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Inhibition of yeast inorganic pyrophosphatase by nucleotidylsulfates
A R C t l I V E S OF B I O C H E M I S T R Y AND B I O P t I Y S I C S
146, 71-75 (1971)
Inhibition of Yeast Inorganic Pyrophosphatase by Nucleotidylsulfates ~ M . S H O Y A B AND W . M A R X
Department of Biochemistry, University of Southern California, School of Medicine, Los Angeles, California 90083 Received February 4, 1971; accepted May 19, 1971 The influence of some purine nucleotides on the activity of yeast inorganic pyrophosphatase was investigated. The nucleotidylsulfates examined were found to exert a significant inhibition. Guanylylsulfate and inosinylsulfate depressed the rate of the pyrophosphatase reaction markedly, adenylylsulfate and its deoxy analog inhibited to a lesser but still significant extent. earlier (1). Adenosine, ADP, ATP, GDP, GTP, IMP, and ITP were purchased from CalBiochem, dAMP, cyclic AMP, and GMP from P-L Biochemicals, Inc., and Tris, AMP, and crystalline bovine serum albumin from Sigma Chemical Company. APS was prepared according to the method of Baddiley et al. from AMP and pyridine sulfate (4), and purified by chromatography on ECTEOLA-celhflose (2). dAPS, GPS, and IPS were prepared and purified in a similar manner. Methods. The inorganic pyrophosphatase was used without further treatment after a 100-fold dilution with cold water containing 1 mg/ml of albumin. The enzyme activity was determined by measuring Pi formation from PPI. The reaction mixture, containing 100 mM Tris-HC1, pH 7.4, 3.8 m• sodium PPi, 2 m~ MgCI2, and 0.01 ml enzyme solution in a total volume of 0.2 ml, was incubated for 5 rain at 37 ~ The reaction was stopped by adding 0.5 ml of 2.5% molybdate in 5 ~ H2SO4. The Pi liberated was measured by the method of Fiske and Subbarow (5) in a total volume of 5 ml. In the control sample, boiled enzyme was used, or enzyme was added after incubation.
Recently, the purification and properties of m o u s e m a s t o c y t o m a A T P sulfurylase ( A T P : sulfate a d e n y l y l t r a n s f e r a s e , E C 2 . 7 . 7 . 4 ) were i n v e s t i g a t e d in our l a b o r a t o r y (1, 2); for a s s a y of t h i s e n z y m e , a m e t h o d of R o b b i n s a n d L i p m a n n (3) w a s u s e d w h i c h involved yeast inorganic pyrophosphatase ( p y r o p h o s p h a t e p h o s p h o h y d r o l a s e , E C 3. 6 . 1 . 1 ) . D u r i n g t h e s e studies, we n o t i c e d that the pyrophosphatase was inhibited by a d e n y l y l s u l f a t e (APS). 2 T h i s p h e n o m e n o n was i n v e s t i g a t e d further, in p a r t i c u l a r , w i t h r e s p e c t t o t h e effects of o t h e r nucleotides. T h e results of these s t u d i e s a r e p r e s e n t e d in this communication. MATERIALS AND METHODS
Materials. Chemicals were C. P. or reagent grade, or the purest preparations available. Sodium pyrophosphate was obtained from Mallinckrodt Chemical Works, and crystalline yeast inorganic pyrophosphatase (1.2 mg protein/ml, corresponding to 820 enzyme U/mg protein) from Worthington Biochemical Corporation. Control experiments demonstrated that the pyrophosphatase was free of ATP sulfurylase activity, as measured according to a technique described
RESULTS AND DISCUSSION
Influence of some nueleotides on pyrophosphatase. T h e influence of some nueleotides a n d a d e n o s i n e on t h e a c t i v i t y of y e a s t inorganic p y r o p h o s p h a t a s e w a s s t u d i e d und e r t h e c o n d i t i o n s d e s c r i b e d above. T h e r e s u l t s of t h e s e e x p e r i m e n t s are shown in T a b l e I. T h e n u c l e o t i d y l sulfates e x a m i n e d were f o u n d t o i n h i b i t t h e e n z y m a t i c h y d r o l y sis of PP~ significantly. I n t h e presence of 1
This investigation was supported, in part, by Research Grant 5 R01 HE 1596 from the National Heart Institute, Public Health Service. z Abbreviations used: APS, adenylylsulfate; dAPS, deoxyadenylylsulfate; GPS, guanylylsulfate; IPS, inosinylsulfate; PAPS, 3t-phospho adenylylsulfate. 71
72
SHOYAB AND MARX TABLE I
I N F L U E N C E OF SOME P U R I N E N U C L E O T I D E S AND ADENOSINE
ON Y E A S T
INORGANIC PYROPttOS-
PHATASE a Addition to incubation mixture
mvmoles Pi formed in 5 min
Percentage inhibition
None GPS IPS dAPS APS AMP GTP GDP Cyclic AMP GMP ADP Adenosine ATP
420 150 156 238 239 326 358 358 359 383 388 410 425
-64 63 44 43 22 15 15 15 9 8 1 0
Determination of pyrophosphatase activity as described in text, except for the addition of the nucleotides or adenosine as indicated at a final concentration of 1 raM. mM GPs or IPS, the reaction rate was depressed by over 60%; APS and dAPS caused an inhibition of over 40 %. Of the adenosine phosphates examined, only A M P was found to inhibit yeast pyrophosphatase; A D P had no significant effect. The other nucleotides tested depressed the activity of yeast pyrophosphatase only to a small extent, or not at all. Our results with A D P fail to confirm observations by Kunitz indicating that "adenosine pyrophosphate" inhibited the yeast enzyme "to a great extent" (6). Nordlie and Lardy reported rat liver pyrophosphatase to be inhibited by A M P , ADP, and ATP, but not b y adenosine (7). When Zn 2+ was substituted for Mg 2+ as cofactor, on the other hand, ATP, ADP, and other nucleoside di- and triphosphates were found b y Schlesinger and Coon to function as substrates for pyrophosphatase rather than as inhibitors (8).
Influence of nuclcotidylsulfate concentration. The influence of varying the concentration of some nucleotidylsulfates on the activity of pyrophosphatase was then studied under the same conditions (Fig. 1). GPS and IPS were again found to inhibit more strongly than APS and dAPS. A depression of the reaction
rate by 50 % was noted in the presence of 0.58 mM GPS or IPS, and of 1.15 m ~ APS or dAPS, respectively. Three mM GPS or IPS caused an inhibition of about 90 %. The curves representing rate as a function of inhibitor concentration were almost identical for GPS and IPS, and for APS and dAPS, respectively. Influence of incubation time. The velocity of the uninhibited enzymatic P P I hydrolysis was found to remain constant for the first 15 rain, and then to decrease with time. After addition of inhibitor, the reaction proceeded at a reduced but constant velocity during the entire time period examined (Fig. 2). Qualitatively, the results observed in the absence of inhibitor confirm findings by Kunitz (6), but the data are not strictly comparable, owing to differences in the concentration of M g 2+ present. Influence of enzyme concentration. At lower pyrophosphatase concentration, the rate of the uninhibited P P i hydrolysis was found to be linearly proportional to the enzyme concentration, in agreement with observations by Kunitz (6). At pyrophosphatase levels above 2 units/m], however, the reaction rate approached a constant value, under the conditions of our experiments. I n the presence of GPS, a marked inhibition was Z tr 4 0 0 Z C3
It.
200
n-
6 E
I
I
I
I
2
3
INHIBITOR CONCENTRATION, mM FIG. 1. Inhibition of yeast inorganic pyrophosphatase by some purine nueleotidylsulfates as a function of inhibitor concentration. Determination of pyrophosphatase activity as described in text, except for the addition of purine nueleotidylsulfates at the concentrations indicated: APS added (-- A -- A --) ; dAPS added (-- O - - O --) ; GPS added (-- . . . . ) ; IPS added (-- 9 -- 9 --).
PYROPHOSPHATASE
73
INHIBITION
z
~(2} I'~ l to
o h E 0.5 r to _3 o
J
E f 5
I0
15
20
25
TIME, MIN
INCUBATION
F i e . 2. I n h i b i t i o n of yeast inorganic p y r o p h o s p h a t a s e b y GPS as a f u n c t i o n of inc u b a t i o n time. D e t e r m i n a t i o n of p y r o p h o s p h a t a s e a c t i v i t y as described in text, except for i n c u b a t i o n time and a d d i t i o n of GPS as i n d i c a t e d : no GPS ( - - O - - 9 - - ) ; 1 m• GPS a d d e d (-- 9 - - 9 - - ) .
L5.
Z u") Z (:3
o
1.0
nO b_
O9 to __1 0 0.5
E
1.0
ENZYME
~0
CONCENTRATION,
units/ml
FIG. 3. I n h i b i t i o n of y e a s t inorganic p y r o p h o s p h a t a s e b y GPS as a function of enzyme c o n c e n t r a t i o n . D e t e r m i n a t i o n of p y r o p h o s p h a t a s e a c t i v i t y as described in text, except for enzyme c o n c e n t r a t i o n a n d a d d i t i o n of GPS as indicated. No GPS ( - - O O--); 1 tara GPS added ( - - 9 - - 9 - - ) .
3.0
74
SHOYAB AND MARX 6OO
Z tO
s
400
nO LL
o
n03 2 0 0
E
t
I
I
!
2,5
5.0
7.5
I0.0
PPi
CONCENTRATION,
mM
Fro. 4. Inhibition of yeast inorganic pyrophosphatase by GPS as a function of PPi concentration. Pyrophosphatase activity determined as described in text, except for PPI concentration and addition of GPS as indicated: no GPS (. . . . . ); 0.5 m~ GPS added (-- 9 1 6 9 --),l.0mMGPSadded ( - - 9 9 again noted, and the P P i hydrolysis as a function of enzyme concentration followed a straight line relationship over the entire concentration range examined (Fig. 3). Influence of substrate concentration. The reaction velocity as a function of the P P i concentration is shown in Fig. 4. In the absence of inhibitor, a linear relationship was observed below 0.8 mM PP~; above this level, the reaction rate increased more slowly, passed through a maximum at 3 mM PPI (molar ratio of P P J M g ~ + : I . 5 ) , and, then, decreased, with further rising substrate concentration. These results confirm data of Bailey and Webb (9) and Heppel and Hilmoe (10) who reported that yeast pyrophosphatase was inhibited by high substrate concentrations. This inhibition might be interpreted on the basis of a reaction between P P I and Mg :+, resulting in complex formation or precipitation, and, thereby removing Mg 2+ from a participation in the enzymatic action, in accord with observations indicating t h a t the inhibition by higher PP~ levels was a function of the Mg 2+ concentration (6), and that M g P P i precipitated at higher concentrations (6, 10). The possibility cannot be excluded, however, that MgPPI was the
true substrate for the pyrophosphatase reaction (11), and that higher PPI concentrations inhibited owing to competition with MgPPI for the active site of the enzyme (12). In the presence of GPS, the reaction velocity was not influenced at PPI concentrations below 0.04 M; at higher substrate concentrations, GPS markedly depressed the activity of pyrophosphatase; after addition of GPS, the reaction rate as a function of the P P i concentration followed a pattern similar to that observed for the uninhibited reaction, except that the maximum was shifted somewhat towards a lower P P I concentration (Fig. 4). At a substrate concentration of 3 mM, the reaction was depressed b y about 67 %, in the presence of 1 mM GPS. The results presented indicate that yeast inorganic pyrophosphatase was inhibited by purine nucleotidylsulfates, such as GPS, IPS, APS, and dAPS. Since it was observed that the enzymatic hydrolysis of PPI did not follow Michaelis-Menten ldnetics, in agreement with findings of Kunitz (6), and in view of the complications mentioned above, it is difficult to interpret the data with respect to the nature of this inhibition.
PYROPHOSPHATASE INHIBITION
75
The significance of the phenomenon described is not understood. The possibility m a y be considered t h a t the inhibition of pyrophosphatase b y APS contributes to the conservation of high energy phosphate, or, perhaps, to the regulation of the synthesis of PAPS (the "active sulfate"), since APS (the precursor of PAPS) and P P i are formed simultaneously during the first step of sulfate activation (13, 14):
2. SHOYAB, M., AND MAn,X, W., unpublished results. a. ROBBINS, P. W., AND LIPMANN, F., J. Biol, Chem. 9.33, 681, 686 (1958). 4. BADDILE~',J., BUCHANAN,J. G., AND LETTI~:RS, R., J. Chem. Sot., 1067 (1957).
A T P + S042- ~ APS + PP~.
7. NORDLIE, R. C., AND LARDY, H. A., Biochim. Biophys. Acta 53, 309 (1961). 8. SCHLENINGER,M. J., AND COON, M. J., Biocairn. Biophys. Acta 41, 30 (1966). 9. BAILEY, K., AND WEBB, E. I., Biochem. J.
I t appears questionable, however, whether APS concentrations in vivo approach levels high enough to play a significant role in either of these postulated mechanisms. I t would be interesting to investigate the effects of APS and its analogs on pyrophosphatases from other sources, as well as on alkaline phosphatases, since Moss et al. (15) have reported results which are consistent with the view t h a t alkaline phosphatases can function also as inorganic pyrophosphatases. ACKNOWLED GMENT The excellent technical assistance of Mrs. Lucia Y. Su is greatly appreciated. REFERENCES 1. SI~OYAB,M., ~.NDMAax, W., Life Sci. 9, 1151 (1970).
5. FISKE, C. H., AND SUBBAROW, Y., J. Biol. Chem. 66, 375 (1925). 6. KIINITZ, M., J. Gen. Physiol. 35, 423 (1951-
1952).
38,394 (1944). 10. HEPPEL, L. n., AN]) HILMOE, R. J., J. Biol. Chem. 199., 87 (1951). 11. BLOcH-FRANKENTttAL, L., Biochem. J. 57, 87 (1954). 12. W ~ , J. L., "Enzyme and Metabolic Inhibitors," Vol. 1, p. 132. Academic Press, New York, 1963. 13. BANDURSKI, R. S., WILSON, L. G., AND SQIJII~s, C. L., J. Amer. Chem. Soc. 78, 6408 (1956). 14. ROBUINS, P. W., AND LIPMANN, F., Or. Amer. Chem. Soc. 78, 6409 (1956). 15. MOSS, D. W., EATON, H., SMITH, J. K., A.ND W~IITBu L. G., Biochem. J. 102, 53 (1967).
146, 71-75 (1971)
Inhibition of Yeast Inorganic Pyrophosphatase by Nucleotidylsulfates ~ M . S H O Y A B AND W . M A R X
Department of Biochemistry, University of Southern California, School of Medicine, Los Angeles, California 90083 Received February 4, 1971; accepted May 19, 1971 The influence of some purine nucleotides on the activity of yeast inorganic pyrophosphatase was investigated. The nucleotidylsulfates examined were found to exert a significant inhibition. Guanylylsulfate and inosinylsulfate depressed the rate of the pyrophosphatase reaction markedly, adenylylsulfate and its deoxy analog inhibited to a lesser but still significant extent. earlier (1). Adenosine, ADP, ATP, GDP, GTP, IMP, and ITP were purchased from CalBiochem, dAMP, cyclic AMP, and GMP from P-L Biochemicals, Inc., and Tris, AMP, and crystalline bovine serum albumin from Sigma Chemical Company. APS was prepared according to the method of Baddiley et al. from AMP and pyridine sulfate (4), and purified by chromatography on ECTEOLA-celhflose (2). dAPS, GPS, and IPS were prepared and purified in a similar manner. Methods. The inorganic pyrophosphatase was used without further treatment after a 100-fold dilution with cold water containing 1 mg/ml of albumin. The enzyme activity was determined by measuring Pi formation from PPI. The reaction mixture, containing 100 mM Tris-HC1, pH 7.4, 3.8 m• sodium PPi, 2 m~ MgCI2, and 0.01 ml enzyme solution in a total volume of 0.2 ml, was incubated for 5 rain at 37 ~ The reaction was stopped by adding 0.5 ml of 2.5% molybdate in 5 ~ H2SO4. The Pi liberated was measured by the method of Fiske and Subbarow (5) in a total volume of 5 ml. In the control sample, boiled enzyme was used, or enzyme was added after incubation.
Recently, the purification and properties of m o u s e m a s t o c y t o m a A T P sulfurylase ( A T P : sulfate a d e n y l y l t r a n s f e r a s e , E C 2 . 7 . 7 . 4 ) were i n v e s t i g a t e d in our l a b o r a t o r y (1, 2); for a s s a y of t h i s e n z y m e , a m e t h o d of R o b b i n s a n d L i p m a n n (3) w a s u s e d w h i c h involved yeast inorganic pyrophosphatase ( p y r o p h o s p h a t e p h o s p h o h y d r o l a s e , E C 3. 6 . 1 . 1 ) . D u r i n g t h e s e studies, we n o t i c e d that the pyrophosphatase was inhibited by a d e n y l y l s u l f a t e (APS). 2 T h i s p h e n o m e n o n was i n v e s t i g a t e d further, in p a r t i c u l a r , w i t h r e s p e c t t o t h e effects of o t h e r nucleotides. T h e results of these s t u d i e s a r e p r e s e n t e d in this communication. MATERIALS AND METHODS
Materials. Chemicals were C. P. or reagent grade, or the purest preparations available. Sodium pyrophosphate was obtained from Mallinckrodt Chemical Works, and crystalline yeast inorganic pyrophosphatase (1.2 mg protein/ml, corresponding to 820 enzyme U/mg protein) from Worthington Biochemical Corporation. Control experiments demonstrated that the pyrophosphatase was free of ATP sulfurylase activity, as measured according to a technique described
RESULTS AND DISCUSSION
Influence of some nueleotides on pyrophosphatase. T h e influence of some nueleotides a n d a d e n o s i n e on t h e a c t i v i t y of y e a s t inorganic p y r o p h o s p h a t a s e w a s s t u d i e d und e r t h e c o n d i t i o n s d e s c r i b e d above. T h e r e s u l t s of t h e s e e x p e r i m e n t s are shown in T a b l e I. T h e n u c l e o t i d y l sulfates e x a m i n e d were f o u n d t o i n h i b i t t h e e n z y m a t i c h y d r o l y sis of PP~ significantly. I n t h e presence of 1
This investigation was supported, in part, by Research Grant 5 R01 HE 1596 from the National Heart Institute, Public Health Service. z Abbreviations used: APS, adenylylsulfate; dAPS, deoxyadenylylsulfate; GPS, guanylylsulfate; IPS, inosinylsulfate; PAPS, 3t-phospho adenylylsulfate. 71
72
SHOYAB AND MARX TABLE I
I N F L U E N C E OF SOME P U R I N E N U C L E O T I D E S AND ADENOSINE
ON Y E A S T
INORGANIC PYROPttOS-
PHATASE a Addition to incubation mixture
mvmoles Pi formed in 5 min
Percentage inhibition
None GPS IPS dAPS APS AMP GTP GDP Cyclic AMP GMP ADP Adenosine ATP
420 150 156 238 239 326 358 358 359 383 388 410 425
-64 63 44 43 22 15 15 15 9 8 1 0
Determination of pyrophosphatase activity as described in text, except for the addition of the nucleotides or adenosine as indicated at a final concentration of 1 raM. mM GPs or IPS, the reaction rate was depressed by over 60%; APS and dAPS caused an inhibition of over 40 %. Of the adenosine phosphates examined, only A M P was found to inhibit yeast pyrophosphatase; A D P had no significant effect. The other nucleotides tested depressed the activity of yeast pyrophosphatase only to a small extent, or not at all. Our results with A D P fail to confirm observations by Kunitz indicating that "adenosine pyrophosphate" inhibited the yeast enzyme "to a great extent" (6). Nordlie and Lardy reported rat liver pyrophosphatase to be inhibited by A M P , ADP, and ATP, but not b y adenosine (7). When Zn 2+ was substituted for Mg 2+ as cofactor, on the other hand, ATP, ADP, and other nucleoside di- and triphosphates were found b y Schlesinger and Coon to function as substrates for pyrophosphatase rather than as inhibitors (8).
Influence of nuclcotidylsulfate concentration. The influence of varying the concentration of some nucleotidylsulfates on the activity of pyrophosphatase was then studied under the same conditions (Fig. 1). GPS and IPS were again found to inhibit more strongly than APS and dAPS. A depression of the reaction
rate by 50 % was noted in the presence of 0.58 mM GPS or IPS, and of 1.15 m ~ APS or dAPS, respectively. Three mM GPS or IPS caused an inhibition of about 90 %. The curves representing rate as a function of inhibitor concentration were almost identical for GPS and IPS, and for APS and dAPS, respectively. Influence of incubation time. The velocity of the uninhibited enzymatic P P I hydrolysis was found to remain constant for the first 15 rain, and then to decrease with time. After addition of inhibitor, the reaction proceeded at a reduced but constant velocity during the entire time period examined (Fig. 2). Qualitatively, the results observed in the absence of inhibitor confirm findings by Kunitz (6), but the data are not strictly comparable, owing to differences in the concentration of M g 2+ present. Influence of enzyme concentration. At lower pyrophosphatase concentration, the rate of the uninhibited P P i hydrolysis was found to be linearly proportional to the enzyme concentration, in agreement with observations by Kunitz (6). At pyrophosphatase levels above 2 units/m], however, the reaction rate approached a constant value, under the conditions of our experiments. I n the presence of GPS, a marked inhibition was Z tr 4 0 0 Z C3
It.
200
n-
6 E
I
I
I
I
2
3
INHIBITOR CONCENTRATION, mM FIG. 1. Inhibition of yeast inorganic pyrophosphatase by some purine nueleotidylsulfates as a function of inhibitor concentration. Determination of pyrophosphatase activity as described in text, except for the addition of purine nueleotidylsulfates at the concentrations indicated: APS added (-- A -- A --) ; dAPS added (-- O - - O --) ; GPS added (-- . . . . ) ; IPS added (-- 9 -- 9 --).
PYROPHOSPHATASE
73
INHIBITION
z
~(2} I'~ l to
o h E 0.5 r to _3 o
J
E f 5
I0
15
20
25
TIME, MIN
INCUBATION
F i e . 2. I n h i b i t i o n of yeast inorganic p y r o p h o s p h a t a s e b y GPS as a f u n c t i o n of inc u b a t i o n time. D e t e r m i n a t i o n of p y r o p h o s p h a t a s e a c t i v i t y as described in text, except for i n c u b a t i o n time and a d d i t i o n of GPS as i n d i c a t e d : no GPS ( - - O - - 9 - - ) ; 1 m• GPS a d d e d (-- 9 - - 9 - - ) .
L5.
Z u") Z (:3
o
1.0
nO b_
O9 to __1 0 0.5
E
1.0
ENZYME
~0
CONCENTRATION,
units/ml
FIG. 3. I n h i b i t i o n of y e a s t inorganic p y r o p h o s p h a t a s e b y GPS as a function of enzyme c o n c e n t r a t i o n . D e t e r m i n a t i o n of p y r o p h o s p h a t a s e a c t i v i t y as described in text, except for enzyme c o n c e n t r a t i o n a n d a d d i t i o n of GPS as indicated. No GPS ( - - O O--); 1 tara GPS added ( - - 9 - - 9 - - ) .
3.0
74
SHOYAB AND MARX 6OO
Z tO
s
400
nO LL
o
n03 2 0 0
E
t
I
I
!
2,5
5.0
7.5
I0.0
PPi
CONCENTRATION,
mM
Fro. 4. Inhibition of yeast inorganic pyrophosphatase by GPS as a function of PPi concentration. Pyrophosphatase activity determined as described in text, except for PPI concentration and addition of GPS as indicated: no GPS (. . . . . ); 0.5 m~ GPS added (-- 9 1 6 9 --),l.0mMGPSadded ( - - 9 9 again noted, and the P P i hydrolysis as a function of enzyme concentration followed a straight line relationship over the entire concentration range examined (Fig. 3). Influence of substrate concentration. The reaction velocity as a function of the P P i concentration is shown in Fig. 4. In the absence of inhibitor, a linear relationship was observed below 0.8 mM PP~; above this level, the reaction rate increased more slowly, passed through a maximum at 3 mM PPI (molar ratio of P P J M g ~ + : I . 5 ) , and, then, decreased, with further rising substrate concentration. These results confirm data of Bailey and Webb (9) and Heppel and Hilmoe (10) who reported that yeast pyrophosphatase was inhibited by high substrate concentrations. This inhibition might be interpreted on the basis of a reaction between P P I and Mg :+, resulting in complex formation or precipitation, and, thereby removing Mg 2+ from a participation in the enzymatic action, in accord with observations indicating t h a t the inhibition by higher PP~ levels was a function of the Mg 2+ concentration (6), and that M g P P i precipitated at higher concentrations (6, 10). The possibility cannot be excluded, however, that MgPPI was the
true substrate for the pyrophosphatase reaction (11), and that higher PPI concentrations inhibited owing to competition with MgPPI for the active site of the enzyme (12). In the presence of GPS, the reaction velocity was not influenced at PPI concentrations below 0.04 M; at higher substrate concentrations, GPS markedly depressed the activity of pyrophosphatase; after addition of GPS, the reaction rate as a function of the P P i concentration followed a pattern similar to that observed for the uninhibited reaction, except that the maximum was shifted somewhat towards a lower P P I concentration (Fig. 4). At a substrate concentration of 3 mM, the reaction was depressed b y about 67 %, in the presence of 1 mM GPS. The results presented indicate that yeast inorganic pyrophosphatase was inhibited by purine nucleotidylsulfates, such as GPS, IPS, APS, and dAPS. Since it was observed that the enzymatic hydrolysis of PPI did not follow Michaelis-Menten ldnetics, in agreement with findings of Kunitz (6), and in view of the complications mentioned above, it is difficult to interpret the data with respect to the nature of this inhibition.
PYROPHOSPHATASE INHIBITION
75
The significance of the phenomenon described is not understood. The possibility m a y be considered t h a t the inhibition of pyrophosphatase b y APS contributes to the conservation of high energy phosphate, or, perhaps, to the regulation of the synthesis of PAPS (the "active sulfate"), since APS (the precursor of PAPS) and P P i are formed simultaneously during the first step of sulfate activation (13, 14):
2. SHOYAB, M., AND MAn,X, W., unpublished results. a. ROBBINS, P. W., AND LIPMANN, F., J. Biol, Chem. 9.33, 681, 686 (1958). 4. BADDILE~',J., BUCHANAN,J. G., AND LETTI~:RS, R., J. Chem. Sot., 1067 (1957).
A T P + S042- ~ APS + PP~.
7. NORDLIE, R. C., AND LARDY, H. A., Biochim. Biophys. Acta 53, 309 (1961). 8. SCHLENINGER,M. J., AND COON, M. J., Biocairn. Biophys. Acta 41, 30 (1966). 9. BAILEY, K., AND WEBB, E. I., Biochem. J.
I t appears questionable, however, whether APS concentrations in vivo approach levels high enough to play a significant role in either of these postulated mechanisms. I t would be interesting to investigate the effects of APS and its analogs on pyrophosphatases from other sources, as well as on alkaline phosphatases, since Moss et al. (15) have reported results which are consistent with the view t h a t alkaline phosphatases can function also as inorganic pyrophosphatases. ACKNOWLED GMENT The excellent technical assistance of Mrs. Lucia Y. Su is greatly appreciated. REFERENCES 1. SI~OYAB,M., ~.NDMAax, W., Life Sci. 9, 1151 (1970).
5. FISKE, C. H., AND SUBBAROW, Y., J. Biol. Chem. 66, 375 (1925). 6. KIINITZ, M., J. Gen. Physiol. 35, 423 (1951-
1952).
38,394 (1944). 10. HEPPEL, L. n., AN]) HILMOE, R. J., J. Biol. Chem. 199., 87 (1951). 11. BLOcH-FRANKENTttAL, L., Biochem. J. 57, 87 (1954). 12. W ~ , J. L., "Enzyme and Metabolic Inhibitors," Vol. 1, p. 132. Academic Press, New York, 1963. 13. BANDURSKI, R. S., WILSON, L. G., AND SQIJII~s, C. L., J. Amer. Chem. Soc. 78, 6408 (1956). 14. ROBUINS, P. W., AND LIPMANN, F., Or. Amer. Chem. Soc. 78, 6409 (1956). 15. MOSS, D. W., EATON, H., SMITH, J. K., A.ND W~IITBu L. G., Biochem. J. 102, 53 (1967).