23 Kinetic and Thermodynamic Studies of Yeast Inorganic Pyrophosphatase Downloaded via TUFTS UNIV on July 12, 2018 at 21:03:53 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
B A R R Y S. C O O P E R M A N Department of Chemistry, University of Pennsylvania, Philadelphia, P A 19104
Despite their widespread distribution and central importance to cellular metabolism, phosphoryl-transfer enzymes remain incompletely understood with respect to their detailed mechanisms, especially when compared with what is known about more well studied enzymes, such as the serine proteases. Our goal is to obtain a detailed understanding of enzymatic catalysis of phosphoryl transfer and toward this end we have chosen to study yeast inorganic pyrophosphatase (PPase) as an example of a phosphoryl transfer enzyme. PPase is a dimer made up of identical subunits of molecular weight 32,000 daltons (1). Its covalent structure has been determined (2), two research groups have reported low-resolution crystal structures (3,4), and a high resolution structure determination is underway (D. Voet, personal communication). PPase catalyzes three different reactions, inorganic pyrophosphate (PPi) hydrolysis, H O-inorganic phosphate (Pi) oxygen exchange, and, considerably more slowly, PPi:Pi equilibration (5,6). PPase requires divalent metal ions for activity. The highest activity is conferred by Mg+, although substantial activity (>5% of that found with Mg+) is also found in the presence of Zn+ > Co = Mn+ (7). In this paper we report on recent findings of ours which have 1) demonstrated that PPase activity requires three divalent metal ions per subunit and 2) allowed formulation of a minimal kinetic scheme for PPase catalysis which accounts quantitatively for the three activities it manifests. 2
2
2
2
2+
2
Binding
Studies
I t had been p r e v i o u s l y shown by Rapoport et al. (8) that nat i v e PPase binds two d i v a l e n t metal ions ( M g , C o , M n ) per subunit. We have now used e q u i l i b r i u m d i a l y s i s t o extend these s t u d i e s , by measuring the e f f e c t o f added P i on Mn + and C o binding ( 9 ) . The r e s u l t s (Figure 1) demonstrate that i n the presence o f P i a t h i r d d i v a l e n t metal i o n i s bound per subunit. F o r Mn2+, one i n t r i n s i c d i s s o c i a t i o n constant c h a r a c t e r i z e s the b i n d 2+
2 +
2+
2
0097-6156/81/0171-0119$05.00/0 © 1981 American Chemical Society Quin and Verkade; Phosphorus Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 1981.
2 +
120
PHOSPHORUS CHEMISTRY
Figure 1. Scatchard plots of metal ion binding to PPase as measured by equilibrium dialysis. A; Mn binding (a) no added Pi, enzyme concentration 7-42μΜ (O); φ) in the presence of 50 μ M Pi, enzyme concentration 7-36 μΜ (·); (c) in the presence of 4mM Pi, enzyme concentration 8-116μΜ (Π). Β: Co * binding (a)no added Pi, enzyme concentration 8-96μΜ (O); φ) in the presence of 2.5mM Pi, enzyme concentration 30-35μΜ (Π) (see Réf. 9). 2+
2
Quin and Verkade; Phosphorus Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 1981.
23.
COOPERMAN
Ύeast
Inorganic
Pyrophosphatase
121
ing i n the absence of P i , and another, lower by about t h r e e - f o l d , c h a r a c t e r i z e s the b i n d i n g i n the presence of P i . For Co +, e s s e n t i a l l y the same i n t r i n s i c d i s s o c i a t i o n constant c h a r a c t e r i z e s the b i n d i n g i n both the presence and absence of P i . These s t u d i e s , along w i t h p a r a l l e l s t u d i e s on d i v a l e n t metal i o n e f f e c t s on P i b i n d i n g (as measured by e q u i l i b r i u m d i a l y s i s p r o t e c t i o n of a c t i v i t y against chemical m o d i f i c a t i o n , 31p and water proton r e l a x a t i o n r a t e s ) have allowed e v a l u a t i o n of a l l of the e q u i l i b r i u m constants i n Scheme I d e s c r i b i n g d i v a l e n t metal i o n (Mn or Co +) and P i b i n d i n g to PPase. 2
2 +
2
K i n e t i c and
Thermodynamic
Studies
We have combined the r e s u l t s of three d i f f e r e n t types of measurement, enzyme-bound P P i , r a t e s of H20-Pi oxygen exchange, and r a t e s of P P i h y d r o l y s i s , to formulate the minimal scheme f o r PPase c a t a l y s i s shown i n equation ( 1 ) , and to evaluate the r a t e constants contained w i t h i n i t (10). We note that a l l constants are apparent f o r pH 7.0
( k l
7
1
H
1
?°*
(
k
3
\ MgE(MgPi)
6s-l
222s-l
(k )
(U)
2
k
< *> 740s-l
2
(k ) 464 -1 7
R
(1) 1.6
χ 105M-VI (k
6>
M
8
E
M
g
P
i
* 9 χ 10*M-1.-1
+
M
(k
tpi* MgPi* (pH
7.0,
+ Pi
8> 25°C)
and f u r t h e r that the complexes shown i n equation (1) are d e f i n e d w i t h respect to s t o i c h i o m e t r y but not with respect to r e l a t i v e p o s i t i o n i n g . Thus, f o r example, Mg2EMgPPi r e f e r s to a complex with three Mg and one P P i , and i s not meant to imply that MgPPi i s n e c e s s a r i l y bound as a complex to Mg2E. The major f e a t u r e s of equation (1) are: 1) the i m p l i e d r e quirement f o r three bound metal ions per a c t i v e subunit; 2) the r e l e a s e of the e l e c t r o p h i l e P i ( i . e . , the phosphoryl group a t tacked n u c l e o p h i l i c a l l y by H2O i n the forward d i r e c t i o n ) p r i o r to the r e l e a s e of the l e a v i n g group P i ; and 3) the numerical evalua t i o n of the r a t e constants. We now d i s c u s s each of these f e a tures i n t u r n . Boyer and h i s co-workers have r e c e n t l y presented evidence that H20-Pi exchange proceeds v i a enzyme-bound P P i (denoted EPPi) formation from P i (6,11). Extending t h e i r r e s u l t s , we have i n v e s t i g a t e d EPPi formation as a f u n c t i o n of MgPi c o n c e n t r a t i o n at d i f -
Quin and Verkade; Phosphorus Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 1981.
122
PHOSPHORUS CHEMISTRY
Scheme I. Relevant equilibria in solutions of enzyme, divalent metal ion, and Pi (see Kef. 9).
Ε ^=
=3t EM Λ
EM^
(EM ) 3
/II
1
\ 4
iEMPi
Φ
• ΕΜ Ρ 3
/ι κ"
^ 3 Ρ
2
1
, Φ (ΕΡ ) 2
ΕΜΡ„
=^ΕΜ Ρ, 0
Quin and Verkade; Phosphorus Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 1981.
23.
COOPERMAN
Y east Inorganic
123
Pyrophosphatase
f e r e n t f i x e d l e v e l s of i M g ] f . These measurements r e q u i r e d development o f two methods f o r q u a n t i t a t i v e l y measuring P P i i n the presence of a ΙΟ^-ΙΟ^ molar excess of P i . The f i r s t i n v o l v e s the s e l e c t i v e e x t r a c t i o n i n t o i s o b u t a n o l from water o f 32pi as a phosphomolybdate complex l e a v i n g behind 32ppi i the aqueous s o l u t i o n . The second i n v o l v e s the separation of P P i from P i by two-dimensional t i c on polyethyleneimine p l a t e s . We found that the dependence o f EPPi formation on [MgPi] obeyed equation (2), where [ E P P i ] and [Ε]χ represent t h e t o t a l concentrations of EPPi and enzyme, r e s p e c t i v e l y , r e e
n
t
[Ε]
τ
[EPPi]
=
t
A [MgPi]
+
2
Β
+
c
(
2
)
[MgPi]
and that whereas the e m p i r i c a l parameters Β and C were independent of [Mg2+] over t h e range studied (10-30mM), parameter A was approximately p r o p o r t i o n a l t o [Mg2+]. The q u a l i t a t i v e s i g n i f i cance of t h i s r e s u l t was that enzyme forms c o n t a i n i n g e i t h e r two bound P i ' s or one bound PPi required one a d d i t i o n a l Mg2+ compared with enzyme forms c o n t a i n i n g one or no P i . Thus, the t h i r d metal ion which i s bound on P i a d d i t i o n (Figure 1) appears necessary f o r EPPi formation. Rates o f H20-Pi oxygen exchange were d e t e r mined by measuring 18 0 r e l e a s e from 1^0-labeled P i using the nmr method of Cohn and Hu (12) , which r e s o l v e s the 31p peaks due t o the f i v e l ^ O - l a b e l e d and unlabeled s p e c i e s . We found that the exchange r a t e has e s s e n t i a l l y the same dependence on [MgPi] as does EPPi formation, thus p r o v i d i n g confirmatory evidence f o r the intermediacy of EPPi i n oxygen exchange. Further, we found, i n agreement with the r e s u l t s of Hackney (13), that the value of t h e p a r t i t i o n c o e f f i c i e n t f o r l ^ O - P i oxygen exchange, Pc, defined as the r a t e a t which enzyme-bound P i l o s e s water i n t h e exchange step d i v i d e d by the sum of t h i s r a t e and the r a t e o f r e l e a s e of P i to the medium, was much l e s s than one (we f i n d 0.23; Hackney r e ports 0.30) and e s s e n t i a l l y independent o f MgPi c o n c e n t r a t i o n over a wide range. This r e s u l t r e q u i r e s that the P i c o n t a i n i n g t h e oxygen from H20 be r e l e a s e d f i r s t , s i n c e , were i t r e l e a s e d second, i t i s c l e a r from equation (1) that Pc should increase with i n c r e a s i n g MgPi u n t i l i t a t t a i n e d a l i m i t i n g value of 1.0. The eight r a t e constants i n equat ion (1) were evaluated using the f o l l o w i n g eight equations. The measured parameters A,B, and C i n equation (2) a l l o w e v a l u a t i o n o f K3 (k3/k4), K5 ( k / k ) , and K7 (ky/kg). Knowing these constants and p r e v i o u s l y determined values f o r the s o l u t i o n e q u i l i b r i u m of P P i ^ 2 P i and f o r Mg2+ binding t o P P i and P i allows c a l c u l a t i o n o f Κχ ( k / k ) , by c l o s i n g a thermodynamic loop. The remaining 4 equations (3-6) were p r o vided by measuring Pc and enzyme-catalyzed r a t e s o f oxygen ex change and of P P i h y d r o l y s i s . An independent check on the v a l i d i t y of the c a l c u l a t i o n i s provided by t h e good agreement 5
1
2
Quin and Verkade; Phosphorus Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 1981.
6
124
PHOSPHORUS
Pc=
c a t , hyd
4Pc
(3)
k k 3
5
cat,ex
Κ m, hyd
+ k (k +k +k ) y
3
4
(4) 3Pc
k [k k +k (k +k )] ?
3
5
2
4
5
(6)
k [ k ^ + k j (k +k +k ) ] ±
5
CHEMISTRY
3
4
5
1
of our c a l c u l a t e d value f o r k ( 6 s ~ l ) and t h e value of 5 s " which can be estimated from a measured value of P P i r e l e a s e from enzyme (6). The r a t e constant v a l u e s shown i n equation (1) lead t o t h e f o l l o w i n g c o n c l u s i o n s regarding the three r e a c t i o n s c a t a l y z e d by PPase; (1) f o r P P i h y d r o l y s i s a l l t h r e e steps f o l l o w i n g P P i b i n d i n g , steps 3,5, and 7, a r e p a r t i a l l y rate-determining; (2) f o r H20-Pi exchange, P P i s y n t h e s i s , step 4, i s almost e x c l u s i v e l y rate-determining; (3) f o r P P i : P i e q u i l i b r a t i o n , P P i r e l e a s e , step 2, i s e x c l u s i v e l y rate-determining. 2
Literature Cited
1. Heinrikson, R. L.; Sterner, R.; Noyes, C.; Cooperman, B. S.; Bruckmann, R. H. J. Biol. Chem. 1973, 248, 2521-8. 2. Cohen, S. A.; Sterner, R.; Keim, P. S.; Heinrikson, R. L.; J. Biol. Chem. 1978, 253, 889-97. 3. Bunick, G.; McKenna, G. P.; Scarbrough, F. E.; Uberbacher, E. C.; Voet, D. Acta Crystallog, Sect. B. 1978, 34, 3210-5. 4. Makhaldiani, V. V.; Smirnova, Ε. Α.; Voronova, A. A.; Kuranova, I. P.; Arutyunyun, E. G.; Vainshtein, Β. K.; Höhne, W. E.; Binwald, B.; Hansen, G. Dokl. Akad. Nauk SSSR 1978, 240 1478-81. 5. Cohn, M. J. Biol. Chem. 1958, 230, 369-79. 6. Janson, C. Α.; Degani, C.; Boyer, P. D. J. Biol. Chem. 1979 254, 3743-9. 7. Butler, L. G.; Sperow, J. W. Bioinorg. Chem. 1977, 7, 141-50. 8. Rapoport, Τ. Α.; Höhne, W. E.; Heitmann, P.; Rapoport, S. M. Eur. J. Biochem. 1973, 33, 341-7. 9. Cooperman, B. S.; Panackal, Α.; Springs, B.; Hamm, D. J. Biochemistry 1981, 20, in press. 10. Springs, B.; Welsh, Κ. M.; Cooperman, B. S. Biochemistry 1981, 20, in press. 11. Hackney, D. D.; Boyer, P. D. Proc. Natl. Acad. Sci. USA 1978, 75, 3133-7. 12. Cohn, M.; Hu, A. Proc. Natl. Acad. Sci. USA 1978, 75, 200-3. 13. Hackney, D. D. J. Biol. Chem. 1980, 255, 5320-8. R E C E I V E D June 30, 1981.
Quin and Verkade; Phosphorus Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 1981.
