ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 180, 19-25 (1977)

Evidence for an Essential Role for Arginyl Residues for Yeast Phosphoglycerate Kinasel

KATHRYN ROGERS AND BRUCE H. WEBER2

Department of Chemistry and Institute for Molecular Biology, California State University, Fullerton, California 92634

Received August 10, 1976

Reaction of yeast phosphoglycerate kinase with either butanedione or cyclohex- anedione can result in modification of up to all 13 arginyl residues with total loss of activity; however, extrapolation to zero activity for partially modified preparations indicates that up to 7 arginyls are essential. Whereas 20 mM 3-phosphoglycerate affords partial protection of activity toward both reagents, 20 mM MgATP affords complete protection of activity and protects 2 arginyls against modification by butanedione and 1 arginyl against modification by cyclohexanedione. With butanedione the modification could be reversed with total recovery of activity, suggesting that only arginyl groups were modified, which is consistent with the amino acid analysis of the modified protein. Only at high cyclohexanedione concentrations or long reaction times was a yellow product obtained that showed loss of lysyl residues. Circular dichroism spectra show that even when all the arginyls are modified by butanedione or up to 10 modified by cyclohexanedione there is no change observed in the far or near ultraviolet, indicating that there is no detectable conformational change concomitant with modification, which is confirmed by hydrodynamic studies. It is concluded that at least one, possibly two, arginyls of yeast phosphoglycerate kinase are essential for its action on ATP.

3-Phosphoglycerate kinase (EC 2.7.2.3) is a glycolytic enzyme that catalyzes the conversion of 1,3-diphosphoglycerate to 3- phosphoglycerate with the concomitant phosphorylation of ADP to ATP. Struc- tural studies on this enzyme are well un- derway (1, 2) and chemical modification experiments have shown an essential role for one carboxyl (3), one tyrosyl, and up to three lysyl residues (4).

At the time this work was initiated, there was no information available on the role of arginyl residues in the action of kinases. The fact that several dehydrogen- ases (5-9) have been shown to have essen- tial arginyl groups for binding NAD and that the X-ray results (10) indicate a simi- larity of three-dimensional structure for

1 Supported by NSF Grant No. GB-37965. Taken in part from the thesis of K.R. submitted in partial fulfillment of the requirements for an M.A. degree, California State University, Fullerton, Calif.

2 To whom inquiries and reprint requests should be addressed.

phosphoglycerate kinase in the nucleotide binding region with that of the NAD-bind- ing domain of the dehydrogenases sug- gests that arginine might serve a similar function in binding ATP for kinases. Re- cently (11, 12), it has been shown for crea- tine kinase that one arginyl per subunit functions in ATP binding. In this paper we report evidence, using the established re- agents for arginine modification, butane- dione (13), and cyclohexanedione (14, 15) that one and possibly two arginyls are es- sential for the action of phosphoglycerate kinase.

MATERIALS

Yeast phosphoglycerate kinase was obtained as a crystalline suspension in 3 M ammonium sulfate, pH 7.0 from Boehringer-Mannheim Corp. or was iso- lated according to the method of Brake and Weber (3). The specific activity varied between 850 and 1000 unitslmg at 30°C at saturating conditions as corrected by the method of Scopes (161 and Larsson- Raznikiewicz (17). Glyceraldehyde J-phosphate de- hydrogenase (crystalline suspension), 3-phospho-

19

Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved. ISSN 0003-9861

20 ROGERS AND WEBER

glycerate, ATP, and NADH were all purchased from Boehringer-Mannheim Corp. Cyclohexanedione and butanedione were obtained from Aldrich Chem- ical Company. The butanedione was distilled just prior to use. All materials used for amino acid anal- ysis were purchased from Pierce Chemical Com- pany. All other reagents were analytical grade. All water used was double-distilled and deionized.

METHODS

Enzyme preparation. Desalting of phosphoglycer- ate kinase was done at 4°C by conventional dialysis or by pressure dialysis employing an Amicon Ultra- filtration cell. Insoluble material was removed by centrifugation at 15,000 rpm using a Beckman J-21 preparative centrifuge.

Protein concentrations were determined from 280 nm absorbance using an extinction coefficient of EP$Z (A,,,) = 0.50 (18). Protein concentrations were adjusted by dilution with the appropriate buffer or by concentration using an Amicon ultrafiltration cell.

Phosphoglycerate kinase assay. The specific activ- ity was determined spectrophotometrically by use of a coupled assay. The assay couples the phospho- glycerate kinase reaction to the reaction catalyzed by glyceraldehyde 3-phosphate dehydrogenase. As an index of activity, the decrease in 340~nm absorb- ance, due to the oxidation of NADH, is followed. The reaction mixture contained 0.1 M Tris-nitrate buffer, pH 7.6, 0.033 M MgS04, 0.32 M 3-phospho- glycerate, 0.0165 M ATP, 0.0086 M NADH, and be- tween 0.1 and 0.5 pg of phosphoglycerate kinase. The decrease in absorbance was followed with a Beckman Acta III spectrophotometer. The assays were done at 25°C and corrected to 30°C and to saturation conditions as described by Scopes (16) and Larsson-Raznikiewicz (17).

Amino acid analysis. Analyses were performed by the method of Spackman et al. (19) and Moore and Stein (20) using an accelerated system (21) on a Beckman Model 120-C amino acid analyzer. Ali- quots of phosphoglycerate kinase containing ap- proximately 1 mg of the protein were hydrolyzed at 110°C in U~CUO in 6 N HCl. Ten microliters of a 5% solution of phenol was added to all samples to pro- tect against the loss of tyrosine. Mercapacetic acid (20 yl) was added to prevent regeneration of argi- nine. After a 24-h hydrolysis period, the samples were dried by rotary evaporation, washed with dou- ble-distilled deonized water, and diluted to 0.5 ml with 0.001 M HCl. Total amino acid compositions were used to quantitate the amount of arginine present in the samples; the yields of other animo acids were used to determine the protein concentra- tion .

Circular dichroism. Circular dichroism spectra were obtained from a modified Beckman CD spectro-

photometer at room temperature. In the range of 403 to 236 nm a l-cm light path length was used, and in the range of 235 nm and below the light path length was 0.1 cm. The spectra obtained represent an aver- age of 16 scans at the rate of one scan per minute.

Sedimentation velocity. Sedimentation coeffi- cients on native and modified enzymes were deter- mined in a Beckman Model E analytical ultracentri- fuge at 60,000 rpm employing double-sector cells in an AnD rotor using schlieren optics at a protein concentration of 10 mg/ml in 0.1 M borate buffer, pH 8.0.

Modification of phosphoglycerate kinase with bu- tanedione. A 4.0-ml solution of phosphoglycerate ki- nase (1.5 mg/ml)r in a 100 mM borate buffer, pH 8.0, was reacted by adding a l.O-ml solution containing the appropriate amount of freshly distilled butane- dione in 100 mM borate buffer, pH 8.0, to bring the concentration of butanedione of the final solution to the desired level. A control at the same phospho- glycerate kinase concentration lacking reagent was run simultaneously. For substrate protection exper- iments, additional reactions were carried out in the presence of substrate; care was taken to adjust ionic strength of the control and the reaction without substrates present to the same value as that for which substrates were added. All reactions were carried out in closed containers at 30°C in a circulat- ing water bath, unless otherwise stated. At various timed intervals, 0.5-ml aliquots were removed from both sample and control, immediately assayed for enzyme activity, and then precipitated with 0.10 ml of 6 N HCl. The precipitated protein was then washed with double-distilled deionized water to re- move excess reagent and HCl and prepared for amino acid analysis. Quantitation of the amount of modified arginine was achieved by measuring the disappearance of arginine.

Reversal of butanedione modification of phospho- glycerate kinase. A solution of phosphoglycerate ki- nase (6 mg/ml) in 100 mM borate buffer at pH 8.0 was modified at 30°C with 5 mM butanedione. Ali- quots were taken out at timed intervals and assayed for loss of enzymatic activity. When modification appeared to be complete, an aliquot was applied to a gel filtration column (0.9 x 15 cm) of Sephadex G-25 equilibrated with 20 mM Veronal-1 M NaCl. Ali- quots of the appropriate eluate fraction were re- moved at timed intervals and assayed for enzymatic activity.

Modification of phosphoglycerute kinase with cy- cloheranedione. A 4.0-ml solution of phosphoglycer- ate kinase (1.5 mg/ml) in 100 mM borate buffer, pH 9.0, was reacted by addition of 1.0 ml of a solution of cyclohexanedione in 100 mM borate buffer to bring the total concentration of cyclohexanedione to the desired level. A control at the same phosphoglycer- ate kinase concentration with no cyclohexanedione

ARGINYL MODIFICATION OF PHOSPHOGLYCERATE KINASE 21

was run simultaneously. When substrate protection of reaction and at 50 mM cyclohexanedione studies were done there were always additional con- 8% residual activity was achieved after 30 trols containing phosphoglycerate kinase but lack- ing substrate, with care being taken to adjust the

min. Initial studies involving variations in

ionic strength to the same level. All reactions were pH and temperature indicated that the

done in closed containers at 30°C in a circulating conditions employed are optimal for modi-

water bath. At various timed intervals 0.5-ml ali- fication of yeast phosphoglycerate kinase.

quots were removed from both the sample and con- Unless high concentrations of cyclohex- trol and assayed for enzyme activity. Each aliquot anedione (above 50 mM) or long reaction was checked for possible lysine modification by look- times are employed (greater than 400 ing for the appearance of a yellow color in the pro- min), there is no indication of a side reac- tein solution and by the solution’s absorbance at 440 tion with lysyl residues as judged by nm (22). The aliquots were subsequently placed in amino acid analysis and absorbance at 440 1.0 ml of a 30% acetic acid solution to halt the nm (22). Except for the loss of lysine ob- reaction. The samples were then dialyzed for ap- proximately 2 h in 2.0 liters of 15, 7.5, and 1.0%

served under extreme reaction conditions,

acetic acid, respectively. The samples were then no other changes in amino acid composi-

freeze-dried for at least 12 h. Each sample was then tion were observed even when all 13 argi-

diluted to 1.0 ml with 6 N HCl. Quantitation of the nyls were modified by cyclohexanedione.

amount of modified arginine was achieved by meas- Plotting residual activity versus moles of uring the disappearance of arginine by amino acid arginine modified (Fig. 1) gives a linear analysis. relationship that suggests that the essen-

RESULTS tial arginyl(s) iscare) in a class of reactiv- ity that includes 7 out of the 13 arginines.

Modification with Butanedione Further reaction of three of the remaining

Reaction of butanedione in borate buffer arginyls can be obtained without concomi-

with yeast phosphoglycerate kinase causes tant reaction of lysyl residues.

rapid inhibition of enzyme activity at 1, 10, Substrate Protection Studies and 25 mM butanedione in 100 mM borate; the reaction time varied from 20 to 60 min

Figures 2 and 3 show the results of the

and the percentage residual activity var- substrate protection experiments for both

ied from 25 to O%, depending upon the butanedione and cyclohexanedione modifi-

concentration of butanedione. Initial stud- cation, respectively. In both cases, 20 mM

ies involving variations of the pH and tem- 3-phosphoglycerate provides only partial

perature of reaction indicated that the con- protection whereas 20 mM MgATP affords

ditions described under Methods are opti- complete protection of activity. Determi-

mal for phosphoglycerate kinase. Upon re- nation of the stoichiometry of arginyls

moval of the reagent and borate by gel modified for enzyme reacted in the pres-

filtration, full enzyme activity is restored ence and absence of MgATP shows that 1.9

within 100 min of completion of the gel mol of arginyl/mol of enzyme are protected

filtration; this reversal of the reaction is by MgATP against reaction with butane-

consistent with arginyl modification in the dione and that 0.9 mol of arginyl/mol of

absence of lysyl side reaction. This is con- enzyme is protected against reaction with

firmed by the observation of no loss of cyclohexanedione; see Table I. Protection

lysine or change in any other amino acid, of activity due to reaction of the reagent

as judged by amino acid analysis, concomi- with MgATP can be excluded (12).

tant with arginine modification. CD Spectra of Modified Proteins.

Modification with Cyclohexanedione The near-uv3 and far-uv CD spectra of

Reaction of cyclohexanedione in borate control and for phosphoglycerate kinase

buffer with yeast phosphoglycerate kinase modified with cyclohexanedione or bu-

also causes rapid inhibition of enzyme ac- tanedione were all identical and agree

tivity; at 15 mM cyclohexanedione 20% re- a Abbreviations used: uv, ultraviolet; CD, circu-

sidual activity was achieved after 400 min lar dichroism.

22 ROGERS AND WEBER

with the published spectra (4). This was true even when all 13 arginyls had been modified by butanedione or up to 10 had been modified by cyclohexanedione. How- ever, alteration in the near-uv spectrum was observed when lysyl residues were also modified by cyclohexanedione (which gives a yellow-colored protein); it is not clear if this difference is the result of a conformational change near aromatic resi-

FIG. 1. Residual activity of yeast phosphoglycer- ate kinase versus moles of arginine modified per mole of enzyme by cyclohexanedione. Combination of data from seven experiments.

dues or is due to the addition of the chro- mophore.

Sedimentation Studies on Modified Pro- teins

The sopo,W value for native yeast phos- phoglycerate kinase is 3.4s. The value of SOZO,W for phosphoglycerate kinase with all 13 arginyls modified by butanedione is also 3.4s. Thus, within the limits of experimen- tal error, there appears to be no change in shape of phosphoglycerate kinase even when all 13 arginyls are modified.

DISCUSSION

Previous studies have shown that one carboxyl (3), one tyrosyl (41, and up to three lysyl residues (4) are essential for the activity of yeast phosphoglycerate ki- nase, whereas modification of the single cysteinyl (4), the three methionyl (4), or the histidyl residues (23) does not affect activity. In evaluating the results pre- sented here for an essential role for arginyl residues, care must be taken to eliminate the possibility that the loss of activity

l . .

mn,a I, TlMI 140

FIG. 2a. Plot of protection of phosphoglycerate kinase activity by 3-phosphoglycerate against inhibition by butanedione. Shown are the time courses of inhibition for control which contains phosphoglycerate kinase with 20 mM Na2HP0, in a 100 mM borate buffer, pH 8.0 (A-A); phosphoglycerate kinase and 5.0 mM butanedione in borate buffer at pH 8.0 with no substrate but with 20 mM Na*HPO, (O-O); and phosphoglycerate kinase with 5.0 mM butanedione in borate buffer at pH 8.0 with 20 mM 3-phosphoglycerate (0-O). All samples were 25 WM in phosphoglycerate kinase. Time is expressed in minutes after start of reaction.

FIG. 2b. Plot of protection of phosphoglycerate kinase activity by MgATP against inhibition by butanedione. Shown are the time courses of inhibition for control which contains phospho- glycerate kinase with 24 mM Mg(NO& and 20 mM Na,HP04 in 100 mM borate buffer at pH 8.0 (A-A), phosphoglycerate kinase and 50 mM butanedione in borate buffer, pH 8.0 with no substrate but with 24 mM Mg(NO,), and 20 mM Na,HPO, (O-O), and phosphoglycerate kinase with 50 mM butanedione in borate buffer at pH 8.0 with 20 mM MgATP (0-O). All samples were 25 PM in phosphoglycerate kinase. The r-axis represents time in minutes after the start of the reaction.

ARGINYL MODIFICATION OF PHOSPHOGLYCERATE KINASE 23

b

TIME TIME

FIG. 3a. Plot of protection of phosphoglycerate kinase activity by 3-phosphoglycerate against inhibition by cyc1ohexanedione.Shov.m are the time courses of inibition for control which contains phosphoglycerate kinase with 20 mM Na,HP04 in a borate buffer, pH 9.0 (A-A); phosphoglycerate kinase and 20 mM cyclohexanedione in 100 mM borate buffer at pH 9.0 with no substrate but with 20 mM Na,HP04 (O-O); and phosphoglycerate kinase with 20 mM 3-phosphoglycerate (0-O). All samples were 25 PM in phosphoglycerate kinase. Time is expressed in minutes after the start of the reaction.

FIG. 3b. Plot of protection of phosphoglycerate kinase activity by MgATP against inhibition by cyclohexanedione. Shown are the same courses of inhibition for control which contains phosphoglycerate kinase with 24 mM Mg(NO,), and 20 mM Na,HPO, in borate buffer at pH 9.0 with no substrate (A-A), phosphoglycerate kinase and 15 mM cyclohexanedione in borate buffer at pH 9.0 with 24 mM Mg(NO& and 20 mM Na*HPO, (O-O), and phosphoglycerate kinase with 15 mM cyclohexanedione in borate buffer at pH 9.0 with 20 mM MgATP (O-O). All samples were 25 PM in phosphoglycerate kinase. Time is expressed in minutes after the start of the reaction.

TABLE I

STOICHIOMETRY OF ARGININE MODIFICATION WITH AND WITHOUT SUBSTRATE F’ROTECTION~

Amino acid Control Butanedione MgATP + Cyclohex- MgATP + butanedione anedione cyclohexane-

dione

Arginine 12.6 6.5 8.4 6.9 7.8 Lysine 40.0 40.1 40.0 39.8 40.0 Glutamate 36.6 37.4 37.2 36.1 36.3 Leucine 38.9 37.9 37.8 38.8 38.8 Glycine 35.8 36.0 35.6 36.5 36.2 Phenylalanine 17.6 16.9 17.1 17.7 17.3

” Results are expressed as micromoles of amino acid recovered per micromole of phosphoglycerate kinase assuming a molecular weight of 44,500 (4). Besides arginine, results for a representative selection of other amino acids are included. Conditions of reactions are described in Figs. 2b and 3b; samples taken for analysis represent the end points on each plot.

might be due to a side reaction with one of the other essential amino acids or due to a change in conformation concomitant with modification.

Of the essential residues for phospho- glycerate kinase, only lysine has been re- ported to react with butanedione or cyclo- hexanedione. Amino acid analysis of the enzyme modified by butanedione shows no loss of lysine under conditions where up to

all the arginines are lost. No other changes are observed in the amino acid composition (within 3%). Further, removal of butanedione and borate gives complete regeneration of enzyme activity. For the cyclohexanedione reaction also there is no change in any other residues when up to 10 arginyls are modified. When the reaction was carried out under conditions of above 50 mM cyclohexanedione or for longer than

24 ROGERS AND WEBER

400 min a product that absorbed at 440 nm was obtained that did show concomitant loss of lo-15% of the lysyl residues as judged by amino acid analysis. Therefore, it is reasonable to conclude that within the experimental limits of detection, only argi- nyl groups were modified by butanedione or cyclohexanedione under the conditions usually employed.

Careful analysis of both the near-uv and far-uv spectra for yeast phosphoglycerate kinase, modified by either butanedione or cyclohexanedione, fails to show any differ- ence, indicating that modification of even all the arginyl residues, in the absence of lysyl side reaction, does not alter the sec- ondary structure of the protein nor the tertiary structure around the aromatic res- idues. The sedimentation coefficient that was observed for phosphoglycerate kinase fully modified by butanedione suggests that there is no evidence for a separation of the lobes of the enzyme that could occur without any concomitant alteration of the secondary and tertiary structure of the en- zyme. Thus, it seems highly unlikely that the inhibition observed could be due to a movement of the lobes but rather is due to arginyl in the active site.

The fact that for both butanedione and cyclohexanedione modification of yeast phosphoglycerate kinase the essential ar- ginyhs) in the active site are in a reaction class containing some of the nonessential arginyls stands in sharp contrast to the results reported for creatine kinase (12) where only 1 of the 18 arginyls per subunit is modified by butanedione. Thus, it would appear that the microenvironment around the arginyls is different for these two ki- nases.

The difference that is seen between the reagents in the stoichiometry of arginyls protected by MgATP, two arginyls pro- tected against butanedione modification, whereas one arginyl protected against cy- clohexanedione modification, suggests that the MgATP is blocking access of the butanedione reagent to two arginyls but that cyclohexanedione reacts, under the conditions employed, readily only with one of these residues. Thus, it would appear that there could be two arginyl residues in

J.

4.

5.

6.

10.

11.

12.

13. 14.

15.

16. 17.

18.

19.

20.

the vicinity of the nucleotide binding site and that one or both may be essential for phosphoglycerate kinase activity.

For creatine kinase (11, 12) and more recently for mitochondrial ATPase (24) it appears that an arginyl residue functions in binding the ATP substrate. That one or two arginyl residues seem to be essential in the action of phosphoglycerate kinase suggests that arginyl side chains may gen- erally be involved in ATP binding or in catalyzing the phosphor-y1 transfer, as has been recently suggested for creatine ki- nase (25). ’

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ARGINYL MODIFICATION OF PHOSPHOGLYCERATE KINASE 25

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