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Rabbit brain purine nucleoside phosphorylase
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 190, No. 2, October, 662-670, 1978
Rabbit Brain Purine Nucleoside
Phosphorylase
Physical and Chemical Properties. Inhibition Studies with Aminopterin, Structurally Related Compounds’ ARTHUR
Folic Acid and
S. LEWIS’
Department of Biochemistry, Albert Einstein College of Medicine, Yeshiva University, Bronx, New York 10461,and the Department of Natural Science, Medgar Evers College, City University of New York, 1150 Carroll Street, Brooklyn, New York 11225 Received January
23,1978: revised June 19,1976
Rabbit brain purine nucleoside phosphorylase used in this study was purified 600%fold to apparent homogeneity and a specific activity 0150 pm01 mir-’ mg -’ protein. A molecular weight of 70.000 daltons was determined for the native enzyme by gel filtration on Sephadex. Electrophoresis on polyacrylamide gel, in presence of sodium dodecyl sulfate, gave a subunit molecular weight of 34,500 daltons, suggesting that the enzyme is dimeric with, probably, identical subunits. The relationship of the structure of certain biologically active substances to their inhibitory action on the enzyme was examined. Folic acid and the compound D,L-6methyl 5,6,7,8-tetrahydropterine, with similar substituents on their primary ring structure, were competitive inhibitors of the enzyme. The inhibition constants calculated were 3.37 x 1O-5 M for folic acid and 3.80 x 10e5 M for n,L-g-methyl 5,6,7,8-tetrahydropterine, Aminopterin and the purine analog 8-aza-2,6-diaminopurine, with similar substituents on their primary ring structure, were noncompetitive inhibitors of the enzyme. Their respective inhibition constants were 1.50 X 10m4 and 1.95 X 10m4M. Erythro-9-(2-hydroxy-3-nonyl) adenine, an adenosine deaminase inhibitor, was also examined for inhibitory pottllcy with mammalian purine nucleoside phosphorylase, and was observed to be a competitive inhibitor of this enzyme, with an inhibition constant of 1.90 x 10m4M. Tile Michaelis constant for the substrate guanosine was near 6.0 x 10m5M. Physical probe of the nature of the functional groups which participate in enzymic catalysis implicated both hiitidine and cysteine as the essential catalytic species. Photooxidation studies suggested a pH-dependem sensitivity of an essential catalytic group, and its probable location at the active site.
The properties of mammalian purine nucleoside phosphorylase (purine nucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1) have not been extensively investigated. Studies which have extended the early observations of Klein (1) and Kalckar (2) have been concerned primarily with the metabolic activities of the enzyme. In recent years, a few reports have dealt with the purification of the mammalian enzyme to homogeneity, and have described some ’ Address correspondence to the City University address. *This work was supported in part by Research Grant AM 17622 from the National Institutes of Arthritis, Metabolic and Digestive Diseases of the National Institutes of Health.
of the properties of the purified enzyme (3-11). The recent implication of this enzyme in the combined immunological deficiency syndrome in humans (12-14) has considerably enhanced its importance in human metabolism. An understanding of the characteristic properties of this enzyme, and its pharmacological response to common agents, may w3l.l be appreciated. This investigation presents, as a novel observation, the inhibitory effects of folic acid and aminopterin on tl- e catalytic activity of mammalian purine nucleoside phosphorylase. The relationship of the structure of these compounds to their inhibitory action on the enzyme was compared, and tht 662
0003-9861/78/1902-0662$02.00/0 Copyright 0 1978 by Academic Press, Inc. All rigbta of reproduction in any form reserved.
RABBIT
BRAIN
PURINE
PHOSPHORYLASE
NUCLEOSIDE
dependence of the specific inhibitory action of folic acid, aminopterin, and structurally related compounds on the nature of the substituents to their primary ring structure, was experimentally demonstrated. Evidence is presented in this study which suggest that rabbit brain purine nucleoside phosphorylase is of a dimeric structure with, probably, identical subunits. Studies are also presented here which attempt to probe the nature of the active center of the enzyme and the essential groups which participate in enzymic catalysis. EXPERIMENTAL
PROCEDURES
Materials Frozen rabbit brains were purchased from Pel-Freez Biologicals. Purine and purine nucleosides were products of Calbiochem. Folic acid, r+-6-methyl 5,6,7,8tetrahydropterine, aminopterin, and 8-aza-2,6diaminopurine were products of Sigma Chemical Co. Erythro-9-(2-hydroxy-3-nonyl) adenine was a product of Burroughs Wellcome Co. DEAE-cellulose and hydroxylapatite were products of Bio-Rad. Sephadex G-100 and G-206 were products of Pharmacia. The buffer system was prepared by mixing equimolar solutions (0.2 M) of dibasic sodium phosphate and monobasic potassium phosphate.
Methods Enzyme assay. The assay for the phosphorolytic reaction of the enzyme has been previously reported (6, 7). All assays in this study were performed at constant ionic strength, in 0.2 M phosphate buffer. Inhibition studies were performed in 0.2 M phosphate buffer, at the pH of optimal activity of the enzyme (pH 6.0). Specific sctivity was recorded as pmol guanosine phosphorylyzed mini’ mg-’ protein, based on molar absorptivities of 11,900 for the substrate guaTABLE
INHIBITION
STUDIES
663
nosine and 9,940 for the product guanine, in 0.2 M phosphate buffer (pH 6.0) at 252 nm. Purification of the enzyme. Except for slight modifications, the enzyme from rabbit brain was purified by procedures almost identical with that reported for the enzyme from rabbit liver (6) and the enzyme from bovine brain (7). The enzvme was homogeneous by disc electrophoresis in presence and absence of sodium dodecyl sulfate, at 6,000-fold purification and a specific activity of 50 pm01 mm’ mg-’ protein. The purification steps are summarized in Table I. Figure 1 is a photograph of the electrophoresed enzyme on polyacrylamide gel. Purification of folic acid and aminopterin. Folic acid and aminopterin used in this study were fust purified by chromatography on DEAE-cellulose. The compounds (250 mg) were each dissolved in a minimal volume of 0.1 M Tris base which was 5 mM with 2mercaptoethanol, and adsorbed on a column of DEAE-cellulose equilibrated against 0.01 M phosphate buffer (pH 7.5). The compounds were each eluted by gradient elution, consisting of 0.025 M phosphate buffer (pH 7.5) in the reservoir feeding the column, and 0.2 M KC1 in the same buffer in the second reservoir. All buffers contained 5 mM L-mercaptoethanol. The inhibitory potency of folic acid was increased severalfold by purifr,ation on DEAE-cellulose. The chromatographic purification of aminopterin did not appear to enhance nor suppress its inhibitory potency.
Determination of ionizable groups in the enzyme. Maximal initial velocities and Michaelis constants were determined from double reciprocal plots of substrate saturation of the enzyme with guanosine as the variable substrate in 0.2 M phosphate buffer, between pH 5.0 and 9.0, at 0.5 pH units. Plots of the negative log of V,, versus pH, and of pK, versus pH, wer? constructed after the method of Dixon (15). Apparent pKa values were determined from inflections of unit slope from the above plots (15).
Determination of the molecular weight of the enzyme. The molecular weight of the native enzyme was I
SUMMARYOFISOLATIONANDPURIFICATIONPROCEDURES Purification
procedures
Homogenate extract First ammonium sulfate fractionation First DEAE-cellulose chromatography Chromatography on hydroxylapatite Gel filtration on Sephadex G-100 Heating at 60°C for 5 min Selective precipitation with saturated
(NH&SO4
Total protein bg)
Specific activity (micromoles/ min/mg protein)
34,029 6,978 694 8.5 2.5 1.8 0.64
0.008 0.040 0.251 10.80 19.80 30.6 50.0
Percentage yield
100 108 67.5 35.3 19.4 22.0 12.0
PWification (-fold)
1 5.3 33 1415 2605 3825 6000
664
ARTHUR
FIG. 1. Photograph of disc gel electrophoresis of the enzyme on polyacrylamide after the final stages of purification. The amount of protein was 35 pg, and the buffer system was Tris-glycine buffer (pH 9.5). determined by 1) Gel filtration on Sephadex G-100. The gel was swollen in distilled water for 48 h and, after packing, the column was intermittently washed with 0.025 M phosphate buffer (pH 7.5) for 48 h. The gel bed dimensions were 1.6 x 42 cm, with a void volume of 43 ml. The protein standards were cytochrome c (12,000)~ myoglobin (17,000), ovalbumin (43,000), bovine serum albumin (66,500), and guanine deaminase (60,000). The enzyme (3.5 mg protein in 1.0 ml buffer) and the protein standards (4.0 mg in 1.0 ml buffer) were filtered successively through the column, and eluted with 0.025 M phosphate buffer (pH 7.5). Fractions of 2.0 ml were collected. The molecular weight was calculated after the method of Andrews (16). 2) Gel filtration on Sephadex G-200. The molecular
S. LEWIS weight of the native enzyme was also determined by gel filtration on Sephadex G-200. The gel bed dimensions were 2.0 x 42 cm with a void volume of 72 ml. Enzyme and protein standards were eluted successively with 0.025 M phosphate buffer, and fractions of 2.5 ml were collected. The molecular weight was calculated as above. Electrophoretic method. The molecular weight of the subunits of rabbit brain purine nucleoside phosphorylase was determined by electrophoresis on polyacrylamide, in presence of sodium dodecyl sulfate, after the method of Weber and Osbom (17). The protein standards were myoglobin, cr-chymotrypsinogen-A, ovalbumin, guanine deaminase, and bovine serum albumin. Photooxidation of the enzyme. The strong susceptibility of the enzyme to photoinactivation in presence of methylene blue made possible the use of simple equipment in this study. The source of illumination was a biology desk lamp fitted with a 100 watt tungsten light bulb and reflector. The lamp was enclosed with white paper within a tripod; and the samples were illuminated at the aperture above in a beaker containing ice and water about 22 cm above the light source. Just before each experiment, the enzyme was diluted with methylene blue solution in 0.1 M phosphate buffer saturated with oxygen at the pH of the experiment. The final concentration of the enzyme was 1 pM, and the final concentration of methylene blue was 10 mg%. The periods of illumination for the experiments varied, and are specified in the legends to the figures. Calculations were based on values determined for light controls which were similarly prepared in 0.1 M phosphate buffer, at the same pH as test samples, and illuminated in absence of methylene blue. Dark controls containing methylene blue, which were prepared and stored in the dark for the duration of each experiment, showed no significant inactivation. Aliquots, which were removed at various time intervals of illumination, were directly transferred to a cuvette contaming 2.5 ml of substrate (100 pM guanosine in 0.2 M phosphate buffer, pH 6.0) and assayed immediately for residual catalytic activity. Preparation of inhibitors for assay. Fixed concentrations of the inhibitors were prepared together with the variable substrate guanosine, in 0.2 M phosphate buffer, at pH 6.0, the pH of optimal activity of the enzyme. Substantial absorbance of folic acid and aminopterin at the wavelength of this investigation (252 nm) necessitated compensatorily low concentrations of the variable substrate. RESULTS
Active Site Studies pH-activity studies: Determination of ionizable catalytic groups on the enzyme. A broad pH-activity profile was determined
RABBIT
BRAIN
PURINE
NUCLEOSIDE
PHOSPHORYLASE
I
INHIBITION
STUDIES
665
I
PH
FIG. 2. Determination of ionizable groups in the enzyme. a), Plot of V,, uersus pH. b), Plot of pK, uersus pH (15). V,, and pK,,, values were determined from double reciprocal plots of substrate saturation of the enzyme with guanosine as the variable substrate, in 0.2 M phosphate buffer, between pH 5.0 and 9.0, at 0.5 pH units. Values for V mar are the negative log of relative POD. pKa values were calculated from inflections of unit slopes of the graphs (15). Other conditions are specified in the methods section.
\.i I
. 5
IO
\ 15
20
Tim*
(mini
25
30
35
I
FIG. 3. Inactivation
of the enzyme by p-chloromercuribenzoate (ClHgBzO). The enzyme (1 CM) was incubated with ClHgBzO (100 pM) in 0.1 M phosphate buffer (pH 7.0) at 25°C; and aliquots were removed and assayed at various time intervals. Total inactivation, as shown of the graph, was obtained by increasing the concentration of the inhibitor to 200 pM after 15 min of incubation. The addition of an excess of 2mercaptoethanol(2-RSH) to the totally inactivated enzyme, as shown above, gave almost immediate and complete regeneration of enzymic activity.
between
pH
5.5 and
8.0 with
a small
maxi-
mum at pH 6.0, which probably reflected the acidic and basic limits of the catalytic groups of the enzyme. Apparent pKa values, which were determined from inflections of unit slopes of the plot of the negative log of V,,, uersus pH (E), corresponded to pH 5.8 and pH 8.5 for groups in complexation with the substrate. Inflec-
tions which were observed with pKa values near pH 6.8 and 7.8, have been attributed to the ionizations of free inorganic orthophosphate, and inorganic orthophosphate which was probably bound at the active site of the enzyme (Fig. 2a). Inorganic orthophosphate was the major component of the buffer system and also a substrate for the enzyme. Apparent pKa values which were similarly determined from a plot of pK, uersus pH, gave inflections of unit slope near pH 5.6 and 7.5 for groups which are complexed at the active site of the enzyme; and near pH 8.2 for a group in the free enzyme (Fig. 2b). These results are strongly suggestive of the participation of hi&dine (pKa, 5.6 and 5.8) and cysteine (pKa 8.2 and 8.5) in enzymic catalysis.
Inhibition studies with p-chloromercuribenzoate. The enzyme Gas strongly susceptible to inactivation by p-chloromercuribenzoate. Incubation of the enzyme at a concentration of 1 PM with an excess of pchloromercuribenzoate (100 PM) in 0.1 M phosphate buffer (pH 7.0) at 25”C, gave a loss of 85% of the enzymic activity within 15 min of incubation. Total inactivation was achieved by increasing the concentration of the inhibitor to 200 PM. The catalytic activity of the inactivated enzyme was completely restored by treatment with an excess of 2-mercaptoethanol (Fig. 3). These
666
ARTHUR
results suggest strongly a reversible interaction of an essential cysteine SH with the sulfhydryl reagent. Photooxidation studies. The enzyme showed a high sensitivity to photooxidation in presence of methylene blue. A loss of almost 95% of the enzymic activity was sustained within 40 min of irradiation (Fig. 4a, slope 3). A first order rate was observed for the disappearance of catalytic activity, suggestive of the oxidation of a group which may be essential for catalysis (see Fig. 4a, slope 2). Substantial protection against inactivation was afforded the enzyme by hypoxanthine, a strong competitive inhibitor of the enzyme (Fig. 4a, slope 1). This latter observation is consistent with the localization of the susceptible group at the active site (18, 19). A progressive increase of photoinactivation of the enzyme was observed as the pH was increased above pH 7.0; suggestive of the dependence on pH of a photosensitive group which may be essential for enzymic catalysis (Fig. 4b). Molecular Weight of the Enzyme Molecular weights of 70,000 daltons were determined for the native enzyme in separate experiments on Sephadex G-100 and Sephadex G-200. A mean molecular weight
5
IO Irrad.
20 time
30
40
S. LEWIS
of 70,000 daltons is reported here for native put-me nucleoside phosphorylase from rabbit brain (Fig. 5a). A molecular weight of 34,500 daltons was determined for the subunits of rabbit brain purine nucleoside phosphorylase by electrophoretic technique (Fig. 5b). These results suggest that the native enzyme may be dimeric in structure, with possibly identical subunits. Competitive inhibition of the enzyme by folic acid and DL-6-methyl 5,6,7,84etrahydropterine. Initial velocity studies in presence of folk acid showed that this compound was a competitive inhibitor of the enzyme (Fig. 6a). Negative cooperative effects (20) were elicited by folic acid at low concentrations of the variable substrate. Initial velocity studies in presence of the compound r&L-8methyl 5,6,7,8-tetrahydropterine, which bears similar substituents to the primary cyclic structure, also gave competitive inhibition of the enzyme (Fig. 6b). Noncompetitive inhibition by aminopterin and 8-aza-2,6-diaminopurine. The folic acid antagonist, aminopterin, bearing primary amine substituents to the primary cyclic structure, was a noncompetitive inhibitor of rabbit brain purine nucleoside phosphorylase. The inhibition by aminopterin manifested itself by suppressive ef-
I
(min)
FIG. 4. Photooxidation of the enzyme. The buffer was 0.1 M phosphate buffer saturated with oxygen. The concentration of enzyme was 1 PM and the concentration of methylene blue was 10 mg/lOO ml. Other conditions are specified under methods. a), These experiments were conducted at pH 7.0. Slope 3 shows loss of enzymic activity as a function of irradiation time. Slope 2 shows first order rate for the loss of enzymic activity. Slope 1 shows the protection afforded the enzyme by hypoxanthine. b), pH Dependence of photooxidation. Tests were irradiated for 2 min in presence of dye and controls were similarly irradiated in absence of dye; in 0.1 M phosphate buffer at each pH. Loss of activity was calculated as a percentage of control.
RABBIT
BRAIN
PURINE
NUCLEOSIDE
PHOSPHORYLASE
INHIBITION
\
32-
= E -
667
b l
34-
STUDIES
cyto.
\
4b-
30 t
i 2
i2 2s i
\
E”
’ 26. c .o; 24.
221
rob. br. PNPas.. -
f
. 4:2
4:4
Pib
Q
]
‘.
4.4-
4.2-
,2
\ ~6myo~‘obi”~~
,4
log mol.wt.
+chymo..P
mobility
FIG. 5. Determination of molecular weight of the enzyme. a), The molecular weight of the native enzyme was determined by gel filtration of Sephadex G-100 and G-200. The graph above shows the results obtained with Sephadex G-100. The conditions are specified at length in the section on methods. b), The subunit molecular weight was determined by electrophoresis on polyacrylamide, in presence of sodium dodecyl sulfate. The conditions are specified at length in the section on methods.
IO 20 [Guanosine]
mM-1
40 [Guanosine]
60
SO mM-1
FIG. 6. Double reciprocal plots of initial velocity versus [s] for competitive inhibition of the enzyme by a, folic acid, and b, n,L-6-methyl5,6,7,8-tetrahydropterine (CHs-THP). Guanosine was the variable substrate. The concentrations of the inhibitors, as indicated on the graphs, were prepared together with the substrate in 0.2 M phosphate buffer (pH 6.0). Due to substantial absorbance of folic acid at 252 mn, the concentrations of folk acid at 50 and 100 F were examined at a maximal substrate concentration of only 100 pM. The amount of enzyme used was 0.65 pg protein in 50 d. Initial velocities are expressed as POD/mm. The inhibition constant is listed in Table II.
fects both on the maximal catalytic rate and on the Michaelis constant for the variable substrate. The primary double reciprocal plots for this inhibition intersected
below the x-axis, so that the apparent dissociation constant for the central complex was significantly smaller than apparent Michaelis constants (Fig. 7a).
ARTHUR
S. LEWIS
/”
1020
40
[guanosine]
80 mM-1
[guanosine]
mM-1
FIG. 7. Double reciprocal plots of initial velocity uersus [s] for noncompetitive inhibition of the enzyme by a, aminopterin, and b, 8-aza-2,6-diaminopurine (8-aza-diaminopurine), with guanosine as the variable substrate in 0.2 M phosphate buffer (pH 6.0). Various concentrations of inhibitors were prepared together with the substrate. The significant absorbance of aminopterin at 100 PM was compensated for by a reduction in the maximal concentration of the variable substrate to only 50 j.tM, as indicated above. The amount of enzyme used was 0.65 ccgprotein in 50 al. Initial velocities were expressed as AOD/min.
L reciprocal plots of initial velocity inhibition of the enzyme by erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA). Guanosine was the variable substrate. Concentrations of the inhibitor were prepared together with the variable substrate in 0.2 M phosphate buffer (pH 6.0). The amount of enzyme used was 0.65 pg in 50 al and initial velocities are shown as relative AOD/min. FIG.
8. Double
versus [s] for competitive
The purine analog, 8-axa-2,6-diaminopurine, bears some similarity to aminopterin in that its primary amine substituents are identically disposed. Initial velocity studies
in presence of this compound indicated that the compound was similarly a noncompetitive inhibitor of purine nucleoside phosphorylase (Fig. 7b). In spite of the grossly dissimilar substituents to carbon-6 of folic acid and D,L-6methyl 5,6,7,8-tetrahydropterine, and also with respect to aminopterin and 8-aza-2,6diaminopurine, a striking similarity was observed in the values for the inhibition constants which were calculated for the respective pairs of competitive and noncompetitive inhibitors. The bulky substituents to carbon 6 of folic acid and aminopterin, may not have sterically hindered nor enhanced their binding capacity relative to their respective counterparts, r&r,-6-methyl5,6,7,8tetrahydropterine and 8-aza-2,6-diaminopurine. The selective pattern of the observed inhibitions may probably be governed by the nature of the interactions of the enzyme with the substituents to the primary cyclic structure of these compounds. Competitive inhibition by erythro-g-(2hydroxy-3-nonyl) adenine. The inhibitory potency of erythro-9-(2-hydroxy-3-nonyl) adenine, an adenosine deaminase inhibitor (21, 22), was also examined with rabbit brain purine nucleoside phosphorylase, and
RABBIT
BRAIN
PURINE
NUCLEOSIDE
PHOSPHORYLASE
TABLE
INHIBITION
STUDIES
669
II
SUMMARY OF KINETIC CONSTANTS FOR INHIBITORS OF RABBIT BRAIN PURINE NUCLEOSIDE PHOSPHORYLASE’ Inhibitor
Folic acid n,L-6-methyl5,6,7,8-tetrahydropterine Aminopterin 8-Axa-2,6-diaminopurine Erythro-9-(2-hydroxy-3-nonyl) adenine
Apparent inhibition constant (M)* 3.37 3.80 1.50 1.95 1.90
x x x x x
m5 N5 1o-4 1o-4 1om4
Type of inhibition Competitive Competitive Noncompetitive Noncompetitive Competitive
a Kinetic constants were determined with guanosine as variable substrate, in 0.2 M phosphate buffer (pH 6.0) at 25°C. The K,,, for guanosine was 6.0 X 10m5M. * Apparent inhibition constants were calculated from a secondary replot of the primary slopes versus the __ concentrations of inhibitor.
was found to inhibit the enzyme competitively (Fig. 8). These effects are not, at this time, properly understood. Adenosine itself was neither a substrate nor an inhibitor for the enzyme. It is possible, however, to postulate that the interactions of this analog with the enzyme may be hydrophobic in nature. The inhibition constants are summarized in Table II. DISCUSSION
Active site studies have implicated histidine and cysteine as the catalytic groups in the enzyme. The pKa values which were determined for the groups which participate in enzymic catalysis are consistent with this conclusion. The total inactivation of the enzyme by p-chloromercuribenzoate, within a relatively short period of incubation, was suggestive of its interaction with an essential cysteine residue in the enzyme. The total reactivation of the completely inactivated enzyme within 5 min of exposure to an excess of 2-mercaptoethanol, argues strongly for a reversible reduction of an essential cysteine residue. The suggested role of cysteine as a participating species in the enzymic catalysis of mammalian purine nucleoside phosphorylase has been amply documented (3-7). A first order rate which was observed for the loss of enzymic activity upon photooxidation, was suggestive of the oxidation of an essential catalytic group in the enzyme. Moreover, the protection afforded the enzyme against photooxidation by hypoxan-
thine, a competitive inhibitor, also suggests that the localization of the susceptible group may be at the active site of the enzyme (18, 19). Studies of the physical properties of this enzyme suggest that the enzyme is dimeric in structure. Comparative studies presently made in this laboratory, suggest tentatively that mammalian purine nucleoside phosphorylase may be quite similar in molecular weight and subunit structure (A, S. Lewis, unpublished observations). The biological role of folk acid derivatives in synthetic metabolic processes has been well established, and the antagonistic effects of the folate analogs, aminopterin and amethopterin, on these metabolic processeshave been amply investigated. A one-site-of-action hypothesis, however, has long been associated with the folate antagonists, as inhibitors of folate reductase. The elegant in vitro studies of Borsa and Whitmore, which have appeared in recent years (23-25), have demonstrated the nonspecific inhibitory action of the folate antagonist amethopterin (methotrexate). Inhibition constants similar to those reported here for folic acid and aminopterin have been reported for the inhibition by amethopterin of soluble purified preparations of thymidylate synthetase from mammalian and bacterial sources (25). Evidence presented in this investigation substantiates the reported findings of the nonspecific inhibitory effects of the folate antagonists. The subtle relationship of the structure of the compounds examined in this study to their inhibitory activity was clearly dem-
670
ARTHUH
on&rated. The structural dissimilarities within the pairs of the compounds examined, were not reflected in their calculated inhibition constants. These results suggest that inhibitory specificity may, probably, be resident in the nature of the substituents to the primary cyclic structure of the compounds. This latter observation may be of pharmacological significance. The enzyme fails to recognize adenosine as substrate or inhibitor, but effectively binds the adenosine analog erythro-9-(2-hydroxy-3-nonyl) adenine, in which a strongly hydrophobic residue is substituted for ribose. Moreover, the enzyme accommodates the bulky and sterically unfavorable structures of folic acid and aminopterin as inhibitors, but fails to recognize as substrates or inhibitors the phosphorylated derivatives of its normal substrates. These observations make possible the postulation of strongly hydrophobic regions within the active center of rabbit brain purine nucleoside phosphorylase. REFERENCES 1. KLEIN, W. (1935) 2. Physiol. Chem. 231,125. 2. KALCKAR, H. (1947) J. Biol. Chem. 167,461-475. 3. KIM, B. K., CHA, S., AND PARKS, R. E., JR. (1968)
J. Biol. Chem. 243, 1763-1770. 4. AGARWAL, R. P., AND PARKS, R. E., JR. (1971) J.
Biol. Chem. 246,3763-3768. 5. LEWIS, A. S. (1973) Ph.D. Thesis, City University of New York. 6. LEWIS, A. S., AND GLANTZ, M. D., (1976) J. Biol. Chem. 261,407-413. 7. LEWIS, A. S., AND GLANTZ, M. D. (1976) Biochem-
S. LEWIS
istry 15,4452-4457. 8. LEWIS, A. S. (1977) J. Biol. Chem. 252, 732-738. 9. EDWARDS, Y. H., EDWARDS, P. A., AND HOPKINSON, D. A. (1973) FEBS Lett 32,235-237. 10. MILMAN, G., ANTON, D. L., AND WEBER, J. S. (1976) Biochemistry 15,4967-4973. 11. KIM, B. K., CHA, S., AND PARKS, R. E., JR. (1968) J. Biol. Chem. 243,1771-1776. 12. GIBLETT, E. R., AMMANN, A. J., SANDMAN, R., WARA, D. W., AND DIAMOND, L. K. (1975) Lancet 1, 1010-1013. 13. COHEN, A., DOYLE, D., MARTIN, D. W., JR., AMMANN, A. J. (1976) N. Engl. J. Med. 295,
1449-1454. 14. STOOP, J. W., ZECERS, B. J. M., HENDRICKS, G. F. M., SIECENBEEK VAN HEUKELOM, L. H., AND BALLIEUX, R. E. (1977) N. En&. J. Med. 296, 651-655. 15. DIXON, M. (1953) Biochem. J. 56, 161-170. 16. ANDREWS, P. (1970) Methods B&hem. Anal. 18, l-53. 17. WEBER, K., AND OSBORN, M. (1969) J. Biol. Chem. 244,4406-4412. 18. WESTHEAD, E. W. (1965) Biochemistry 4, 2139-2144. 19. BRAND, K., TSOLAS, O., AND HORECKER, B. L. (1969) Arch. Biochem. Biophys. 130, 521-529. 20. KOSHLAND, D. E., JR., (1970) The Molecular Basis In “The Enzymes” for Enzyme Regulation. (Boyer, P. D., ed.), Ed. 3, Vol. 1, pp. 341-396, Academic Press, New York. 21. SCHAEFFER, H. J., AND SCHWENDER, C. F. (1974) J. Med. Chem. l?:, 6-8. 22. LAPI, L., AND COHEN, S. S. (1977) Biochem. Phar-
macol. 26, 71-76. 23. BORSA, J., AND WHITMORE, G. F. (1969) Cancer
Res. 29,737-747. 24. BORSA, J., AND WHITMORE, G. F. (1969) Mol.
Pharmacol. 5,303-317. 25. BORSA, J., AND WHITMORE, G. F. (1969) Mol.
Pharmacol. 5,318-332.
Rabbit Brain Purine Nucleoside
Phosphorylase
Physical and Chemical Properties. Inhibition Studies with Aminopterin, Structurally Related Compounds’ ARTHUR
Folic Acid and
S. LEWIS’
Department of Biochemistry, Albert Einstein College of Medicine, Yeshiva University, Bronx, New York 10461,and the Department of Natural Science, Medgar Evers College, City University of New York, 1150 Carroll Street, Brooklyn, New York 11225 Received January
23,1978: revised June 19,1976
Rabbit brain purine nucleoside phosphorylase used in this study was purified 600%fold to apparent homogeneity and a specific activity 0150 pm01 mir-’ mg -’ protein. A molecular weight of 70.000 daltons was determined for the native enzyme by gel filtration on Sephadex. Electrophoresis on polyacrylamide gel, in presence of sodium dodecyl sulfate, gave a subunit molecular weight of 34,500 daltons, suggesting that the enzyme is dimeric with, probably, identical subunits. The relationship of the structure of certain biologically active substances to their inhibitory action on the enzyme was examined. Folic acid and the compound D,L-6methyl 5,6,7,8-tetrahydropterine, with similar substituents on their primary ring structure, were competitive inhibitors of the enzyme. The inhibition constants calculated were 3.37 x 1O-5 M for folic acid and 3.80 x 10e5 M for n,L-g-methyl 5,6,7,8-tetrahydropterine, Aminopterin and the purine analog 8-aza-2,6-diaminopurine, with similar substituents on their primary ring structure, were noncompetitive inhibitors of the enzyme. Their respective inhibition constants were 1.50 X 10m4 and 1.95 X 10m4M. Erythro-9-(2-hydroxy-3-nonyl) adenine, an adenosine deaminase inhibitor, was also examined for inhibitory pottllcy with mammalian purine nucleoside phosphorylase, and was observed to be a competitive inhibitor of this enzyme, with an inhibition constant of 1.90 x 10m4M. Tile Michaelis constant for the substrate guanosine was near 6.0 x 10m5M. Physical probe of the nature of the functional groups which participate in enzymic catalysis implicated both hiitidine and cysteine as the essential catalytic species. Photooxidation studies suggested a pH-dependem sensitivity of an essential catalytic group, and its probable location at the active site.
The properties of mammalian purine nucleoside phosphorylase (purine nucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1) have not been extensively investigated. Studies which have extended the early observations of Klein (1) and Kalckar (2) have been concerned primarily with the metabolic activities of the enzyme. In recent years, a few reports have dealt with the purification of the mammalian enzyme to homogeneity, and have described some ’ Address correspondence to the City University address. *This work was supported in part by Research Grant AM 17622 from the National Institutes of Arthritis, Metabolic and Digestive Diseases of the National Institutes of Health.
of the properties of the purified enzyme (3-11). The recent implication of this enzyme in the combined immunological deficiency syndrome in humans (12-14) has considerably enhanced its importance in human metabolism. An understanding of the characteristic properties of this enzyme, and its pharmacological response to common agents, may w3l.l be appreciated. This investigation presents, as a novel observation, the inhibitory effects of folic acid and aminopterin on tl- e catalytic activity of mammalian purine nucleoside phosphorylase. The relationship of the structure of these compounds to their inhibitory action on the enzyme was compared, and tht 662
0003-9861/78/1902-0662$02.00/0 Copyright 0 1978 by Academic Press, Inc. All rigbta of reproduction in any form reserved.
RABBIT
BRAIN
PURINE
PHOSPHORYLASE
NUCLEOSIDE
dependence of the specific inhibitory action of folic acid, aminopterin, and structurally related compounds on the nature of the substituents to their primary ring structure, was experimentally demonstrated. Evidence is presented in this study which suggest that rabbit brain purine nucleoside phosphorylase is of a dimeric structure with, probably, identical subunits. Studies are also presented here which attempt to probe the nature of the active center of the enzyme and the essential groups which participate in enzymic catalysis. EXPERIMENTAL
PROCEDURES
Materials Frozen rabbit brains were purchased from Pel-Freez Biologicals. Purine and purine nucleosides were products of Calbiochem. Folic acid, r+-6-methyl 5,6,7,8tetrahydropterine, aminopterin, and 8-aza-2,6diaminopurine were products of Sigma Chemical Co. Erythro-9-(2-hydroxy-3-nonyl) adenine was a product of Burroughs Wellcome Co. DEAE-cellulose and hydroxylapatite were products of Bio-Rad. Sephadex G-100 and G-206 were products of Pharmacia. The buffer system was prepared by mixing equimolar solutions (0.2 M) of dibasic sodium phosphate and monobasic potassium phosphate.
Methods Enzyme assay. The assay for the phosphorolytic reaction of the enzyme has been previously reported (6, 7). All assays in this study were performed at constant ionic strength, in 0.2 M phosphate buffer. Inhibition studies were performed in 0.2 M phosphate buffer, at the pH of optimal activity of the enzyme (pH 6.0). Specific sctivity was recorded as pmol guanosine phosphorylyzed mini’ mg-’ protein, based on molar absorptivities of 11,900 for the substrate guaTABLE
INHIBITION
STUDIES
663
nosine and 9,940 for the product guanine, in 0.2 M phosphate buffer (pH 6.0) at 252 nm. Purification of the enzyme. Except for slight modifications, the enzyme from rabbit brain was purified by procedures almost identical with that reported for the enzyme from rabbit liver (6) and the enzyme from bovine brain (7). The enzvme was homogeneous by disc electrophoresis in presence and absence of sodium dodecyl sulfate, at 6,000-fold purification and a specific activity of 50 pm01 mm’ mg-’ protein. The purification steps are summarized in Table I. Figure 1 is a photograph of the electrophoresed enzyme on polyacrylamide gel. Purification of folic acid and aminopterin. Folic acid and aminopterin used in this study were fust purified by chromatography on DEAE-cellulose. The compounds (250 mg) were each dissolved in a minimal volume of 0.1 M Tris base which was 5 mM with 2mercaptoethanol, and adsorbed on a column of DEAE-cellulose equilibrated against 0.01 M phosphate buffer (pH 7.5). The compounds were each eluted by gradient elution, consisting of 0.025 M phosphate buffer (pH 7.5) in the reservoir feeding the column, and 0.2 M KC1 in the same buffer in the second reservoir. All buffers contained 5 mM L-mercaptoethanol. The inhibitory potency of folic acid was increased severalfold by purifr,ation on DEAE-cellulose. The chromatographic purification of aminopterin did not appear to enhance nor suppress its inhibitory potency.
Determination of ionizable groups in the enzyme. Maximal initial velocities and Michaelis constants were determined from double reciprocal plots of substrate saturation of the enzyme with guanosine as the variable substrate in 0.2 M phosphate buffer, between pH 5.0 and 9.0, at 0.5 pH units. Plots of the negative log of V,, versus pH, and of pK, versus pH, wer? constructed after the method of Dixon (15). Apparent pKa values were determined from inflections of unit slope from the above plots (15).
Determination of the molecular weight of the enzyme. The molecular weight of the native enzyme was I
SUMMARYOFISOLATIONANDPURIFICATIONPROCEDURES Purification
procedures
Homogenate extract First ammonium sulfate fractionation First DEAE-cellulose chromatography Chromatography on hydroxylapatite Gel filtration on Sephadex G-100 Heating at 60°C for 5 min Selective precipitation with saturated
(NH&SO4
Total protein bg)
Specific activity (micromoles/ min/mg protein)
34,029 6,978 694 8.5 2.5 1.8 0.64
0.008 0.040 0.251 10.80 19.80 30.6 50.0
Percentage yield
100 108 67.5 35.3 19.4 22.0 12.0
PWification (-fold)
1 5.3 33 1415 2605 3825 6000
664
ARTHUR
FIG. 1. Photograph of disc gel electrophoresis of the enzyme on polyacrylamide after the final stages of purification. The amount of protein was 35 pg, and the buffer system was Tris-glycine buffer (pH 9.5). determined by 1) Gel filtration on Sephadex G-100. The gel was swollen in distilled water for 48 h and, after packing, the column was intermittently washed with 0.025 M phosphate buffer (pH 7.5) for 48 h. The gel bed dimensions were 1.6 x 42 cm, with a void volume of 43 ml. The protein standards were cytochrome c (12,000)~ myoglobin (17,000), ovalbumin (43,000), bovine serum albumin (66,500), and guanine deaminase (60,000). The enzyme (3.5 mg protein in 1.0 ml buffer) and the protein standards (4.0 mg in 1.0 ml buffer) were filtered successively through the column, and eluted with 0.025 M phosphate buffer (pH 7.5). Fractions of 2.0 ml were collected. The molecular weight was calculated after the method of Andrews (16). 2) Gel filtration on Sephadex G-200. The molecular
S. LEWIS weight of the native enzyme was also determined by gel filtration on Sephadex G-200. The gel bed dimensions were 2.0 x 42 cm with a void volume of 72 ml. Enzyme and protein standards were eluted successively with 0.025 M phosphate buffer, and fractions of 2.5 ml were collected. The molecular weight was calculated as above. Electrophoretic method. The molecular weight of the subunits of rabbit brain purine nucleoside phosphorylase was determined by electrophoresis on polyacrylamide, in presence of sodium dodecyl sulfate, after the method of Weber and Osbom (17). The protein standards were myoglobin, cr-chymotrypsinogen-A, ovalbumin, guanine deaminase, and bovine serum albumin. Photooxidation of the enzyme. The strong susceptibility of the enzyme to photoinactivation in presence of methylene blue made possible the use of simple equipment in this study. The source of illumination was a biology desk lamp fitted with a 100 watt tungsten light bulb and reflector. The lamp was enclosed with white paper within a tripod; and the samples were illuminated at the aperture above in a beaker containing ice and water about 22 cm above the light source. Just before each experiment, the enzyme was diluted with methylene blue solution in 0.1 M phosphate buffer saturated with oxygen at the pH of the experiment. The final concentration of the enzyme was 1 pM, and the final concentration of methylene blue was 10 mg%. The periods of illumination for the experiments varied, and are specified in the legends to the figures. Calculations were based on values determined for light controls which were similarly prepared in 0.1 M phosphate buffer, at the same pH as test samples, and illuminated in absence of methylene blue. Dark controls containing methylene blue, which were prepared and stored in the dark for the duration of each experiment, showed no significant inactivation. Aliquots, which were removed at various time intervals of illumination, were directly transferred to a cuvette contaming 2.5 ml of substrate (100 pM guanosine in 0.2 M phosphate buffer, pH 6.0) and assayed immediately for residual catalytic activity. Preparation of inhibitors for assay. Fixed concentrations of the inhibitors were prepared together with the variable substrate guanosine, in 0.2 M phosphate buffer, at pH 6.0, the pH of optimal activity of the enzyme. Substantial absorbance of folic acid and aminopterin at the wavelength of this investigation (252 nm) necessitated compensatorily low concentrations of the variable substrate. RESULTS
Active Site Studies pH-activity studies: Determination of ionizable catalytic groups on the enzyme. A broad pH-activity profile was determined
RABBIT
BRAIN
PURINE
NUCLEOSIDE
PHOSPHORYLASE
I
INHIBITION
STUDIES
665
I
PH
FIG. 2. Determination of ionizable groups in the enzyme. a), Plot of V,, uersus pH. b), Plot of pK, uersus pH (15). V,, and pK,,, values were determined from double reciprocal plots of substrate saturation of the enzyme with guanosine as the variable substrate, in 0.2 M phosphate buffer, between pH 5.0 and 9.0, at 0.5 pH units. Values for V mar are the negative log of relative POD. pKa values were calculated from inflections of unit slopes of the graphs (15). Other conditions are specified in the methods section.
\.i I
. 5
IO
\ 15
20
Tim*
(mini
25
30
35
I
FIG. 3. Inactivation
of the enzyme by p-chloromercuribenzoate (ClHgBzO). The enzyme (1 CM) was incubated with ClHgBzO (100 pM) in 0.1 M phosphate buffer (pH 7.0) at 25°C; and aliquots were removed and assayed at various time intervals. Total inactivation, as shown of the graph, was obtained by increasing the concentration of the inhibitor to 200 pM after 15 min of incubation. The addition of an excess of 2mercaptoethanol(2-RSH) to the totally inactivated enzyme, as shown above, gave almost immediate and complete regeneration of enzymic activity.
between
pH
5.5 and
8.0 with
a small
maxi-
mum at pH 6.0, which probably reflected the acidic and basic limits of the catalytic groups of the enzyme. Apparent pKa values, which were determined from inflections of unit slopes of the plot of the negative log of V,,, uersus pH (E), corresponded to pH 5.8 and pH 8.5 for groups in complexation with the substrate. Inflec-
tions which were observed with pKa values near pH 6.8 and 7.8, have been attributed to the ionizations of free inorganic orthophosphate, and inorganic orthophosphate which was probably bound at the active site of the enzyme (Fig. 2a). Inorganic orthophosphate was the major component of the buffer system and also a substrate for the enzyme. Apparent pKa values which were similarly determined from a plot of pK, uersus pH, gave inflections of unit slope near pH 5.6 and 7.5 for groups which are complexed at the active site of the enzyme; and near pH 8.2 for a group in the free enzyme (Fig. 2b). These results are strongly suggestive of the participation of hi&dine (pKa, 5.6 and 5.8) and cysteine (pKa 8.2 and 8.5) in enzymic catalysis.
Inhibition studies with p-chloromercuribenzoate. The enzyme Gas strongly susceptible to inactivation by p-chloromercuribenzoate. Incubation of the enzyme at a concentration of 1 PM with an excess of pchloromercuribenzoate (100 PM) in 0.1 M phosphate buffer (pH 7.0) at 25”C, gave a loss of 85% of the enzymic activity within 15 min of incubation. Total inactivation was achieved by increasing the concentration of the inhibitor to 200 PM. The catalytic activity of the inactivated enzyme was completely restored by treatment with an excess of 2-mercaptoethanol (Fig. 3). These
666
ARTHUR
results suggest strongly a reversible interaction of an essential cysteine SH with the sulfhydryl reagent. Photooxidation studies. The enzyme showed a high sensitivity to photooxidation in presence of methylene blue. A loss of almost 95% of the enzymic activity was sustained within 40 min of irradiation (Fig. 4a, slope 3). A first order rate was observed for the disappearance of catalytic activity, suggestive of the oxidation of a group which may be essential for catalysis (see Fig. 4a, slope 2). Substantial protection against inactivation was afforded the enzyme by hypoxanthine, a strong competitive inhibitor of the enzyme (Fig. 4a, slope 1). This latter observation is consistent with the localization of the susceptible group at the active site (18, 19). A progressive increase of photoinactivation of the enzyme was observed as the pH was increased above pH 7.0; suggestive of the dependence on pH of a photosensitive group which may be essential for enzymic catalysis (Fig. 4b). Molecular Weight of the Enzyme Molecular weights of 70,000 daltons were determined for the native enzyme in separate experiments on Sephadex G-100 and Sephadex G-200. A mean molecular weight
5
IO Irrad.
20 time
30
40
S. LEWIS
of 70,000 daltons is reported here for native put-me nucleoside phosphorylase from rabbit brain (Fig. 5a). A molecular weight of 34,500 daltons was determined for the subunits of rabbit brain purine nucleoside phosphorylase by electrophoretic technique (Fig. 5b). These results suggest that the native enzyme may be dimeric in structure, with possibly identical subunits. Competitive inhibition of the enzyme by folic acid and DL-6-methyl 5,6,7,84etrahydropterine. Initial velocity studies in presence of folk acid showed that this compound was a competitive inhibitor of the enzyme (Fig. 6a). Negative cooperative effects (20) were elicited by folic acid at low concentrations of the variable substrate. Initial velocity studies in presence of the compound r&L-8methyl 5,6,7,8-tetrahydropterine, which bears similar substituents to the primary cyclic structure, also gave competitive inhibition of the enzyme (Fig. 6b). Noncompetitive inhibition by aminopterin and 8-aza-2,6-diaminopurine. The folic acid antagonist, aminopterin, bearing primary amine substituents to the primary cyclic structure, was a noncompetitive inhibitor of rabbit brain purine nucleoside phosphorylase. The inhibition by aminopterin manifested itself by suppressive ef-
I
(min)
FIG. 4. Photooxidation of the enzyme. The buffer was 0.1 M phosphate buffer saturated with oxygen. The concentration of enzyme was 1 PM and the concentration of methylene blue was 10 mg/lOO ml. Other conditions are specified under methods. a), These experiments were conducted at pH 7.0. Slope 3 shows loss of enzymic activity as a function of irradiation time. Slope 2 shows first order rate for the loss of enzymic activity. Slope 1 shows the protection afforded the enzyme by hypoxanthine. b), pH Dependence of photooxidation. Tests were irradiated for 2 min in presence of dye and controls were similarly irradiated in absence of dye; in 0.1 M phosphate buffer at each pH. Loss of activity was calculated as a percentage of control.
RABBIT
BRAIN
PURINE
NUCLEOSIDE
PHOSPHORYLASE
INHIBITION
\
32-
= E -
667
b l
34-
STUDIES
cyto.
\
4b-
30 t
i 2
i2 2s i
\
E”
’ 26. c .o; 24.
221
rob. br. PNPas.. -
f
. 4:2
4:4
Pib
Q
]
‘.
4.4-
4.2-
,2
\ ~6myo~‘obi”~~
,4
log mol.wt.
+chymo..P
mobility
FIG. 5. Determination of molecular weight of the enzyme. a), The molecular weight of the native enzyme was determined by gel filtration of Sephadex G-100 and G-200. The graph above shows the results obtained with Sephadex G-100. The conditions are specified at length in the section on methods. b), The subunit molecular weight was determined by electrophoresis on polyacrylamide, in presence of sodium dodecyl sulfate. The conditions are specified at length in the section on methods.
IO 20 [Guanosine]
mM-1
40 [Guanosine]
60
SO mM-1
FIG. 6. Double reciprocal plots of initial velocity versus [s] for competitive inhibition of the enzyme by a, folic acid, and b, n,L-6-methyl5,6,7,8-tetrahydropterine (CHs-THP). Guanosine was the variable substrate. The concentrations of the inhibitors, as indicated on the graphs, were prepared together with the substrate in 0.2 M phosphate buffer (pH 6.0). Due to substantial absorbance of folic acid at 252 mn, the concentrations of folk acid at 50 and 100 F were examined at a maximal substrate concentration of only 100 pM. The amount of enzyme used was 0.65 pg protein in 50 d. Initial velocities are expressed as POD/mm. The inhibition constant is listed in Table II.
fects both on the maximal catalytic rate and on the Michaelis constant for the variable substrate. The primary double reciprocal plots for this inhibition intersected
below the x-axis, so that the apparent dissociation constant for the central complex was significantly smaller than apparent Michaelis constants (Fig. 7a).
ARTHUR
S. LEWIS
/”
1020
40
[guanosine]
80 mM-1
[guanosine]
mM-1
FIG. 7. Double reciprocal plots of initial velocity uersus [s] for noncompetitive inhibition of the enzyme by a, aminopterin, and b, 8-aza-2,6-diaminopurine (8-aza-diaminopurine), with guanosine as the variable substrate in 0.2 M phosphate buffer (pH 6.0). Various concentrations of inhibitors were prepared together with the substrate. The significant absorbance of aminopterin at 100 PM was compensated for by a reduction in the maximal concentration of the variable substrate to only 50 j.tM, as indicated above. The amount of enzyme used was 0.65 ccgprotein in 50 al. Initial velocities were expressed as AOD/min.
L reciprocal plots of initial velocity inhibition of the enzyme by erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA). Guanosine was the variable substrate. Concentrations of the inhibitor were prepared together with the variable substrate in 0.2 M phosphate buffer (pH 6.0). The amount of enzyme used was 0.65 pg in 50 al and initial velocities are shown as relative AOD/min. FIG.
8. Double
versus [s] for competitive
The purine analog, 8-axa-2,6-diaminopurine, bears some similarity to aminopterin in that its primary amine substituents are identically disposed. Initial velocity studies
in presence of this compound indicated that the compound was similarly a noncompetitive inhibitor of purine nucleoside phosphorylase (Fig. 7b). In spite of the grossly dissimilar substituents to carbon-6 of folic acid and D,L-6methyl 5,6,7,8-tetrahydropterine, and also with respect to aminopterin and 8-aza-2,6diaminopurine, a striking similarity was observed in the values for the inhibition constants which were calculated for the respective pairs of competitive and noncompetitive inhibitors. The bulky substituents to carbon 6 of folic acid and aminopterin, may not have sterically hindered nor enhanced their binding capacity relative to their respective counterparts, r&r,-6-methyl5,6,7,8tetrahydropterine and 8-aza-2,6-diaminopurine. The selective pattern of the observed inhibitions may probably be governed by the nature of the interactions of the enzyme with the substituents to the primary cyclic structure of these compounds. Competitive inhibition by erythro-g-(2hydroxy-3-nonyl) adenine. The inhibitory potency of erythro-9-(2-hydroxy-3-nonyl) adenine, an adenosine deaminase inhibitor (21, 22), was also examined with rabbit brain purine nucleoside phosphorylase, and
RABBIT
BRAIN
PURINE
NUCLEOSIDE
PHOSPHORYLASE
TABLE
INHIBITION
STUDIES
669
II
SUMMARY OF KINETIC CONSTANTS FOR INHIBITORS OF RABBIT BRAIN PURINE NUCLEOSIDE PHOSPHORYLASE’ Inhibitor
Folic acid n,L-6-methyl5,6,7,8-tetrahydropterine Aminopterin 8-Axa-2,6-diaminopurine Erythro-9-(2-hydroxy-3-nonyl) adenine
Apparent inhibition constant (M)* 3.37 3.80 1.50 1.95 1.90
x x x x x
m5 N5 1o-4 1o-4 1om4
Type of inhibition Competitive Competitive Noncompetitive Noncompetitive Competitive
a Kinetic constants were determined with guanosine as variable substrate, in 0.2 M phosphate buffer (pH 6.0) at 25°C. The K,,, for guanosine was 6.0 X 10m5M. * Apparent inhibition constants were calculated from a secondary replot of the primary slopes versus the __ concentrations of inhibitor.
was found to inhibit the enzyme competitively (Fig. 8). These effects are not, at this time, properly understood. Adenosine itself was neither a substrate nor an inhibitor for the enzyme. It is possible, however, to postulate that the interactions of this analog with the enzyme may be hydrophobic in nature. The inhibition constants are summarized in Table II. DISCUSSION
Active site studies have implicated histidine and cysteine as the catalytic groups in the enzyme. The pKa values which were determined for the groups which participate in enzymic catalysis are consistent with this conclusion. The total inactivation of the enzyme by p-chloromercuribenzoate, within a relatively short period of incubation, was suggestive of its interaction with an essential cysteine residue in the enzyme. The total reactivation of the completely inactivated enzyme within 5 min of exposure to an excess of 2-mercaptoethanol, argues strongly for a reversible reduction of an essential cysteine residue. The suggested role of cysteine as a participating species in the enzymic catalysis of mammalian purine nucleoside phosphorylase has been amply documented (3-7). A first order rate which was observed for the loss of enzymic activity upon photooxidation, was suggestive of the oxidation of an essential catalytic group in the enzyme. Moreover, the protection afforded the enzyme against photooxidation by hypoxan-
thine, a competitive inhibitor, also suggests that the localization of the susceptible group may be at the active site of the enzyme (18, 19). Studies of the physical properties of this enzyme suggest that the enzyme is dimeric in structure. Comparative studies presently made in this laboratory, suggest tentatively that mammalian purine nucleoside phosphorylase may be quite similar in molecular weight and subunit structure (A, S. Lewis, unpublished observations). The biological role of folk acid derivatives in synthetic metabolic processes has been well established, and the antagonistic effects of the folate analogs, aminopterin and amethopterin, on these metabolic processeshave been amply investigated. A one-site-of-action hypothesis, however, has long been associated with the folate antagonists, as inhibitors of folate reductase. The elegant in vitro studies of Borsa and Whitmore, which have appeared in recent years (23-25), have demonstrated the nonspecific inhibitory action of the folate antagonist amethopterin (methotrexate). Inhibition constants similar to those reported here for folic acid and aminopterin have been reported for the inhibition by amethopterin of soluble purified preparations of thymidylate synthetase from mammalian and bacterial sources (25). Evidence presented in this investigation substantiates the reported findings of the nonspecific inhibitory effects of the folate antagonists. The subtle relationship of the structure of the compounds examined in this study to their inhibitory activity was clearly dem-
670
ARTHUH
on&rated. The structural dissimilarities within the pairs of the compounds examined, were not reflected in their calculated inhibition constants. These results suggest that inhibitory specificity may, probably, be resident in the nature of the substituents to the primary cyclic structure of the compounds. This latter observation may be of pharmacological significance. The enzyme fails to recognize adenosine as substrate or inhibitor, but effectively binds the adenosine analog erythro-9-(2-hydroxy-3-nonyl) adenine, in which a strongly hydrophobic residue is substituted for ribose. Moreover, the enzyme accommodates the bulky and sterically unfavorable structures of folic acid and aminopterin as inhibitors, but fails to recognize as substrates or inhibitors the phosphorylated derivatives of its normal substrates. These observations make possible the postulation of strongly hydrophobic regions within the active center of rabbit brain purine nucleoside phosphorylase. REFERENCES 1. KLEIN, W. (1935) 2. Physiol. Chem. 231,125. 2. KALCKAR, H. (1947) J. Biol. Chem. 167,461-475. 3. KIM, B. K., CHA, S., AND PARKS, R. E., JR. (1968)
J. Biol. Chem. 243, 1763-1770. 4. AGARWAL, R. P., AND PARKS, R. E., JR. (1971) J.
Biol. Chem. 246,3763-3768. 5. LEWIS, A. S. (1973) Ph.D. Thesis, City University of New York. 6. LEWIS, A. S., AND GLANTZ, M. D., (1976) J. Biol. Chem. 261,407-413. 7. LEWIS, A. S., AND GLANTZ, M. D. (1976) Biochem-
S. LEWIS
istry 15,4452-4457. 8. LEWIS, A. S. (1977) J. Biol. Chem. 252, 732-738. 9. EDWARDS, Y. H., EDWARDS, P. A., AND HOPKINSON, D. A. (1973) FEBS Lett 32,235-237. 10. MILMAN, G., ANTON, D. L., AND WEBER, J. S. (1976) Biochemistry 15,4967-4973. 11. KIM, B. K., CHA, S., AND PARKS, R. E., JR. (1968) J. Biol. Chem. 243,1771-1776. 12. GIBLETT, E. R., AMMANN, A. J., SANDMAN, R., WARA, D. W., AND DIAMOND, L. K. (1975) Lancet 1, 1010-1013. 13. COHEN, A., DOYLE, D., MARTIN, D. W., JR., AMMANN, A. J. (1976) N. Engl. J. Med. 295,
1449-1454. 14. STOOP, J. W., ZECERS, B. J. M., HENDRICKS, G. F. M., SIECENBEEK VAN HEUKELOM, L. H., AND BALLIEUX, R. E. (1977) N. En&. J. Med. 296, 651-655. 15. DIXON, M. (1953) Biochem. J. 56, 161-170. 16. ANDREWS, P. (1970) Methods B&hem. Anal. 18, l-53. 17. WEBER, K., AND OSBORN, M. (1969) J. Biol. Chem. 244,4406-4412. 18. WESTHEAD, E. W. (1965) Biochemistry 4, 2139-2144. 19. BRAND, K., TSOLAS, O., AND HORECKER, B. L. (1969) Arch. Biochem. Biophys. 130, 521-529. 20. KOSHLAND, D. E., JR., (1970) The Molecular Basis In “The Enzymes” for Enzyme Regulation. (Boyer, P. D., ed.), Ed. 3, Vol. 1, pp. 341-396, Academic Press, New York. 21. SCHAEFFER, H. J., AND SCHWENDER, C. F. (1974) J. Med. Chem. l?:, 6-8. 22. LAPI, L., AND COHEN, S. S. (1977) Biochem. Phar-
macol. 26, 71-76. 23. BORSA, J., AND WHITMORE, G. F. (1969) Cancer
Res. 29,737-747. 24. BORSA, J., AND WHITMORE, G. F. (1969) Mol.
Pharmacol. 5,303-317. 25. BORSA, J., AND WHITMORE, G. F. (1969) Mol.
Pharmacol. 5,318-332.