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Evidence for the proximity of two sulfhydryl groups at the active site of 6-phosphogluconate dehydrogenase
ARCWIVEB
OF
BIOCHEMIWRY
AND BIOPHYSICS
Vol. 186, No. 2, March, pp. 406-410, 1978
Evidence MARIO
for the Proximity of Two Sulfhydryl Groups at the Active Site of 6-Phosphogluconate Dehydrogenasel RIPPA,
MARCO SIGNORINI, ALESSANDRO FRANC0 DALLOCCHIO
Istituto di Chimica Biologica,
PERNICI,
AND
Universitci, Ferrara, Italy
Received September 26, 1977; revised November 22, 1977 The reaction between 6-phosphogluconate dehydrogenase from Candidn utilis and 5,5’-dithiobis(2-nitrobenzoate) results in the inactivation of the enzyme. At pH 6.0 the inactivation can be correlated with the modification of only one SH group per enzyme subunit. The modified SH group can react with another SH group forming an intramolecular disulfide bridge. Since the modified enzymes, either with an SH group modified or with a cystine disulfide bridge, are still able to bind the substrate and the coenzyme, gross conformational changes seem unlikely to have occurred. The results obtained suggest that the SH poups of two cysteine residues are located close to each other in the three-dimensional structure of the active site of the enzyme.
6-Phosphogluconate dehydrogenase from Candidu utilis is composed of two subunits with a molecular weight of 50,000 (1) and has two coenzyme (1) and two substrate (2) binding sites. The active site of the enzyme contains residues of lysine (3, 4), histidine (2, 5-7), tyrosine (8, 9), and cysteine (9-12). In the active site of the enzyme a lysine residue is close to a histidine residue (6) and at a measurable distance from a phosphate binding site of the protein (3). More recently it has been reported that a cysteine residue of the active site is close to a tyrosine residue (9). To obtain additional information on the three-dimensional structure of the active site of the enzyme, we studied the reaction between the enzyme and DTNB2 and the properties of the modified enzyme. The data reported here indicate that two sulfhydryl groups are sufficiently close in the three-dimensional structure of the active site that an intramolecular disulfide bridge can easily be formed. 1 This work was supported by Grant CT76.01313.04 from the Italian Conaiglio Nazionale delle Ricerche. * Abbreviations used: 6PG, 6-phosphogluconate; DTNB, 5,5’-dithiobis(2-nitrobenzoate); TNB-, 5thio-2.nitrobenzoate; DTT, dithiothreitol. 406 0003-9861/78/1862-0406$02.00/O Copyright 0 1978 by Academic F’reas, Inc. All rights of reproduction in any form reserved.
MATERIALS AND METHODS 6-Phosphogluconate dehydrogenase (6-phosphoD-gluconat.e:NADP+ 3-oxidoreductase, decarboxylating, EC 1.1.1.44), Type I, from Candida utilis was prepared and assayed as previously described (13). The ammonium sulfate suspension of enzyme crystals was centrifuged and the crystals were suspended in 50 mM acetate buffer, pH 6.0. The protein was freed from ammonium sulfate by gel filtration through a column of Sephadex G-50 equilibrated either with 50 mM acetate buffer, pH 6.0, or with Tris-HCl buffer, pH 7.5, both buffers containing 0.1 mM EDTA. The enzyme was then diluted with the same buffer to the desired protein concentration, which was determined spectrophotometrically (13). 6-Phosphogluconate (6PG), NADP+, DTNB, and DTT were purchased fmm Sigma (St. Louis, Missouri). DTNB concentration was determined spectrophotometrically (14) at 412 nm after reaction with an excess of mercaptoethanol. Enzyme inactivation. All operations, unless otherwise stated, were carried out at room temperature (20°C). The enzyme solution was treated with an excess of DTNB and the absorbance at 412 nm was recorded. At given time intervals, enzyme aliquota were removed, diluted in acetate buffer, and immediately tested for residual enzymatic activity. Fluorescence measurements were made with a Perkin-Elmer MPF 3L recording spectrofluorimeter in a-ml cuvettes (l-cm light path) at 20°C. The fluorescence values were corrected for the inner filter effect according to Parker (15).
6-PHOSPHOGLUCONATE
DEHYDROGENASE
STUDIES
407
RESULTS
Kinetics and Stoichiometry of the Znactiuation The incubation of 6-phosphogluconate dehydrogenase with DTNB at pH 6.0 causes a decrease of the catalytic activity of the enzyme (Fig. 1) and the release into the medium of TNB- ions (Fig. 2) due to the reaction of the inhibitor with the
20
‘\
o-+NADP+
Yl
--__
.-.
5
15
10
INCUBATION
TIME
-0
J
= 60 3-
FIG. 3. Correlation between inactivation and modification of SH groups. The data reported were obtained from the graphs of Figs. 1 and 2. Symbols are as in Fig. 1.
20
(min)
FIG. 1. Inactivation of 6-phosphogluconate dehydrogenase by DTNB. A solution containing the enzyme (20 nmol of subunit/ml) in 50 mM acetate buffer, pH 6.0, 0.1 mM EDTA was treated with DTNB (final concentration, 180 nmol/ml). At the time intervals indicated, aliquota were removed, diluted in acetate buffer, and immediately tested for residual enzymatic activity. 0, no other additions; 0, incubation with DTNB in the presence of 0.46 mM NADP+; 0, in the presence of 1.40 mM 6PG. 2.0 t
10 5 INCUBATION
80
0.5 1.0 1.5 SH GROUPSMODIFIED ( RESIDUES/SUBUNIT 1
--.=__
Y\
3
15
20
TlMElmln)
FIG. 2. Modification of SH groups by DTNB. The absorbance of the incubation mixture at 412 nm described in the legend to Fig. 1 was recorded as a function of the incubation time. The data are plotted as number of SH groups modified per enzyme subunit. Symbols are as in Fig. 1.
sulfhydryl groups of the enzyme. Almos, complete inactivation of the enzyme was observed when one sulfhydryl group per enzyme subunit was modified (Fig. 3). The presence of NADP+ in the reaction mixture decreased significantly both the rate of the modification of the SH groups (Fig. 2) and inactivation (Fig. 3). In the presence of 6PG, on the other hand, the rate of the modification of the SH groups was drastically reduced (Fig. 2) and the modification of one SH group per subunit caused less than a 30% inactivation (Fig. 3); hence a strong protective effect of the substrate was observed (Fig. 1). At pH 6.0 only two or three SH groups can be titrated with DTNB in a short time, the fourth group reacting very slowly. At pH 7.5 the rate of reaction is higher and all four SH groups are quickly titrated, even in the absence of denaturing agents. But at this pH the reaction is less selective and complete inactivation of the enzyme requires the modification of more than two SH groups per enzyme subunit. Formation of a Dilsulfide Bridge Between Two Cysteine Residues at the Active Site The enzyme was treated at pH 6.0 with DTNB until the modification of one SH
408
RIPPA ETAL.
group per subunit was obtained. The protein, which was 20% active, was immediately freed from excess DTNB and TNBions by gel filtration at pH 6.0 and then brought to pH 7.5. Upon standing the absorbance of the protein solution at 412 nm increased, indicating the release of 0.82 TNB- ion per enzyme subunit (Fig. 4). This release indicates that the modified SH group at the active site of the enzyme reacts at pH 7.5 with a nearby SH group by a thiol-disulfide exchange reaction (Scheme I, reaction b) with the formation of a disulfide bridge. The formation of a disultide bridge did not cause a further decrease of the enzymatic activity.
Reversibility of the Inactivation When the enzyme, with one SH group modified per subunit and possessing 20% of the original activity, was treated with 1
I
,
I
I
06 0.L
20
LO 60
INCUBATION
80
100
TIME (min)
FIG. 4. Formation of a cystine disulfide bridge at the active site of the enzyme. A solution of the enzyme (60 nmol of subunit/ml) in 50 rnM acetate buffer, pH 6.0, was treated with DTNB (final concentration, 180 nmollml). When the modification of one SH group per subunit was obtained, with 80% inactivation of the enzyme, the protein was freed from excess DTNB and TNB- ions by gel filtration at pH 6.0, treated aa indicated with 0.46 m&z NADP+ or 1.4 mM 6PG (final concentrations), and brought to pH 7.5. The absorbance of the solution at 412 nm was recorded 88 a function of time. 0, no additions; Cl, in the presence of 0.46 mM NADP+; 0, in the presence of 1.40 mM 6PG.
t
I
10
20
INCUBATION
I
30
LO 50
TlME(min1
FIG. 5. Reactivation of the modified enzyme by treatment with D’M’. The enzyme was treated with DTNB aa indicated in the legend to Fig. 1. When one SH per subunit was modified, DTT (at a final concentration of 10 mM) was added. Aliquots were taken, diluted, and assayed for enzymatic activity (0). The enzyme with 0.82 cystine disulfide bridge per subunit, prepared aa described in the legend to Fig. 4, waz treated as above with Dll’ and the catalytic activity was assayed after dilution (0).
an excess of DTT, there was an almost instantaneous recovery of enzymatic activity: In less than 2 min 92% of the original activity was recovered (Fig. 5). When the treatment with DTT was performed on the disulfide form of the enzyme the recovery was slower and only 63% of the original activity was recovered in 50 min. The two modified enzymes are two chemically different species, in only one of which is there the electron-withdrawing nitrobenzoate group. The presence of this group and/or a different accessibility of the S-S bridge to the D’M? could explain the observed difference in the reactivation rate. Evidence Formed
that the Disulfide Bridge within the Same Subunit
Is
The native enzyme, treated with maleic anhydride, dissociates into two subunits (1). To establish if the cystine disulfide bridge is formed within the same subunit or between the two subunits, both the native and the cystine-containing enzymes were treated with maleic anhydride and subjected to disc gel electrophoresis as previously described (1). Both maleylated proteins showed the same electrophoretic
6-PHOSPHOGLUCONATE
DEHYDROGENASE
mobility, indicating that the disulfide bridge is formed with the same subunit. Binding of the Substrate and of the Coenzyme to the Modified Enzymes
The enzyme containing one SH group modified per subunit is still able to bind 6PH and NADP+: Indeed, the formation of the disulfide bridge is slower when carried out in the presence of either 6PG or NADP+ (Fig. 4). Each subunit of the cystine-containing enzyme still has two SH groups titratable with DTNB; however, the rate of this titration was slower in the presence of 6PG or NADP+, thus indicating that the cystine enzyme is also able to bind both substrate and coenzyme. Binding of NADP+ to the enzyme causes a quenching of the protein fluorescence (Dallocchio and Rippa, unpublished work). The addition of the same amount of NADP+, either to the native enzyme or to the enzyme containing one SH group modified or 0.82 cystine residue per subunit, yielded the same quenching of the protein fluorescence (Table I). This indicates that the two modified proteins bind the coenzyme with affinity comparable to that of the native enzyme. DISCUSSION
The formation of cystine residues upon treatment with DTNB has also been reTABLE
I
QUENCHING OF THE PROTEIN FLUORESCENCE NATIVE AND MODIFIED ENZYME BY ADDITION
NADP+ (mM) 0 0.123 0.246 0.369
NADP+” Protein fluorescence (arbitrary
OF OF
units)
A
B
C
D
100 63 49 39
100 65 47 40
100 63 51 42
100 69 51 43
(1The fluorescence of a protein solution (9 nmol of subunit/ml) was excited at 280 nm and read at 330 nm before and after the additions of NADP+ at the indicated final concentrations. A, native enzyme in 50 mM acetate buffer, pH 6.0; B, enzyme with one SH group blocked per subunit, in the same buffer; C, native enzyme in 50 mM Tris buffer, pH 7.5; D, enzyme with 0.82 disulfide bridge per subunit in 50 mM Tris buffer, pH 7.5.
STUDIES
409
ported for other enzymes (16-22). In the present case the cystine-containing enzyme can still be dissociated into two sep arate subunits; thus, it may be concluded that this disulfide bridge is formed within the same subunit. Also, the disulfide enzyme is able to bind both 6PG and NADP+. Since the formation of the disulfide bridge occurs readily at pH values where the enzyme is more active (the pH optimum for the activity is 7.7-8) and in a protein which, although inactive, was not subjected to gross conformational changes (is still able to bind 6PG and NADP+), it may be hypothesized that the two cysteine residues involved in the formation of the disulfide bridge are close in the three-dimensional structure of the active site of the enzyme. REFERENCES 1. RIPPA, M., SIGNORINI, M., AND PONTBEMOLI, S. (1969) Ital. J. B&hem. 18, 174;184. 2. RIPPA, M., SIGNORINI, M., AND PONTREMOLI, S. (1972) Arch. Biochem. Bbphys. 150, 503-510. 3. RIPPA, M., SPANIO, L., AND PONTREMOLI, S. (1967) Arch. Biochem. Biophys. 118, 46-57. 4. DALLOCCHIO, F., NEGFUNI, R., SIGNORINI, M., AND RIPPA, M. (1976) Biechim. Btiphys. Acts 249, 629-634. 5. RIPPA, M., AND PONTREMOLI, S. (1968) Biochemistry 7, 1514-1518. 6. RIPPA, M., AND PONTREMOLI, S. (1969) Arch. B&hem. Biqhys. 133, 112-118. 7. RIPPA, M., PICCO, C., AND PONTBEMOLI, S. (1970) J. Biol. Chem. 245, 4977-4981. a. R~~PA, M., hcco, C., SIGNOBINI, M., AND PONTREMOLI, S. (1971) Arch. Biochem. Biophys. 147, 487-492. 9. DALLOCCHIO, F., SIGNOEINI, M., AND RIPPA, M. (1978) Arch. B&hem. Biophys., 165, 57. 10. G~AZI, E., RIPPA, M., AND PONTREMOLI, S. (1965) J. Biol. Chem. 240, 234-237. 11. RIPPA, M., GWI, E., AND PONTREMOLI, S. (1966) J. Biol. Chem. 241, 1632-1635. 12. DALLOCCHIO, F., AND RIPPA, M. (1976) Bull. Mol. Biol. Med. 1, 12-17. 13. RIPPA, M., SIGNORINI, M., AND PICCO, C. (1970) Ital. J. Biochem. 19, 391-396. 14. ELLMAN, G. L. (1958) Arch. Biochem. Biophys. 74, 430-450. 15. PARKER, C. A. (1968) Photoluminescence of Solutions, p. 222, Elsevier, New York. 16. CONNELLAN, J. M., AND FOLK, J. E. (1969) J. Biol. Chem. 244, 3173-3181. 17. VERGER, R., SARDA, L., AND DESNUELLE, P.
410
RIPPA
(1971) B&him. Biophys. Acta 242,580~592. 18. GIIAESETTI, D. R., AND MUSSEY, J. F. (1967) Arch. Biochem. Biophys. 119, 41-49. 19. FLASHNEB, M., HOLLENBEB, P. F., AND COON, M. J. C. (1972)J. Bid. Chem. 247, 8114-8121. 20. Boaoss, L. (1969)Actu B&him. Biophys. Acad.
ET
AL.
Sci. Hung. 4, 57-69. 21. OKABE, K., JACOBS, H. K., AND KUBY, S. A. (1970) J. Bid. Chem. 245, 6498-6510. 22. BRAGINBKY, J. E., FRANZEN, J. S., AND CHUNG, A. E. (1970)Biochem. Biophys. Res. Commun. 38, 644-650.
OF
BIOCHEMIWRY
AND BIOPHYSICS
Vol. 186, No. 2, March, pp. 406-410, 1978
Evidence MARIO
for the Proximity of Two Sulfhydryl Groups at the Active Site of 6-Phosphogluconate Dehydrogenasel RIPPA,
MARCO SIGNORINI, ALESSANDRO FRANC0 DALLOCCHIO
Istituto di Chimica Biologica,
PERNICI,
AND
Universitci, Ferrara, Italy
Received September 26, 1977; revised November 22, 1977 The reaction between 6-phosphogluconate dehydrogenase from Candidn utilis and 5,5’-dithiobis(2-nitrobenzoate) results in the inactivation of the enzyme. At pH 6.0 the inactivation can be correlated with the modification of only one SH group per enzyme subunit. The modified SH group can react with another SH group forming an intramolecular disulfide bridge. Since the modified enzymes, either with an SH group modified or with a cystine disulfide bridge, are still able to bind the substrate and the coenzyme, gross conformational changes seem unlikely to have occurred. The results obtained suggest that the SH poups of two cysteine residues are located close to each other in the three-dimensional structure of the active site of the enzyme.
6-Phosphogluconate dehydrogenase from Candidu utilis is composed of two subunits with a molecular weight of 50,000 (1) and has two coenzyme (1) and two substrate (2) binding sites. The active site of the enzyme contains residues of lysine (3, 4), histidine (2, 5-7), tyrosine (8, 9), and cysteine (9-12). In the active site of the enzyme a lysine residue is close to a histidine residue (6) and at a measurable distance from a phosphate binding site of the protein (3). More recently it has been reported that a cysteine residue of the active site is close to a tyrosine residue (9). To obtain additional information on the three-dimensional structure of the active site of the enzyme, we studied the reaction between the enzyme and DTNB2 and the properties of the modified enzyme. The data reported here indicate that two sulfhydryl groups are sufficiently close in the three-dimensional structure of the active site that an intramolecular disulfide bridge can easily be formed. 1 This work was supported by Grant CT76.01313.04 from the Italian Conaiglio Nazionale delle Ricerche. * Abbreviations used: 6PG, 6-phosphogluconate; DTNB, 5,5’-dithiobis(2-nitrobenzoate); TNB-, 5thio-2.nitrobenzoate; DTT, dithiothreitol. 406 0003-9861/78/1862-0406$02.00/O Copyright 0 1978 by Academic F’reas, Inc. All rights of reproduction in any form reserved.
MATERIALS AND METHODS 6-Phosphogluconate dehydrogenase (6-phosphoD-gluconat.e:NADP+ 3-oxidoreductase, decarboxylating, EC 1.1.1.44), Type I, from Candida utilis was prepared and assayed as previously described (13). The ammonium sulfate suspension of enzyme crystals was centrifuged and the crystals were suspended in 50 mM acetate buffer, pH 6.0. The protein was freed from ammonium sulfate by gel filtration through a column of Sephadex G-50 equilibrated either with 50 mM acetate buffer, pH 6.0, or with Tris-HCl buffer, pH 7.5, both buffers containing 0.1 mM EDTA. The enzyme was then diluted with the same buffer to the desired protein concentration, which was determined spectrophotometrically (13). 6-Phosphogluconate (6PG), NADP+, DTNB, and DTT were purchased fmm Sigma (St. Louis, Missouri). DTNB concentration was determined spectrophotometrically (14) at 412 nm after reaction with an excess of mercaptoethanol. Enzyme inactivation. All operations, unless otherwise stated, were carried out at room temperature (20°C). The enzyme solution was treated with an excess of DTNB and the absorbance at 412 nm was recorded. At given time intervals, enzyme aliquota were removed, diluted in acetate buffer, and immediately tested for residual enzymatic activity. Fluorescence measurements were made with a Perkin-Elmer MPF 3L recording spectrofluorimeter in a-ml cuvettes (l-cm light path) at 20°C. The fluorescence values were corrected for the inner filter effect according to Parker (15).
6-PHOSPHOGLUCONATE
DEHYDROGENASE
STUDIES
407
RESULTS
Kinetics and Stoichiometry of the Znactiuation The incubation of 6-phosphogluconate dehydrogenase with DTNB at pH 6.0 causes a decrease of the catalytic activity of the enzyme (Fig. 1) and the release into the medium of TNB- ions (Fig. 2) due to the reaction of the inhibitor with the
20
‘\
o-+NADP+
Yl
--__
.-.
5
15
10
INCUBATION
TIME
-0
J
= 60 3-
FIG. 3. Correlation between inactivation and modification of SH groups. The data reported were obtained from the graphs of Figs. 1 and 2. Symbols are as in Fig. 1.
20
(min)
FIG. 1. Inactivation of 6-phosphogluconate dehydrogenase by DTNB. A solution containing the enzyme (20 nmol of subunit/ml) in 50 mM acetate buffer, pH 6.0, 0.1 mM EDTA was treated with DTNB (final concentration, 180 nmol/ml). At the time intervals indicated, aliquota were removed, diluted in acetate buffer, and immediately tested for residual enzymatic activity. 0, no other additions; 0, incubation with DTNB in the presence of 0.46 mM NADP+; 0, in the presence of 1.40 mM 6PG. 2.0 t
10 5 INCUBATION
80
0.5 1.0 1.5 SH GROUPSMODIFIED ( RESIDUES/SUBUNIT 1
--.=__
Y\
3
15
20
TlMElmln)
FIG. 2. Modification of SH groups by DTNB. The absorbance of the incubation mixture at 412 nm described in the legend to Fig. 1 was recorded as a function of the incubation time. The data are plotted as number of SH groups modified per enzyme subunit. Symbols are as in Fig. 1.
sulfhydryl groups of the enzyme. Almos, complete inactivation of the enzyme was observed when one sulfhydryl group per enzyme subunit was modified (Fig. 3). The presence of NADP+ in the reaction mixture decreased significantly both the rate of the modification of the SH groups (Fig. 2) and inactivation (Fig. 3). In the presence of 6PG, on the other hand, the rate of the modification of the SH groups was drastically reduced (Fig. 2) and the modification of one SH group per subunit caused less than a 30% inactivation (Fig. 3); hence a strong protective effect of the substrate was observed (Fig. 1). At pH 6.0 only two or three SH groups can be titrated with DTNB in a short time, the fourth group reacting very slowly. At pH 7.5 the rate of reaction is higher and all four SH groups are quickly titrated, even in the absence of denaturing agents. But at this pH the reaction is less selective and complete inactivation of the enzyme requires the modification of more than two SH groups per enzyme subunit. Formation of a Dilsulfide Bridge Between Two Cysteine Residues at the Active Site The enzyme was treated at pH 6.0 with DTNB until the modification of one SH
408
RIPPA ETAL.
group per subunit was obtained. The protein, which was 20% active, was immediately freed from excess DTNB and TNBions by gel filtration at pH 6.0 and then brought to pH 7.5. Upon standing the absorbance of the protein solution at 412 nm increased, indicating the release of 0.82 TNB- ion per enzyme subunit (Fig. 4). This release indicates that the modified SH group at the active site of the enzyme reacts at pH 7.5 with a nearby SH group by a thiol-disulfide exchange reaction (Scheme I, reaction b) with the formation of a disulfide bridge. The formation of a disultide bridge did not cause a further decrease of the enzymatic activity.
Reversibility of the Inactivation When the enzyme, with one SH group modified per subunit and possessing 20% of the original activity, was treated with 1
I
,
I
I
06 0.L
20
LO 60
INCUBATION
80
100
TIME (min)
FIG. 4. Formation of a cystine disulfide bridge at the active site of the enzyme. A solution of the enzyme (60 nmol of subunit/ml) in 50 rnM acetate buffer, pH 6.0, was treated with DTNB (final concentration, 180 nmollml). When the modification of one SH group per subunit was obtained, with 80% inactivation of the enzyme, the protein was freed from excess DTNB and TNB- ions by gel filtration at pH 6.0, treated aa indicated with 0.46 m&z NADP+ or 1.4 mM 6PG (final concentrations), and brought to pH 7.5. The absorbance of the solution at 412 nm was recorded 88 a function of time. 0, no additions; Cl, in the presence of 0.46 mM NADP+; 0, in the presence of 1.40 mM 6PG.
t
I
10
20
INCUBATION
I
30
LO 50
TlME(min1
FIG. 5. Reactivation of the modified enzyme by treatment with D’M’. The enzyme was treated with DTNB aa indicated in the legend to Fig. 1. When one SH per subunit was modified, DTT (at a final concentration of 10 mM) was added. Aliquots were taken, diluted, and assayed for enzymatic activity (0). The enzyme with 0.82 cystine disulfide bridge per subunit, prepared aa described in the legend to Fig. 4, waz treated as above with Dll’ and the catalytic activity was assayed after dilution (0).
an excess of DTT, there was an almost instantaneous recovery of enzymatic activity: In less than 2 min 92% of the original activity was recovered (Fig. 5). When the treatment with DTT was performed on the disulfide form of the enzyme the recovery was slower and only 63% of the original activity was recovered in 50 min. The two modified enzymes are two chemically different species, in only one of which is there the electron-withdrawing nitrobenzoate group. The presence of this group and/or a different accessibility of the S-S bridge to the D’M? could explain the observed difference in the reactivation rate. Evidence Formed
that the Disulfide Bridge within the Same Subunit
Is
The native enzyme, treated with maleic anhydride, dissociates into two subunits (1). To establish if the cystine disulfide bridge is formed within the same subunit or between the two subunits, both the native and the cystine-containing enzymes were treated with maleic anhydride and subjected to disc gel electrophoresis as previously described (1). Both maleylated proteins showed the same electrophoretic
6-PHOSPHOGLUCONATE
DEHYDROGENASE
mobility, indicating that the disulfide bridge is formed with the same subunit. Binding of the Substrate and of the Coenzyme to the Modified Enzymes
The enzyme containing one SH group modified per subunit is still able to bind 6PH and NADP+: Indeed, the formation of the disulfide bridge is slower when carried out in the presence of either 6PG or NADP+ (Fig. 4). Each subunit of the cystine-containing enzyme still has two SH groups titratable with DTNB; however, the rate of this titration was slower in the presence of 6PG or NADP+, thus indicating that the cystine enzyme is also able to bind both substrate and coenzyme. Binding of NADP+ to the enzyme causes a quenching of the protein fluorescence (Dallocchio and Rippa, unpublished work). The addition of the same amount of NADP+, either to the native enzyme or to the enzyme containing one SH group modified or 0.82 cystine residue per subunit, yielded the same quenching of the protein fluorescence (Table I). This indicates that the two modified proteins bind the coenzyme with affinity comparable to that of the native enzyme. DISCUSSION
The formation of cystine residues upon treatment with DTNB has also been reTABLE
I
QUENCHING OF THE PROTEIN FLUORESCENCE NATIVE AND MODIFIED ENZYME BY ADDITION
NADP+ (mM) 0 0.123 0.246 0.369
NADP+” Protein fluorescence (arbitrary
OF OF
units)
A
B
C
D
100 63 49 39
100 65 47 40
100 63 51 42
100 69 51 43
(1The fluorescence of a protein solution (9 nmol of subunit/ml) was excited at 280 nm and read at 330 nm before and after the additions of NADP+ at the indicated final concentrations. A, native enzyme in 50 mM acetate buffer, pH 6.0; B, enzyme with one SH group blocked per subunit, in the same buffer; C, native enzyme in 50 mM Tris buffer, pH 7.5; D, enzyme with 0.82 disulfide bridge per subunit in 50 mM Tris buffer, pH 7.5.
STUDIES
409
ported for other enzymes (16-22). In the present case the cystine-containing enzyme can still be dissociated into two sep arate subunits; thus, it may be concluded that this disulfide bridge is formed within the same subunit. Also, the disulfide enzyme is able to bind both 6PG and NADP+. Since the formation of the disulfide bridge occurs readily at pH values where the enzyme is more active (the pH optimum for the activity is 7.7-8) and in a protein which, although inactive, was not subjected to gross conformational changes (is still able to bind 6PG and NADP+), it may be hypothesized that the two cysteine residues involved in the formation of the disulfide bridge are close in the three-dimensional structure of the active site of the enzyme. REFERENCES 1. RIPPA, M., SIGNORINI, M., AND PONTBEMOLI, S. (1969) Ital. J. B&hem. 18, 174;184. 2. RIPPA, M., SIGNORINI, M., AND PONTREMOLI, S. (1972) Arch. Biochem. Bbphys. 150, 503-510. 3. RIPPA, M., SPANIO, L., AND PONTREMOLI, S. (1967) Arch. Biochem. Biophys. 118, 46-57. 4. DALLOCCHIO, F., NEGFUNI, R., SIGNORINI, M., AND RIPPA, M. (1976) Biechim. Btiphys. Acts 249, 629-634. 5. RIPPA, M., AND PONTREMOLI, S. (1968) Biochemistry 7, 1514-1518. 6. RIPPA, M., AND PONTREMOLI, S. (1969) Arch. B&hem. Biqhys. 133, 112-118. 7. RIPPA, M., PICCO, C., AND PONTBEMOLI, S. (1970) J. Biol. Chem. 245, 4977-4981. a. R~~PA, M., hcco, C., SIGNOBINI, M., AND PONTREMOLI, S. (1971) Arch. Biochem. Biophys. 147, 487-492. 9. DALLOCCHIO, F., SIGNOEINI, M., AND RIPPA, M. (1978) Arch. B&hem. Biophys., 165, 57. 10. G~AZI, E., RIPPA, M., AND PONTREMOLI, S. (1965) J. Biol. Chem. 240, 234-237. 11. RIPPA, M., GWI, E., AND PONTREMOLI, S. (1966) J. Biol. Chem. 241, 1632-1635. 12. DALLOCCHIO, F., AND RIPPA, M. (1976) Bull. Mol. Biol. Med. 1, 12-17. 13. RIPPA, M., SIGNORINI, M., AND PICCO, C. (1970) Ital. J. Biochem. 19, 391-396. 14. ELLMAN, G. L. (1958) Arch. Biochem. Biophys. 74, 430-450. 15. PARKER, C. A. (1968) Photoluminescence of Solutions, p. 222, Elsevier, New York. 16. CONNELLAN, J. M., AND FOLK, J. E. (1969) J. Biol. Chem. 244, 3173-3181. 17. VERGER, R., SARDA, L., AND DESNUELLE, P.
410
RIPPA
(1971) B&him. Biophys. Acta 242,580~592. 18. GIIAESETTI, D. R., AND MUSSEY, J. F. (1967) Arch. Biochem. Biophys. 119, 41-49. 19. FLASHNEB, M., HOLLENBEB, P. F., AND COON, M. J. C. (1972)J. Bid. Chem. 247, 8114-8121. 20. Boaoss, L. (1969)Actu B&him. Biophys. Acad.
ET
AL.
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