial hog liver PEPCK. Recent work from our laboratory (V. Quifiones, M. V. Enci- nas, and E. Cardemil, unpublished) also suggests a low polarity for the active site of S. cerevisiae PEPCK, thus pointing to similarites in the active site environment of PEPCK of different quaternary struc- ture.

The results presented in this paper pro- vide a simple and specific way of introduc- ing a radioactive label at the active site of Saccharomyces cerevisiae PEPCK, which is the first step in obtaining active site pep- tides suitable for amino acid sequencing.

Work is in progress in our laboratory to- ward this goal.

ACKNOWLEDGMENT

We thank Dr. Ana Maria Jabalquinto for sharing ideas and results and offering advice.

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