Inactivation of purine nucleoside phosphorylase by modification of arginine residues

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS

Vol. 190, No. 2, October, pp. 699-704, 1978

Inactivation of Purine Naicleoside Phosphorylase by Modification of Arginine Residues

FRANK JORDAN’ and ANNIE WU

Carl A. Olson Chemistry Laboratories, Rutgers University, Newark, New Jersey 07102

Received January 31,1978; revised May 31, 1978

Treatment of purine nucleoside phosphorylase (EC 2.4.2.1), from either calf spleen or human erythrocytes, with 2,3-butanedione in borate buffer or with phenylglyoxal in Tris buffer markedly decreased the enzyme activity. At pH 8.0 in 60 min, 95% of the catalytic activity was destroyed upon treatment with 33 mM phenylglyoxal and 62% of the activity was lost with 33 tll~ 2,3-butanedione. Inorganic phosphate, ribose-l-phosphate, arsenate, and inosine when added prior to chemical modification all afforded protection from inactivation. No apparent decrease in enzyme catalytic activity was observed upon treatment with maleic anhydride, a lysine-specific reagent. Inactivation of electrophoretically homogeneous calf-spleen purine nucleoside phosphorylase by butanedione was accompanied by loss of arginine residues and of no other amino acid residues. A statistical analysis of the inactivation data vis-h-uis the fraction of arginines modified suggested that one essential arginine residue was being modified.

Purine nucleoside phosphorylase (EC 2.4.2.1; purine nucleoside: orthophosphate ribosyltransferase) cleaves the nucleoside glycosyl bond (1) according to the following reaction:

Purine nucleoside + Pi breakdown

synthesis purine base + ribose-1-P [l]

The equilibrium favors nucleoside synthesis (2, 3). The widespread occurrence of PNPase2 in as diverse sources as Esche- richia co& liver, thymus, human erythrocytes, fish skin, etc.’ (2), suggests an impor- tant role for this enzyme in purine metab- olism, probably in purine salvage.

One of the outstanding questions in the reaction presented in Eq. 1 concerns the nature of the binding site for the negatively charged Pi and ribose-1-P. We wish to present evidence indicating that chemical modification of arginine leads to inactivation of both human erythrocytic and calf-spleen PNPase. The presence of a lysine specific reagent has no effect on enzymic activity.

1 To whom correspondence should be addressed. * Abbreviations used: PNPase, purine nucleoside

phosphorylase; ribose-l-P, a-n-ribose-l-phosphate.

A statistical treatment of the arginine inactivation data suggests that one essential arginine residue is being modified.

MATERIALS AND METHODS

PNPase from human erythrocytes was purified according to the method of Tsuboi and Hudson (4). Adsorption on calcium phosphate gel and elution with trisodium citrate provided protein with specific activity of 10.9 units/mg of protein. Prior to use the enzyme was diluted with 0.001 M EDTA and 0.01% Triton X- 100 (4). PNPase from calf spleen was purchased from Sigma, St. Louis, MO. (lot 86c-0109) as a crystalline suspension in 3.2 M (NH&S04 and had a specitic activity of 25 units/mg of protein. PNPase from calf- spleen was also obtained from Boehringer-Mannheim Biochemicals, Indianapolii, Ind. (lot 1067126) as a crystalline suspension in 3.2 M (NH&SO, and had a specific activity of 26.4 unita/mg of protein. According to polyacrylamide disc-gel electrophoresis this enzyme was homogeneous. Xanthine oxidase isolated from buttermilk was purchased from Sigma, St. Louis, MO., as a suspension in 2.3 M (NH&S01 and had a specific activity of 1.68 unita/mg of protein. Inosine and phenylglyoxal were obtained from Aldrich Chemicals, butanedione from Eastman Organic Chemicals, and maleic anhydride from the Fisher Scientific Co.

Enzyme assays. PNPase activity was measured spectrophotometrically by the coupled xanthine oxidase method following Kalckar’s procedure (5) and

699 0003-9861/78/1902-9699$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

700 JORDAN AND WU

monitoring the amount of urate formed at 293 nm (E293 = 1.2 x 104)

Inosine + P- purine nucleoside phosphorylase

hypoxanthine + ribose-1-P [2]

Hypoxanthine xanthine oxidase

burate + Hz02

A typical reaction mixture contained 96 mM phosphate buffer at pH 7.4, 0.2 mM inosine, 0.04 unit of xanthine oxidase, and 10 4 of purine nucleoside phosphorylase in 3 ml total volume. UV measurements were performed on a Beckman Acta III spectro- photometer.

The enzyme activity was also monitored by the orcinol reaction (6). Employing arsenate instead of phosphate drives the reaction towards nucleoside breakdown as ribose-l-arsenate is hydrolytically labile. A typical reaction mixture contained 5 pmol of inosine, 50 pm01 of arsenate, 100 pm01 of buffer at pH 7.4 and enzyme (diluted to allow for reaction of only 15% of the substrate during the incubation time) in a final volume of 0.75 ml and was incubated at 37°C for 30 min. The reaction was quenched by the addition of 0.5 ml of 7% HCIOl containing 60 mg of activated charcoal as a suspension to remove unutilized nucleoside. Next the reaction was left at room temperature for about JO min and shaken occasionally. The mixture was cen- trifuged and an aliquot of the supernatant solution was used for assay of the ribose employing the orcinol reaction. In a control reaction 0.5 ml of 7% HCIOl and 60 mg of activated charcoal were added to the enzyme before the addition of substrate and the usual protocol was followed from there on. The two assay methods gave nearly identical results.

Inactivation by 2,3- butanedione. The 0.3-ml reaction mixtures containing 1.19 pg of PNPase and 33 mu 2,3-butanedione in 16.5 mu, pH 8.0, borate buffer were incubated at room temperature for different time periods. At the end of the incubation 5 pmol of inosine, 50 pm01 of arsenate, and 100 pmol of acetate buffer were added to a final volume of 1.0 ml. The enzyme activity was measured by the orcinol method upon 30-min incubation at 37°C with added substrate.

Inactivation by phenylglyoxal. Phenyl- glyoxal(33 mu) in 16.5 mu Tris buffer (pH = 8.0) was employed, otherwise the exper- iment followed the protocol outlined above for 2,3-butanedione.

The experiments with the calf spleen PNPase employed 50 ~1 of commercially available enzyme diluted 1:20 with 0.001 M EDTA and 0.01% Triton X-100. This solution was treated with 50 mu phenylglyoxal dissolved in 50 mu Tris (pH = 8.0) buffer. The activity was checked after various incubation times by the orcinol method.

The effect of incubation with substrates prior to chemical modification was studied in several ways.

Method 1. The 0.15~ml reaction mixtures containing 1.19 pg PNPase treated with 33 mu NazHAsOr (or 26 mM inosine) at pH 8.0 were incubated at 25°C for 30 min before 0.05 ml 0.1 M butanedione dissolved in 0.1 M (pH 8.5) borate buffer was added. After various reaction times 5 pmol of inosine, 50 pmol of arsenate, and 100 pmol of acetate buffer (pH 6.0) were added (to the 20 ~1 total volume already present in any test tube) to bring the final volume to 0.8 ml. The activity was measured by the orcinol method (6).

Method 2. The 0.06~ml reaction mixtures containing 0.95 pg of PNPase treated with 33.3 mu phosphate (pH 8.0) (or 33.3 ~I-IM ribose-1-P in 16.7 mu Tris buffer (pH 8.2)) were incubated at 25°C for 30 min before 0.02 ml of 0.1 M butanedione (dissolved in borate buffer, pH 8.5) was added. After various reaction times 8 pmol of inosine and 0.1 unit of xanthine oxidase in 3 ml 0.1 M phosphate buffer (pH 7.5) were added (to the 0.080 ml already present in any test tube) and the activity was measured by the coupled enzyme assay procedure (5).

Method 3. A few experiments were performed to compare the effects of pretreat- ment with substrate on inactivation in- duced by phenylglyoxal. For example, 1.19 pg of PNPase was treated with 66.7 mu substrate in a total volume of 30 ~1 at 25°C for 30 min before 10 d 0.1 M butanedione (dissolved in 0.05 M borate buffer, pH 8.5) was added. After incubation for 30 min longer 3.0 ml of 0.1 M phosphate (pH 7.4) containing 0.63 ~01 of inosine and 0.94

ARGININE IN NUCLEOSIDE PHOSPHORYLASE 701

unit of xanthine oxidase were added and the activity was determined by the coupled enzyme assay procedure (5). In the experiments employing phenylglyoxal, 10 ~1 of 0.1 M phenylglyoxal (dissolved in 0.05 M Tris buffer, pH 8.5) was added for 15 min prior to assay but otherwise the protocol was that followed in the experiments with butanedione.

Incubation with maleic anhydride. The 0.3~ml reaction mixtures containing 1.19 pg PNPase and 33 mu maleic anhydride in 16.5 mu borate buffer (pH 8.5) were incubated at room temperature. The enzyme activity was measured by the orcinol method as described above after various reaction times. Maleic anhydride (66 mM) was also employed in some of the studies.

Amino acid analysis. A 1.13-mg sample of the electrophoretically homogeneous calf spleen enzyme (from Boehringer-Mann- heim) was exhaustively dialysed against 10 mu Tris buffer at pH 8.0. To the dialysed enzyme 76 pmol of butanedione and 38 pmol of borate buffer (pH 8.0) were added to result in a final volume of 1.3 ml. The resulting mixture was incubated at 25°C for 0, 3, 6, and 21 h. At the end of the incubation, after removal of a lo-p1 aliquot for enzyme activity assay (see above), the protein was precipitated out by 6 N HCl. The precipitate was washed with distilled water several times to remove excess butanedione and was suspended in 1 ml of 6 N HCl. Hydrolysis was carried out in sealed, evac- uated ampoules at 110°C for 23 h. Amino acid analysis was performed on a Beckman 120C amino acid analyzer according to the method of Spa&man et al. (7).

RESULTS

Figure 1 shows the time course of inactivation of human erythrocytic PNPase by 2,3-butanedione (8) in borate buffer at pH 8.0 (tI12 = 40.3 min), by phenylglyoxal(9) in Tris buffer at pH 8.0 (tIlz = 19.2 min), and of the enzyme isolated from calf spleen modified with phenylglyoxal in Tris buffer at pH 8.0 (tip = 16.5 min). 1,2-Cyclohexane- dione and its dioxime were also employed in i few studies. Both of them modified PNPase less effectively than butanedione (results of time course of inactivation ex-

periments). Methylglyoxal and glyoxal are reported also to react with the e-amino group of lysine (10). Therefore, maleic anhydride, a reagent known to be specific for lysine (11) was also employed. Incubation with 33 to 66 mM maleic anhydride at pH 8.5-9.0 over periods of time varying from 30 min to 17 h led to no perceivable loss of enzymic activity (Fig. 1).

The inactivation of calf spleen PNPase by butanedione was also monitored by amino acid analysis after acid hydrolysis, conditions under which the butanedione adduct is destroyed (8). Figure 2 compares the loss of arginyl residues and of enzyme activity. Within experimental error there was no loss of any other residues (Table I).

d $2 k $0 Go Id REACTION TIME, min

FIG. 1. The time course of inactivation of PNPase with butanedione and phenylglyoxal (at pH 8.0) and with maleic anhydride (at pH 8.5). See experimental details under Materials and Methods.

FIG. 2. The time course of inactivation, and of the number of arginines destroyed in the reaction of calf spleen PNPase with butanedione.

JORDAN AND WU

The remaining experiments were de- signed to answer the question whether any of the modified arginines are essential for enzymatic activity. Figure 3 demonstrates that the presence of ribose-l-phosphate, HPOd2-, HAsOd’-, or inosine affords partial protection of the enzyme from chemical modification. The most impressive protection is exhibited by ribose-l-phosphate. In separate experiments (Table II) the relative efficacies of the substrates and of HAsOd’- in protecting the enzyme against modification by butanedione and phenylglyoxal were examined.

A statistical method of analysis was applied to the data on modification of PNPase

TABLE I AMINO ACID ANALYSIS~ OF PNPASE FROM CALF

SPLEEN AS A FUNCTION OF THE TIME OF CHEMICAL MODIFICATION WITH BUTANEDIONE

Amino aci&

Time of modification (h)

0 3 6 21

LYS 248 243 242 271 His 130 143 141 164 &Y= 377 224 144 90

a Expressed in nanomoles/ml, see details under Ma- terial and Methods.

*The results here reported were obtained with a short column on the basic amino acids. The results obtained with the long column indicated no change in the amount of any of the amino acids analyzed over the 21-h period.

’ In a totally different set of experiments on a different batch of enzyme from the same source in 6 h 36% of the arginine remained (compared to 38% in this table) and again apparently no other amino acids were destroyed.

from calf-spleen by butanedione to deter- mine the number of essential arginines in the enzyme (12). The data given in Fig. 2 lend themselves to such analysis. One has to quantitate the number of groups modified vs. the residual activity remaining in the partially modified enzyme. The formula employed is

aI/i _ llX - (n-p-s) -.

P where a is the fraction of activity remaining,

TABLE II PROTECTION OF HUMAN ERYTHROCYTIC PNPASE BY SUBSTRATES AGAINST CHEMICAL MODIFICATION AT

PH 8.5

Chlep2a& gdi- Substrate added, Percentage in preliminary enzyme ac- treatment, 66.7 tivity”

rnM

- - 100

2,3-Butanedione (30 - 39 min reaction time) P, 68.3

HAsO:- 70.0 Ribose-1-P 69.5 Inosine 60.9

Phenylglyoxal (15 min - 51.5 reaction time) Pi 72.7

HAsOd*- 65.6 Ribose-1-P 75.6 Inosine 83.3

a The % enzyme activities were measured spectrophotometrically by the coupled xanthine oxidase method at pH = 7.4, 25°C and the activity of native enzyme was taken as 100% see Method 3 under Ma- terials and Methods.

v i L I A 1 I,. 3. 0 0 20 40 60 80 100 0 20 40 60 80 xx)

TIME COURSE OF INACTIVATION,min

FIG. 3. Time course of inactivat,ion of calf-spleen PNPase by butanedione (0) in the presence of: (A) phosphate (0) and ribose-l-phosphate (A) (see Method 2 under Materials and Methods); (B) arsenate and inosine (A) (data coincide within experimental error) (see Method 1 under Materials and Methods).

ARGININE IN NUCLEOSIDE PHOSPHORYLASE 703

FIG. 4. Fraction of activity remaining, a, vs. fraction jof arginine remaining, x, for the reaction of calf-spleen PNPase with butanedione. Experimental data are :kom Fig. 2. The data are plotted for i = 1 (O), i = 2 (A), i = 3 (0). The straight line drawn through the points for i = 1 is that given by linear least-square statistics (R = 0.995).

n is the total number of arginines, i is the number of arginines essential for activity, x is the fraction of unmodified arginines remaining, s is the number of arginines that may react faster than the essential one(s), and p is the number of arginines reacting slower than s, i of which are essential The quantity (n-p-s) is the total number of arginines that remain unreacted. Application of this method to the present problem is illustrated in Fig. 4. The knowledge of the total number of arginines (13), 34 per tri- merit holoenzyme molecule, allows one to conclude that the best linear fit is obtained for i = 1 (linear correlation coefficient of 10.995). The slope and the intercept of this tine allow determination of s = 0.2, p = 8.9 and the quantity (n-p-s) = 2 per monomeric <subunit. Assuming 33 arginines (a multiple of three) instead of 34 does not change these results appreciably.

DISCUSSION

Our results on PNPase isolated from both :human erythrocytes and calf-spleen clearly (demonstrate kinetic inactivation of the en- :zyme by butanedione and phenylglyoxal (Fig. 1). The amino acid analysis suggests that the inactivation is due to covalent modification of arginine residues only (Fig. 2 and Table I). The lack of inactivation by maleic anhydride (supposedly would form

a maleylated lysine) also suggests that either there is no essential lysine residue present or that the essential Jysine residue is buried and does not come in contact with the chemical modifier. Since the reaction time for maleylation was very long (17 h) the second argument does not appear to be a likely explanation.

The question of the essential nature of some of the modified arginines can be an- swered with a tentative yes (short of X-ray studies) on two grounds. First of all, prein- cubation of the enzyme with any of its substrates afforded significant protection from chemical modification (Fig. 3). This protection was most impressive with ribose- l-phosphate, but was also in clear evidence with inosine. Such results of such experiments have been presented as supporting evidence for the essential nature of the modified residue (14, 15). The fact that phenylglyoxal is more efficient in inactivat- ing the enzyme than butanedione also suggests modification of an essential residue as the phenyl ring resembles the purine ring more than does the methyl group. Inosine offers greater protection to modification by phenylglyoxal than by butanedione (Table II). This finding again suggests modification of an essential residue(s) since in the presence of inosine, the approach to the essential arginine of the bulkier phenylglyoxal may be more hindered than is the approach of butanedione. The second argument in favor of an essential arginine being modified is based on a statistical analysis of the data as suggested by Tsou Chen- Lu (12). This method has been previously applied by Paterson and Knowles to the determination of two essential carboxyl groups in pepsin (16) and more recently by Rogers et al. to the determination of one essential arginine in transferrin (17). Em- ploying the data in Fig. 2, the method (see Fig. 4) suggests that there is one essential arginine per subunit in PNPase from calf spleen and that all arginines being modified have similar reactivities.

Preliminary results from this laboratory indicate that the arginine modification here employed also affects the rate of enzyme mediated 32Pi exchange into ribose-l-phosphate (2). The results here reported suggest

704 JORDAN AND WU

that arginine may serve as the anionic rec- ognition site on this enzyme, a role attrib- uted to arginine in several other enzymes (Refs. 14, 17, 18, and references therein). A similar role for arginines in other enzymes participating in the purine salvage pathway is an attractive possibility.

Finally, in addition to the arginine here implicated, cysteine (4, 19,20) and histidine (20) have been suggested to be essential in the action of PNPase from various sources.

ACKNOWLEDGMENT

We want to thank Professor Joseph Fu and his associates of the College of Medicine and Dentistry of New Jersey and to Hoffmann-La Roche Inc., Nutley, New Jersey, for their help with the amino acid analy- ses. Financial support by the Rutgers Research Coun- cil and the Charles and Johanna Busch Fund as well as the NIH Biomedical Research Support is gratefully acknowledged.

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Inactivation of purine nucleoside phosphorylase by modification of arginine residues