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Nucleotidases in plants
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
133, i%-59 (1969)
Nucleotidases III. Effect of Metabolites
in Plants
on the Enzyme Hydrolyzing
Dinucleotide
of Biochemistry, Received
Adenine
(FAD) from Phaseolus radiatus1~2
S. D. RAVINDRANATH Department
Flavine
March
AND
Indian Institute
N. APPAJI
RAO
of Science, Bangalore-lb,
28, 1969; accepted
May
India
9, 1969
The highly purified enzyme from mung bean seedlings hydrolyzing FAD at pH 9.4 and temperature 49”, functioned with an initial fast rate followed by a second slower rate. The activity was linear with enzyme concentration over a small range of concentration and was dependent on the time of incubation. Inhibition of enzyme activity with increasing concentrations of AMP was sigmoid; concentrations less than 1 X 10-G M were without effect, concentrations between 1 X 10-C and 8 X 10-b M inhibited by 20% and concentrations beyond 8 X 10m5M caused progressive inhibition. Concentrations beyond 1 X 1OV M inhibited the activity completely. Preincubation of the eniyme with PCMB or NEM, or aging, or reversible denaturation with urea abolished the inhibitory effect of AMP at concentrations lower than 8 X 10-5 M. The aged enzyme could be reactivated by ADP.
of FAD and the regulatory effects of adenine nucleotides on a number of metabolic reactions, it was of interest to investigate the effect of adenine nucleotides on the highly purified enzyme hydrolyzing FAD (6). In this communication we report the inhibition by AMP on the hydrolysis of FAD catalyzed by the enzyme functioning optimally at pH 9.4, desensitization of the initial inhibitory effect of AMP, and the activation of the partially inactivated enzyme by ADP.
Biosynthesis of flavine coenzyme nucleotides in plants proceeds by the phosphorylation of riboflavine, followed by reaction with another molecule of ATP to yield flavine adenine dinucleotide (FAD) (1, 2). The degradation of these nucleotides in plants is catalyzed by specific enzymes (3-6). FAD hydrolysis in plants is catalyzed by enzymes functioning optimally at pH values of 3.8 (unpublished), 7.4 (4, 5), and at 9.4 (6). The enzyme hydrolyzing FAD at pH 9.4 was extensively purified from mung bean seedlings and was inhibited by metal chelators tetraacetate such as ethylenediamine (EDTA). This inhibition could specifically be reversed by Zn2+ and Co2+ ions (6). The enzymes hydrolyzing FAD at acid pH values were inhibited by AMP (7-9). In view of the fact that AMP is a product of the hydrolysis
MATERIALS
AND
METHODS
Materials. FAD, FMN, ATP, ADP, AMP, and p-chloromercuribenzoate (PCMB) were obtained f rom Sigma Chemical Company, St. Louis, Missouri. FAD, FMN, and ADP were purified on diethyl aminoethyl (DEAE) cellulose columns. All other chemicals used were of analytical reagent grade. The seeds were purchased from local market. Methods. The enzyme was prepared as reported earlier (6). Hydrolysis of FAD was determined as follows: One milliliter of the assay mixture contained 40 rmoles of sodium veronal-hydrochloric acid buffer pH 9.4, 0.5 pmole of FAD, and a suit-
1 This research work has been financed in part by Grant FG-In-392 made by the United St,ates Agriculture Department under US P.L.-480. 2 This work was presented at the Annual meeting of the Society of Biological Chemists, India, at Hyderabad in Feb. 1969. 54
ENZYME
HYDROLYZING
able amount of the enzyme. Appropriate cont’rols were always run. After incubation at 49”, the reaction was stopped by the addition of 2 vol of ethanol. An aliquot of the reaction mixture was subjected to circular paper chromatography on Whatman No. 3 filter paper using butanol: acetic acid:water (4:1:5 v/v) as the developing solvent (10). The chromatograms were air-dried in dark, and the fluorescent spots of FAD and FMN were located under an ultraviolet lamp (Woods filter Phillips lamp HPW 125 W type 57202). The flavines were eluted into 5 ml of glass-distilled water, and fluorescence of the eluates were measured in a Klett fluorimeter with primary B1 (440 rnp) and a secondary orange filter (520 mp). An additional method of assaying the activity was by direct fluorimetry. At neutral pH, FMN and riboflavine are about ten times as fluorescent as FAD (11). Aliquots of the reaction mixture were made up to 5 ml and the amount of FMN was determined by measuring the increase in fluorescence in a Klett fluorimeter as described above. Units of activity. Millimicromoles of FMN formed per minute per milliliter at pH 9.4 and at 49”. Specific activity is defined as units per milligram protein. Protein assays were performed according to the procedure of Lowry et al. (12) using crystalline bovine serum albumin as the standard. RESULTS
Time course of the reaction. In order to determine the rate of the reaction at different time intervals, the reaction was scaled up to 3 ml, incubated at 49”, and 0.2,5-ml aliquots were withdrawn at the time intervals specified in Fig. 1. The amount of FMK formed was determined by direct fluorimetry as well as after chromatographic separation from FAD. From the figure it is obvious that the enzyme functions with a fast rate up to 60 set followed by a second, linear slower rate up to 7 min. These results suggest that either one or both the products of the reaction are inhibitory. Another explanation would be that a reversible steady state is achieved. All attempts at measuring the reaction in the reverse direction were unsuccessful. E$ect of enzyme concentration. In Fig. 2 is presented the effect of increasing enzyme concentration on the activity of enzyme assayed for 1, 3, and 10 min. All activities were normalized to 1 min.
FAD
35
TIME (MIN
)
FIG. 1. Time course of FAD hydrolysis
by the enzyme. The reaction mixture was scaled up to 3 ml and the reaction mixture contained 48 pg of the protein (sp. act. 4000). The reaction mixture (0.25 ml) was withdrawn at time intervals indicated and added into a test tube containing 0.5 ml of ethanol. The activity was assayed as described in test.
From Fig. 2 it is evident that at the enzyme concentrations tested the specific activity is proportional to enzyme concentration, when assayed for 1 min. When the assay time was increased to 3 min the large amount of product(s) formed might have resulted in partial inhibition of the enzyme activity. This partial inhibition was reflected in the nonlinearity of the specific activity with increasing enzyme concentration. On further increase in assay time to 10 min a new linearity at lowered specific activity was established. These results lend further support to the hypothesis that one or both the products at low concentration partially inhibited the reaction. E$ect of FMN and AMP. Increasing concentrations of FMN added to the reaction mixture prior to the addition of FAD had no effect on the activity measured by the disappearance of FAD or by the estimation of the additional FMN formed. Preincubation of the above enzyme with varying concentrations of FMiY also had no effect on the activity. Figure 3 shows the effect of different concentrations of AMP on the enzyme activity. AMP concentrations below 1 X lo-” M were without effect and between 1 X 10e6 and 8 X 10-j ni inhibited the RC-
RAVINDRANATH
I
4
I
8 pg
I
I
12 PROTEIN
16
I
20
I m,
FIG. 2. Effect of time of incubation and protein concentration on the specific activity of the enzyme hydrolyzing FAD. Enzyme protein (sp. act. 4200) indicated in the figure was incubated at 49” and pH 9.4, for 1 (O-O-O-O), 3 (Q--O--O--O), and 10 (X-X-X-X) min and assayed as described in the text. All the activities were normalized to specific activity.
AND
RAO
inhibition (20%) by low concentration of AMP was abolished. Attempts at desensitizing the major inhibitory effect of AMP by increasing concentration of PCMB or controlled heat denaturation or urea denaturation-renaturation or by aging were unsuccessful. All these treatments resulted in the desensitization of the small inhibition by AMP. Preincubation with N-ethyl maleimide (NEM) resulted in a similar desensitization. The effect of varying concentrations of substrate at different fixed concentrations of AMP, showed that at low concentrations of AMP, the K, value for FAD was altered slightly but was without effect on V,,, . At high concentration of AMP, both V,,, as well as Km were altered. The Km and V,,, for the enzyme without any treatment were 2 X 10m4M and 20 mpmoles/min, respectively. E$ect of ADP. Aging of the diluted enzyme (below 200 pg/ml) at 4” for 12 hr or
tivity by about 20%. Increasing the AMP concentration above 8 X lo+ M resulted in a progressive inhibition and complete inhibition was observed at a concentration of 1 X l(k3
M.
To confirm this effect of AMP on the initial and the overall rates of the reaction the enzyme was preincubated with 5 X lo+ M AMP for 5 min at 20” and assayed for the time intervals indicated in Fig. 4A. Addition of AMP to the assay tubes just prior to the addition of the substrate gave similar results. The enzyme was preincubated with 5 X 1W4 M PCMB for 20 min at 20” and assayed for time intervals indicated in Fig. 4B. It is clear that preincubation with PCMB resulted in the reaction proceeding at the initial fast rate for a longer period of time. Preincubation of the enzyme with 5 X 10U5 M AMP and 5 X lOA M PCMB also resulted in the enzyme functioning at the initial rate for a longer period of time. The effect of PCMB on the inhibition of the activity by increasing concentrations of AMP showed (Fig. 3) that the small
AMP
CONC LOG(M)
3. Effect of AMP concentration on the freshly prepared, aged, and PCMB-treated enzyme hydrolyzing FAD. Each assay tube contained 18.4 ag of enzyme protein (sp. act. 4200) and AMP at concentration (final) indicated in the figure. A reaction mixture without AMP served as the control and all assays were performed as described in the text. Freshly prepared enzyme (X-X-X); freshly prepared enzyme was preincubated with 5 X 10-’ M PCMB at 20” for 20 min (O-O-O); and aged enzyme-freshly prepared enzyme kept at 4’ for 15 days (0-0-o). FIG.
ENZYME
TIME
(MIN
HYDROLYZING
57
FAD
I
I
I
I
I
1
2
3
4
5
TIME
)
( MINI
FIG. 4. Effect of AMP and PCMB on the time course of FAD hydrolysis by the enzyme. A, 64 pg of the enzyme (3000 sp act.)/3 ml of the reaction mixture was preincubated with AMP (5 X W6 M) at 20” for 5 min. Aliquots of the reaction mixture were withdrawn at time intervals indicated in Fig. 4A and assayed for activity as described in text. Control enzyme (O--O--O--O); AMP-treated enzyme (O-O-0-0). B, 400rg of enzyme (sp. act. 35OO)/ml was preincubated with 5 X 1O-4 M PCMB at 20” for 20 min. An aliquot, equivalent to 60 rg of protein, was used for the determination of the act,ivity. An enzyme similarly treated without PCMB served as the control. Three-milliliter reactions were run and aliquots were withdrawn at t,ime intervals specified and assayed. Control (O-0-0); PCMB-t,reated enzyme (O-O-O).
longer resulted in appreciable (about 60 %) loss of activity. Addition of ADP resulted in the restoration of the activity. Figure 5 shows the effect of varying concentrations of ADP on the enzyme activity. Increasing the ADP concentration up to 1 X 1OW M resulted in almost a 2.5-fold increase in activity but further increase in ADP concentrations resulted in inhibition. Concentration of ADP above 5 X 1O-4 M resulted in inhibition. The inhibited activity being less than the activity of the control enzyme. This inhibition was not due to the presence of AMP contamination. Effect of other metabolites. UMP, GMP, CI\IP, IMP, riboflavine, and adenosine at the concentrations tested (Table II) had no effect on the enzyme activity. At 1 X 10e4 M ATP, both K, and V,,, were unaltered, whereas at concentration 3 X 10m4M, the K, for FAD was 4 X lOA M and V,,, 12.8 mpmoles/min (Table I). At
TABLE EFFECT
OF
Treatment
Fresh AMP AMP ATP ATP
I
AMP AND ATP ON THE K, OF FAD FOR THE ENZYMES
enzyme (1 X 1O-4 M) (6 X 1O-4 M) (3 X lo-’ M) (6 X 1OW M)
Km x 10-n I
V,,,
AND
V,,,
(mpmoles of FMN formed)
2 4 10 4
20.0 20.0 14.0 12.8
4
8.4
a 18.4 pg of the enzyme (4200 sp. act.) was preincubated with AMP and ATP. The activity was determined at six different concentrations of substrate (1 X 10-4-l X 10-a M). K, and V,,, were calculated from Lineweaver-Burke plots. b V,,, was normalized per minute per milliliter of the reaction mixture.
6 X lop4 M ATP, the K, was 4 X lop4 M and V,,, was 8.4 mpmoles. Thus, it is clear that ATP inhibits the enzyme activity noncompetitively.
58
RAVINDRANATH
AND RAO DISCUSSION
The biosynthesis of FAD involves the initial phosphorylation of riboflavine by a kinase type of reaction followed by adenylate transfer to FMN. Both these reactions require ATP as one of the substrates. In the reaction catalyzed by flavokinase, ADP is a product. FMN and FAD are degraded by phosphatase and nucleotide pyrophosphatase, respectively. Our earlier observations showed that both these reactions are irreversible and specific for these substrates. The FMN-hydrolyzing enzyme is specific for FMN whereas the dinucleotide pyrophosphatase hydrolyzed NAD and NADH in addition to FAD (6). Mitsuda (9) postu--3
lated that
-4
75L
I AOPCONC.
LOG(M)
FIG. 5. Effect of ADP concentration on the diluted and aged enzyme hydrolyzing FAD. Freshly prepared enzyme was diluted to a protein concentration of 200 pg/ml and kept at 4’ for 12-15 hr. Reaction mixture without added ADP served as control and the activity obtained was assumed as 100. ADP was added to a final concentration indicated in the figure. Activity was assayed as described in the text and expressed as percentage of control enzyme.
TABLE II EFFECT OF METABOLITES ON THE ENZYME HYDROLYZING FADa Metabolites
UMP GMP CMP IMP FMN Sodium pyrophosphateb Ribo5avine
% Inhibition Concentration(a6) 1 x lo-’ 1 x 10-a 0 0 0 0 0 0 0
12 0 15 0 0 60 0
0 18.4 pg of the enzyme (sp. act. 4200) was preincubated with metabolites at concentrations indicated in Table for 5 min at 20”. The activity was assayed as described in the text. b Sodium pyrophosphate at 1 X 10-t M completely inhibited the activity.
adenine
nucleotides
exert
a regu-
latory effect on the degradation of flavine nucleotides. From the results presented here it is obvious that AMP, a product of the reaction exerts an inhibitors effect on the rate of FAD hydrolysis, Prkincubation of the enzyme with low concentrations of AMP results in the decrease of the initial fast, rate. This suggests a delicately balanced control on the hydrolysis of FAD, preventing its rapid concentration
degradation. Over a large range A?(/IP has no further
effect. Concentrations above inhibitory 1 X 10e4 M of AMP results in a marked increase in inhibition. Nonlinearity of AMP induced inhibition with increasing concentration suggests that this may not be a simple product inhibition. This biphasic nature of the inhibition gests that An!lP must
curve (Fig. 3) sugbe acting at more
than one site. This hypothesis is supported by the fact that one of the sites can be preferentially desensitized with PCXB, or NEM, or aging, or reversible denaturation with urea. The other mononucleotides, such as UMP, GMP, CMP, I?(IIP, and F?tlN, and riboflavine
and
adenosine
were
without
effect. However, attempts at desensitizing the other AMP site were unsuccessful. These results on the highly purified enzyme hydrolyzing FAD from mung beans suggest possible mechanisms regulating the biosynthesis of flavine nucleotides. ATP, which is involved in the two biosynthetic steps, acts as a noncompetitive inhibitor for the hydrolytic enzyme. ADP, a product
ENZYME
HYDROLYZING
of the first step, activates the partially inactive enzyme. A1\IP, on the other hand, has a finer control on the hydrolytic activity. Undoubtedly these effects will have to be investigated in greater detail at the moleculnr level before a regulatory function on the biosynthetic pathway is ascribed to any or all of these adenine nucleotides. ACKNOWLEDGRIENTS The aat,hors are indebted to Dr. C. S. T’aidyanathan for his help in preparing t,he manuscript and in the c*olwse of this investigation. REFERENCES 1. GIRI, K. N. A., 2. &RI, K. KIXAR, 3. RAO, N.
V., KRISHNASWAYY,
P. R., AND Rao,
Biochem. J. 70,66 (1958). V., RAO, N. A., CAMA, H. R., AND S. 8., Biochem. J. 76,381 (1960). A., CAMA, H. R., KUMAR, S. A., AND
59
FAD
VAIDYANATHAN, C. S., Bioch,im. Biophys. Acta 73, 87 (1963). 4. KORNBERG,
A. AND PRICER, IV.
E.,
JR., J.
Biol. Chem. 182, 763 (1950). 5. KcMaR, S. A., Rao, N. A., AND T’AIDYANATHAN, C. S., Arch. Biochem. Biophys. 111, 646 (1965). 6. RAVINDRANATH, 8. D. AND XAO, N. A., Indian J. Biochem. 6, 137 (1968). 7. BRIOHTWELL, It. AND TAPPEL, A. L., Awh. Biochem. Biovhvs. 124,333 (1968). 8. RAGAB, ?\,I. H.,*B&GHTWELL, h., AND TAPPEL, A. L., Arch. Biochem. Biophys. 123, 179 (1968). 9. ~VITSUDA. H.. Proc. Javan Acarl. 42,940 (1966). 10. GIRI, K.‘V.‘AND KR~HNASJJ-AMY; P. k., j. Indian Inst. Sci. 33,232 (1956). 11. BESSEY, 0. A., LOWRY, 0. H., AND LOVE, R. H., J. Biol. Chem. 180,755 (1949). 12. LOWRT, 0. H., ROSEBROUGH, N. J., FARR, A. L., *END R~XDALL, R. J., J. Viol. Chem. 193, 265 (1951).
OF
BIOCHEMISTRY
AND
BIOPHYSICS
133, i%-59 (1969)
Nucleotidases III. Effect of Metabolites
in Plants
on the Enzyme Hydrolyzing
Dinucleotide
of Biochemistry, Received
Adenine
(FAD) from Phaseolus radiatus1~2
S. D. RAVINDRANATH Department
Flavine
March
AND
Indian Institute
N. APPAJI
RAO
of Science, Bangalore-lb,
28, 1969; accepted
May
India
9, 1969
The highly purified enzyme from mung bean seedlings hydrolyzing FAD at pH 9.4 and temperature 49”, functioned with an initial fast rate followed by a second slower rate. The activity was linear with enzyme concentration over a small range of concentration and was dependent on the time of incubation. Inhibition of enzyme activity with increasing concentrations of AMP was sigmoid; concentrations less than 1 X 10-G M were without effect, concentrations between 1 X 10-C and 8 X 10-b M inhibited by 20% and concentrations beyond 8 X 10m5M caused progressive inhibition. Concentrations beyond 1 X 1OV M inhibited the activity completely. Preincubation of the eniyme with PCMB or NEM, or aging, or reversible denaturation with urea abolished the inhibitory effect of AMP at concentrations lower than 8 X 10-5 M. The aged enzyme could be reactivated by ADP.
of FAD and the regulatory effects of adenine nucleotides on a number of metabolic reactions, it was of interest to investigate the effect of adenine nucleotides on the highly purified enzyme hydrolyzing FAD (6). In this communication we report the inhibition by AMP on the hydrolysis of FAD catalyzed by the enzyme functioning optimally at pH 9.4, desensitization of the initial inhibitory effect of AMP, and the activation of the partially inactivated enzyme by ADP.
Biosynthesis of flavine coenzyme nucleotides in plants proceeds by the phosphorylation of riboflavine, followed by reaction with another molecule of ATP to yield flavine adenine dinucleotide (FAD) (1, 2). The degradation of these nucleotides in plants is catalyzed by specific enzymes (3-6). FAD hydrolysis in plants is catalyzed by enzymes functioning optimally at pH values of 3.8 (unpublished), 7.4 (4, 5), and at 9.4 (6). The enzyme hydrolyzing FAD at pH 9.4 was extensively purified from mung bean seedlings and was inhibited by metal chelators tetraacetate such as ethylenediamine (EDTA). This inhibition could specifically be reversed by Zn2+ and Co2+ ions (6). The enzymes hydrolyzing FAD at acid pH values were inhibited by AMP (7-9). In view of the fact that AMP is a product of the hydrolysis
MATERIALS
AND
METHODS
Materials. FAD, FMN, ATP, ADP, AMP, and p-chloromercuribenzoate (PCMB) were obtained f rom Sigma Chemical Company, St. Louis, Missouri. FAD, FMN, and ADP were purified on diethyl aminoethyl (DEAE) cellulose columns. All other chemicals used were of analytical reagent grade. The seeds were purchased from local market. Methods. The enzyme was prepared as reported earlier (6). Hydrolysis of FAD was determined as follows: One milliliter of the assay mixture contained 40 rmoles of sodium veronal-hydrochloric acid buffer pH 9.4, 0.5 pmole of FAD, and a suit-
1 This research work has been financed in part by Grant FG-In-392 made by the United St,ates Agriculture Department under US P.L.-480. 2 This work was presented at the Annual meeting of the Society of Biological Chemists, India, at Hyderabad in Feb. 1969. 54
ENZYME
HYDROLYZING
able amount of the enzyme. Appropriate cont’rols were always run. After incubation at 49”, the reaction was stopped by the addition of 2 vol of ethanol. An aliquot of the reaction mixture was subjected to circular paper chromatography on Whatman No. 3 filter paper using butanol: acetic acid:water (4:1:5 v/v) as the developing solvent (10). The chromatograms were air-dried in dark, and the fluorescent spots of FAD and FMN were located under an ultraviolet lamp (Woods filter Phillips lamp HPW 125 W type 57202). The flavines were eluted into 5 ml of glass-distilled water, and fluorescence of the eluates were measured in a Klett fluorimeter with primary B1 (440 rnp) and a secondary orange filter (520 mp). An additional method of assaying the activity was by direct fluorimetry. At neutral pH, FMN and riboflavine are about ten times as fluorescent as FAD (11). Aliquots of the reaction mixture were made up to 5 ml and the amount of FMN was determined by measuring the increase in fluorescence in a Klett fluorimeter as described above. Units of activity. Millimicromoles of FMN formed per minute per milliliter at pH 9.4 and at 49”. Specific activity is defined as units per milligram protein. Protein assays were performed according to the procedure of Lowry et al. (12) using crystalline bovine serum albumin as the standard. RESULTS
Time course of the reaction. In order to determine the rate of the reaction at different time intervals, the reaction was scaled up to 3 ml, incubated at 49”, and 0.2,5-ml aliquots were withdrawn at the time intervals specified in Fig. 1. The amount of FMK formed was determined by direct fluorimetry as well as after chromatographic separation from FAD. From the figure it is obvious that the enzyme functions with a fast rate up to 60 set followed by a second, linear slower rate up to 7 min. These results suggest that either one or both the products of the reaction are inhibitory. Another explanation would be that a reversible steady state is achieved. All attempts at measuring the reaction in the reverse direction were unsuccessful. E$ect of enzyme concentration. In Fig. 2 is presented the effect of increasing enzyme concentration on the activity of enzyme assayed for 1, 3, and 10 min. All activities were normalized to 1 min.
FAD
35
TIME (MIN
)
FIG. 1. Time course of FAD hydrolysis
by the enzyme. The reaction mixture was scaled up to 3 ml and the reaction mixture contained 48 pg of the protein (sp. act. 4000). The reaction mixture (0.25 ml) was withdrawn at time intervals indicated and added into a test tube containing 0.5 ml of ethanol. The activity was assayed as described in test.
From Fig. 2 it is evident that at the enzyme concentrations tested the specific activity is proportional to enzyme concentration, when assayed for 1 min. When the assay time was increased to 3 min the large amount of product(s) formed might have resulted in partial inhibition of the enzyme activity. This partial inhibition was reflected in the nonlinearity of the specific activity with increasing enzyme concentration. On further increase in assay time to 10 min a new linearity at lowered specific activity was established. These results lend further support to the hypothesis that one or both the products at low concentration partially inhibited the reaction. E$ect of FMN and AMP. Increasing concentrations of FMN added to the reaction mixture prior to the addition of FAD had no effect on the activity measured by the disappearance of FAD or by the estimation of the additional FMN formed. Preincubation of the above enzyme with varying concentrations of FMiY also had no effect on the activity. Figure 3 shows the effect of different concentrations of AMP on the enzyme activity. AMP concentrations below 1 X lo-” M were without effect and between 1 X 10e6 and 8 X 10-j ni inhibited the RC-
RAVINDRANATH
I
4
I
8 pg
I
I
12 PROTEIN
16
I
20
I m,
FIG. 2. Effect of time of incubation and protein concentration on the specific activity of the enzyme hydrolyzing FAD. Enzyme protein (sp. act. 4200) indicated in the figure was incubated at 49” and pH 9.4, for 1 (O-O-O-O), 3 (Q--O--O--O), and 10 (X-X-X-X) min and assayed as described in the text. All the activities were normalized to specific activity.
AND
RAO
inhibition (20%) by low concentration of AMP was abolished. Attempts at desensitizing the major inhibitory effect of AMP by increasing concentration of PCMB or controlled heat denaturation or urea denaturation-renaturation or by aging were unsuccessful. All these treatments resulted in the desensitization of the small inhibition by AMP. Preincubation with N-ethyl maleimide (NEM) resulted in a similar desensitization. The effect of varying concentrations of substrate at different fixed concentrations of AMP, showed that at low concentrations of AMP, the K, value for FAD was altered slightly but was without effect on V,,, . At high concentration of AMP, both V,,, as well as Km were altered. The Km and V,,, for the enzyme without any treatment were 2 X 10m4M and 20 mpmoles/min, respectively. E$ect of ADP. Aging of the diluted enzyme (below 200 pg/ml) at 4” for 12 hr or
tivity by about 20%. Increasing the AMP concentration above 8 X lo+ M resulted in a progressive inhibition and complete inhibition was observed at a concentration of 1 X l(k3
M.
To confirm this effect of AMP on the initial and the overall rates of the reaction the enzyme was preincubated with 5 X lo+ M AMP for 5 min at 20” and assayed for the time intervals indicated in Fig. 4A. Addition of AMP to the assay tubes just prior to the addition of the substrate gave similar results. The enzyme was preincubated with 5 X 1W4 M PCMB for 20 min at 20” and assayed for time intervals indicated in Fig. 4B. It is clear that preincubation with PCMB resulted in the reaction proceeding at the initial fast rate for a longer period of time. Preincubation of the enzyme with 5 X 10U5 M AMP and 5 X lOA M PCMB also resulted in the enzyme functioning at the initial rate for a longer period of time. The effect of PCMB on the inhibition of the activity by increasing concentrations of AMP showed (Fig. 3) that the small
AMP
CONC LOG(M)
3. Effect of AMP concentration on the freshly prepared, aged, and PCMB-treated enzyme hydrolyzing FAD. Each assay tube contained 18.4 ag of enzyme protein (sp. act. 4200) and AMP at concentration (final) indicated in the figure. A reaction mixture without AMP served as the control and all assays were performed as described in the text. Freshly prepared enzyme (X-X-X); freshly prepared enzyme was preincubated with 5 X 10-’ M PCMB at 20” for 20 min (O-O-O); and aged enzyme-freshly prepared enzyme kept at 4’ for 15 days (0-0-o). FIG.
ENZYME
TIME
(MIN
HYDROLYZING
57
FAD
I
I
I
I
I
1
2
3
4
5
TIME
)
( MINI
FIG. 4. Effect of AMP and PCMB on the time course of FAD hydrolysis by the enzyme. A, 64 pg of the enzyme (3000 sp act.)/3 ml of the reaction mixture was preincubated with AMP (5 X W6 M) at 20” for 5 min. Aliquots of the reaction mixture were withdrawn at time intervals indicated in Fig. 4A and assayed for activity as described in text. Control enzyme (O--O--O--O); AMP-treated enzyme (O-O-0-0). B, 400rg of enzyme (sp. act. 35OO)/ml was preincubated with 5 X 1O-4 M PCMB at 20” for 20 min. An aliquot, equivalent to 60 rg of protein, was used for the determination of the act,ivity. An enzyme similarly treated without PCMB served as the control. Three-milliliter reactions were run and aliquots were withdrawn at t,ime intervals specified and assayed. Control (O-0-0); PCMB-t,reated enzyme (O-O-O).
longer resulted in appreciable (about 60 %) loss of activity. Addition of ADP resulted in the restoration of the activity. Figure 5 shows the effect of varying concentrations of ADP on the enzyme activity. Increasing the ADP concentration up to 1 X 1OW M resulted in almost a 2.5-fold increase in activity but further increase in ADP concentrations resulted in inhibition. Concentration of ADP above 5 X 1O-4 M resulted in inhibition. The inhibited activity being less than the activity of the control enzyme. This inhibition was not due to the presence of AMP contamination. Effect of other metabolites. UMP, GMP, CI\IP, IMP, riboflavine, and adenosine at the concentrations tested (Table II) had no effect on the enzyme activity. At 1 X 10e4 M ATP, both K, and V,,, were unaltered, whereas at concentration 3 X 10m4M, the K, for FAD was 4 X lOA M and V,,, 12.8 mpmoles/min (Table I). At
TABLE EFFECT
OF
Treatment
Fresh AMP AMP ATP ATP
I
AMP AND ATP ON THE K, OF FAD FOR THE ENZYMES
enzyme (1 X 1O-4 M) (6 X 1O-4 M) (3 X lo-’ M) (6 X 1OW M)
Km x 10-n I
V,,,
AND
V,,,
(mpmoles of FMN formed)
2 4 10 4
20.0 20.0 14.0 12.8
4
8.4
a 18.4 pg of the enzyme (4200 sp. act.) was preincubated with AMP and ATP. The activity was determined at six different concentrations of substrate (1 X 10-4-l X 10-a M). K, and V,,, were calculated from Lineweaver-Burke plots. b V,,, was normalized per minute per milliliter of the reaction mixture.
6 X lop4 M ATP, the K, was 4 X lop4 M and V,,, was 8.4 mpmoles. Thus, it is clear that ATP inhibits the enzyme activity noncompetitively.
58
RAVINDRANATH
AND RAO DISCUSSION
The biosynthesis of FAD involves the initial phosphorylation of riboflavine by a kinase type of reaction followed by adenylate transfer to FMN. Both these reactions require ATP as one of the substrates. In the reaction catalyzed by flavokinase, ADP is a product. FMN and FAD are degraded by phosphatase and nucleotide pyrophosphatase, respectively. Our earlier observations showed that both these reactions are irreversible and specific for these substrates. The FMN-hydrolyzing enzyme is specific for FMN whereas the dinucleotide pyrophosphatase hydrolyzed NAD and NADH in addition to FAD (6). Mitsuda (9) postu--3
lated that
-4
75L
I AOPCONC.
LOG(M)
FIG. 5. Effect of ADP concentration on the diluted and aged enzyme hydrolyzing FAD. Freshly prepared enzyme was diluted to a protein concentration of 200 pg/ml and kept at 4’ for 12-15 hr. Reaction mixture without added ADP served as control and the activity obtained was assumed as 100. ADP was added to a final concentration indicated in the figure. Activity was assayed as described in the text and expressed as percentage of control enzyme.
TABLE II EFFECT OF METABOLITES ON THE ENZYME HYDROLYZING FADa Metabolites
UMP GMP CMP IMP FMN Sodium pyrophosphateb Ribo5avine
% Inhibition Concentration(a6) 1 x lo-’ 1 x 10-a 0 0 0 0 0 0 0
12 0 15 0 0 60 0
0 18.4 pg of the enzyme (sp. act. 4200) was preincubated with metabolites at concentrations indicated in Table for 5 min at 20”. The activity was assayed as described in the text. b Sodium pyrophosphate at 1 X 10-t M completely inhibited the activity.
adenine
nucleotides
exert
a regu-
latory effect on the degradation of flavine nucleotides. From the results presented here it is obvious that AMP, a product of the reaction exerts an inhibitors effect on the rate of FAD hydrolysis, Prkincubation of the enzyme with low concentrations of AMP results in the decrease of the initial fast, rate. This suggests a delicately balanced control on the hydrolysis of FAD, preventing its rapid concentration
degradation. Over a large range A?(/IP has no further
effect. Concentrations above inhibitory 1 X 10e4 M of AMP results in a marked increase in inhibition. Nonlinearity of AMP induced inhibition with increasing concentration suggests that this may not be a simple product inhibition. This biphasic nature of the inhibition gests that An!lP must
curve (Fig. 3) sugbe acting at more
than one site. This hypothesis is supported by the fact that one of the sites can be preferentially desensitized with PCXB, or NEM, or aging, or reversible denaturation with urea. The other mononucleotides, such as UMP, GMP, CMP, I?(IIP, and F?tlN, and riboflavine
and
adenosine
were
without
effect. However, attempts at desensitizing the other AMP site were unsuccessful. These results on the highly purified enzyme hydrolyzing FAD from mung beans suggest possible mechanisms regulating the biosynthesis of flavine nucleotides. ATP, which is involved in the two biosynthetic steps, acts as a noncompetitive inhibitor for the hydrolytic enzyme. ADP, a product
ENZYME
HYDROLYZING
of the first step, activates the partially inactive enzyme. A1\IP, on the other hand, has a finer control on the hydrolytic activity. Undoubtedly these effects will have to be investigated in greater detail at the moleculnr level before a regulatory function on the biosynthetic pathway is ascribed to any or all of these adenine nucleotides. ACKNOWLEDGRIENTS The aat,hors are indebted to Dr. C. S. T’aidyanathan for his help in preparing t,he manuscript and in the c*olwse of this investigation. REFERENCES 1. GIRI, K. N. A., 2. &RI, K. KIXAR, 3. RAO, N.
V., KRISHNASWAYY,
P. R., AND Rao,
Biochem. J. 70,66 (1958). V., RAO, N. A., CAMA, H. R., AND S. 8., Biochem. J. 76,381 (1960). A., CAMA, H. R., KUMAR, S. A., AND
59
FAD
VAIDYANATHAN, C. S., Bioch,im. Biophys. Acta 73, 87 (1963). 4. KORNBERG,
A. AND PRICER, IV.
E.,
JR., J.
Biol. Chem. 182, 763 (1950). 5. KcMaR, S. A., Rao, N. A., AND T’AIDYANATHAN, C. S., Arch. Biochem. Biophys. 111, 646 (1965). 6. RAVINDRANATH, 8. D. AND XAO, N. A., Indian J. Biochem. 6, 137 (1968). 7. BRIOHTWELL, It. AND TAPPEL, A. L., Awh. Biochem. Biovhvs. 124,333 (1968). 8. RAGAB, ?\,I. H.,*B&GHTWELL, h., AND TAPPEL, A. L., Arch. Biochem. Biophys. 123, 179 (1968). 9. ~VITSUDA. H.. Proc. Javan Acarl. 42,940 (1966). 10. GIRI, K.‘V.‘AND KR~HNASJJ-AMY; P. k., j. Indian Inst. Sci. 33,232 (1956). 11. BESSEY, 0. A., LOWRY, 0. H., AND LOVE, R. H., J. Biol. Chem. 180,755 (1949). 12. LOWRT, 0. H., ROSEBROUGH, N. J., FARR, A. L., *END R~XDALL, R. J., J. Viol. Chem. 193, 265 (1951).