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Aspects of the Presteady State Hydrolysis of ATP by Na,K-ATPase
CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19
Aspects of the Presteady State Hydrolysis of ATP by Na,K-ATPase A . G.LO WEAND L. A. REEVE Deparimenr of Biochemistry University of Manchester Manchester. England
I.
INTRODUCTION
An e a r l y b u r s t i n t h e h y d r o l y s i s of low (micromolar) c o n c e n t r a t i o n s of ATP by t h e Na,K-ATPase has been r e p o r t e d w i t h enzymes p r e p a r e d from b o t h e l e c t r i c e e l ( F r o e h l i c h e t a l . , 1 9 7 6 ) and p i g b r a i n (Lowe and Smart, 1 9 7 8 ) . T h i s phenomenon i s most simply e x p l a i n e d by p o s t u l a t i n g t h a t d u r i n g a s i n g l e c y c l e of enzyme act i v i t y t h e release of i n o r g a n i c phosphate ( P i ) from ATP i s a r e l a t i v e l y f a s t p r o c e s s and t h a t t h i s i s followed by a s u b s t a n t i a l l y slower s t e p which i s r a t e - l i m i t i n g I f the cycle f o r t h e s t e a d y - s t a t e h y d r o l y s i s of ATP. of ATPase a c t i v i t y i s r e p r e s e n t e d a s f o l l o w s :
577
Copyright 0 1983 by Academic Press, Inc. All rights of reproductionin any form nscrvcd. ISBN 0-12-153319-0
-*
578
ATP
EIATP
I
E,
+ E2-Pi
“77-
A. G.LOWE AND L. A. REEVE
ADP El-P
Jt -
.. E2-P
this hypothesis demands that all steps from El up to and including formation of E2-Pi are fast, whereas one of the steps E20Pi + E and E2 -t E is much slower under conditions in whic2 the early h r s t of Pi release is observed. In keeping with this, the fluorescence studies of Karlish et al. (1976) have shown that the transition from E2 (which is stabilized by K+) to El (stabilized by Na+) is a slow process likely to be rate-limiting for ATPase activity at low trinucleotide concentrations. Post et al. (1972) also suggested that the rate-limiting step in hydrolysis involves the ATPpromoted dissociation of K+ from an occluded E2.K+ complex.
11.
DISCUSSION
Further investi ations have revealed antagonistic effects of Na+ and Kq consistent with the above interpretation. Pre-exposure of the pig brain Na,K-ATPase to 20 mM KC1 before mixing with [y-32P]ATP has been found to suppress the early burst of Pi release and this effect of K+ is decreased b simultaneous preexposure of the enzyme to both Ky and Na+. Reversal of K+ suppression by Na+ is concentration-dependent, 80 mM Na+ being sufficient to restore half of the early burst of Pi in the presence of 20 mM K+. These effects of Na+ and K+ on the early burst of Pi release are mirrored by their effects on the p-nitrophenylphosphatase (pNPPase) activity of the same enzyme since Na+ inhibits K+-dependent pNPPase activity with a ~ 0 . 5 of about 40 mM in the presence of 10 mM KC1. This result is satisfactorily accounted for if the E2 form of the enzyme is active as a pNPPase, whereas the El form has no pNPPase activity.
ASPECTS OF PRESTEADY STATE HYDROLYSIS OF ATP
579
While the rate of the transition E2 to El can satisfactorily account for the early burst of Pi release, the question of the enzymic precursor of Pi is less easily resolved by the results of pre-steady-state experiments. Using a simple model for ATP hydrolysis,
El
+
ATP
- %1 kl
k-l
EIATP
k- 2
ADP
:s pi
E2-P
E2
k- 3
k4
7
k- 4
Lowe and Smart (1978) derived integrated rate equations and showed that the early burst of Pi must be accompanied by a complementary overshoot in its precursor, and that the magnitude of this predicted overshoot was greater than that actually observed for the phosphoenzyme. This finding raises the possibility that ATP hydrolysis can occur by a branched or parallel pathway in which the immediate precursor of Pi can be either E2P or another unidentified intermediate. We have investigated this possibility by comparing the early time courses of ( i ) formation of phosphoenzyme, ( i i ) the release of [ 32P] Pi from [ y-32P]ATP after quenching with perchloric acid, and ( i i i ) the release of [32P]Pi from [y-32P]ATP after quenching with excess unlabeled ATP and allowing hydrolysis of any enzymic intermediates (including phosphoenzyme) before addition of perchloric acid. The results of these experiments have shown that the amount of [32P]Pi released after quenching with cold ATP exceeds the sum of the amounts of phosphoenzyme found and [32P]Pi released on quenching with perchloric acid 10-30 msec after mixing a NaI- and deoxycholateextracted pig brain Na,K-ATPase with [y-32P]ATP. This result is consistent with the existence of an enzymic precursor of [ 32P]Pi other than the phosphoenzyme, although the errors in experiments of this type make it uncertain whether the sum of the phosphoenzyme and the new precursor can account quantitatively for the size of the early burst of Pi release. The nature of this new putative intermediate is not known, but a relatively tightly bound enzyme-ATP complex (E'ATP) (cf. tightly bound ATP in the hydrolysis of ATP by myosin) and an acid-labile form of phosphoenzyme are two possibilities. An intermediate of this type does not necessarily demand a substantial adjustment in the interpretation of Na+ and K+ transport in terms of conformational changes in the Na,K-ATPase. E'ATP could be incorporated into conventional schemes as follows:
A. G. LOWE AND L. A. REEVE
580
Jl E2
rj
F ADP
E2*Pi
+E2-P
E2'Pi
I n such a scheme there w o u l d be t w o precursors of P i and N a + t r a n s p o r t m i g h t occur d u r i n g e i t h e r of t h e t r a n s i t i o n s , E l - P -t E2-P and E i A T P E;.Pi. -f
REFERENCES F r o e h l i c h , J. P . , A l b e r s , R. W . , Koval, G. J . , Goebel, R . , and Berman, M. (1976). Evidence f o r a new i n t e r m e d i a t e s t a t e i n t h e mechanism of (Na' + K+) -adenosine t r i p h o s p h a t a s e . J. B i o l . C h e m . 2 5 1 , 2186-2188. K a r l i s h , S . J. D . , Glynn, I . M., and Yates, D. W. (1976). T r a n s i e n t k i n e t i c s of ( N a + + K+)-ATPase s t u d i e d w i t h a f l u o r e s c e n t probe. N a t u r e (London) 263, 251-253. Lowe , A. G . , and Smart, J. W. (1978). The pre-steady s t a t e hyd r o l y s i s of ATP by p o r c i n e b r a i n ( N a + + Kf)-dependent ATPase. B i o c h i r n . B i o p h y s . Acta 4 8 1 , 695-705. P o s t , R. L . , Hegyvary, C . , and K u m e , S. (1972). A c t i v a t i o n by adenosine t r i p h o s p h a t e i n t h e p h o s p h o r y l a t i o n k i n e t i c s of sodium and potassium i o n t r a n s p o r t ade nos i n e t r ipho spha t a s e . J. Biol. Chern. 2 4 7 , 6530-6540.
-
Aspects of the Presteady State Hydrolysis of ATP by Na,K-ATPase A . G.LO WEAND L. A. REEVE Deparimenr of Biochemistry University of Manchester Manchester. England
I.
INTRODUCTION
An e a r l y b u r s t i n t h e h y d r o l y s i s of low (micromolar) c o n c e n t r a t i o n s of ATP by t h e Na,K-ATPase has been r e p o r t e d w i t h enzymes p r e p a r e d from b o t h e l e c t r i c e e l ( F r o e h l i c h e t a l . , 1 9 7 6 ) and p i g b r a i n (Lowe and Smart, 1 9 7 8 ) . T h i s phenomenon i s most simply e x p l a i n e d by p o s t u l a t i n g t h a t d u r i n g a s i n g l e c y c l e of enzyme act i v i t y t h e release of i n o r g a n i c phosphate ( P i ) from ATP i s a r e l a t i v e l y f a s t p r o c e s s and t h a t t h i s i s followed by a s u b s t a n t i a l l y slower s t e p which i s r a t e - l i m i t i n g I f the cycle f o r t h e s t e a d y - s t a t e h y d r o l y s i s of ATP. of ATPase a c t i v i t y i s r e p r e s e n t e d a s f o l l o w s :
577
Copyright 0 1983 by Academic Press, Inc. All rights of reproductionin any form nscrvcd. ISBN 0-12-153319-0
-*
578
ATP
EIATP
I
E,
+ E2-Pi
“77-
A. G.LOWE AND L. A. REEVE
ADP El-P
Jt -
.. E2-P
this hypothesis demands that all steps from El up to and including formation of E2-Pi are fast, whereas one of the steps E20Pi + E and E2 -t E is much slower under conditions in whic2 the early h r s t of Pi release is observed. In keeping with this, the fluorescence studies of Karlish et al. (1976) have shown that the transition from E2 (which is stabilized by K+) to El (stabilized by Na+) is a slow process likely to be rate-limiting for ATPase activity at low trinucleotide concentrations. Post et al. (1972) also suggested that the rate-limiting step in hydrolysis involves the ATPpromoted dissociation of K+ from an occluded E2.K+ complex.
11.
DISCUSSION
Further investi ations have revealed antagonistic effects of Na+ and Kq consistent with the above interpretation. Pre-exposure of the pig brain Na,K-ATPase to 20 mM KC1 before mixing with [y-32P]ATP has been found to suppress the early burst of Pi release and this effect of K+ is decreased b simultaneous preexposure of the enzyme to both Ky and Na+. Reversal of K+ suppression by Na+ is concentration-dependent, 80 mM Na+ being sufficient to restore half of the early burst of Pi in the presence of 20 mM K+. These effects of Na+ and K+ on the early burst of Pi release are mirrored by their effects on the p-nitrophenylphosphatase (pNPPase) activity of the same enzyme since Na+ inhibits K+-dependent pNPPase activity with a ~ 0 . 5 of about 40 mM in the presence of 10 mM KC1. This result is satisfactorily accounted for if the E2 form of the enzyme is active as a pNPPase, whereas the El form has no pNPPase activity.
ASPECTS OF PRESTEADY STATE HYDROLYSIS OF ATP
579
While the rate of the transition E2 to El can satisfactorily account for the early burst of Pi release, the question of the enzymic precursor of Pi is less easily resolved by the results of pre-steady-state experiments. Using a simple model for ATP hydrolysis,
El
+
ATP
- %1 kl
k-l
EIATP
k- 2
ADP
:s pi
E2-P
E2
k- 3
k4
7
k- 4
Lowe and Smart (1978) derived integrated rate equations and showed that the early burst of Pi must be accompanied by a complementary overshoot in its precursor, and that the magnitude of this predicted overshoot was greater than that actually observed for the phosphoenzyme. This finding raises the possibility that ATP hydrolysis can occur by a branched or parallel pathway in which the immediate precursor of Pi can be either E2P or another unidentified intermediate. We have investigated this possibility by comparing the early time courses of ( i ) formation of phosphoenzyme, ( i i ) the release of [ 32P] Pi from [ y-32P]ATP after quenching with perchloric acid, and ( i i i ) the release of [32P]Pi from [y-32P]ATP after quenching with excess unlabeled ATP and allowing hydrolysis of any enzymic intermediates (including phosphoenzyme) before addition of perchloric acid. The results of these experiments have shown that the amount of [32P]Pi released after quenching with cold ATP exceeds the sum of the amounts of phosphoenzyme found and [32P]Pi released on quenching with perchloric acid 10-30 msec after mixing a NaI- and deoxycholateextracted pig brain Na,K-ATPase with [y-32P]ATP. This result is consistent with the existence of an enzymic precursor of [ 32P]Pi other than the phosphoenzyme, although the errors in experiments of this type make it uncertain whether the sum of the phosphoenzyme and the new precursor can account quantitatively for the size of the early burst of Pi release. The nature of this new putative intermediate is not known, but a relatively tightly bound enzyme-ATP complex (E'ATP) (cf. tightly bound ATP in the hydrolysis of ATP by myosin) and an acid-labile form of phosphoenzyme are two possibilities. An intermediate of this type does not necessarily demand a substantial adjustment in the interpretation of Na+ and K+ transport in terms of conformational changes in the Na,K-ATPase. E'ATP could be incorporated into conventional schemes as follows:
A. G. LOWE AND L. A. REEVE
580
Jl E2
rj
F ADP
E2*Pi
+E2-P
E2'Pi
I n such a scheme there w o u l d be t w o precursors of P i and N a + t r a n s p o r t m i g h t occur d u r i n g e i t h e r of t h e t r a n s i t i o n s , E l - P -t E2-P and E i A T P E;.Pi. -f
REFERENCES F r o e h l i c h , J. P . , A l b e r s , R. W . , Koval, G. J . , Goebel, R . , and Berman, M. (1976). Evidence f o r a new i n t e r m e d i a t e s t a t e i n t h e mechanism of (Na' + K+) -adenosine t r i p h o s p h a t a s e . J. B i o l . C h e m . 2 5 1 , 2186-2188. K a r l i s h , S . J. D . , Glynn, I . M., and Yates, D. W. (1976). T r a n s i e n t k i n e t i c s of ( N a + + K+)-ATPase s t u d i e d w i t h a f l u o r e s c e n t probe. N a t u r e (London) 263, 251-253. Lowe , A. G . , and Smart, J. W. (1978). The pre-steady s t a t e hyd r o l y s i s of ATP by p o r c i n e b r a i n ( N a + + Kf)-dependent ATPase. B i o c h i r n . B i o p h y s . Acta 4 8 1 , 695-705. P o s t , R. L . , Hegyvary, C . , and K u m e , S. (1972). A c t i v a t i o n by adenosine t r i p h o s p h a t e i n t h e p h o s p h o r y l a t i o n k i n e t i c s of sodium and potassium i o n t r a n s p o r t ade nos i n e t r ipho spha t a s e . J. Biol. Chern. 2 4 7 , 6530-6540.
-