18 Hydrolysis of Adenosine 5'-Triphosphate An Isotope-Labeling Study S E Y M O U R M E Y E R S O N and E U G E N E S. K U H N Downloaded by UNIV OF CALIFORNIA SAN DIEGO on February 3, 2016 | http://pubs.acs.org Publication Date: November 11, 1981 | doi: 10.1021/bk-1981-0171.ch018
Research Department, Standard Oil Company (Indiana), Naperville, IL 60566 F A U S T O R A M I R E Z and J A M E S F. M A R E C E K Department of Chemistry, State University of New York, Stony Brook, N Y 11794
The key role played by adenosine 5'-triphosphate (ATP) in biophosphorus chemistry has prompted numerous investigations of its mechanism of hydrolysis(1-6), but many questions remain unanswered(7). In principle the nonenzymetic hydrolysis of ATP can occur via four primary pathways: H 0 H0 H0 H0 2
2
2
2
+ + + +
ΑΤΡ(Ρ ) ΑΤΡ(Ρβ) ΑΤΡ(Ρβ) ΑΤΡ(Ρ ) γ
α
= = = =
ADP + P Ρ + ADP ΑMΡ + PP ΡΡ + AMP i
i
i
i
(1) (2) (3) (4)
Further hydrolysis of the ADP can proceed via two different pathways: H 0 + ΑDΡ(Ρβ) = AMP + P H 0 + A D P ( Ρ ) = Ρ + AMP 2
i
2
i
α
(5) (6)
A recent paper(8) reported a study of the hydrolysis of ATP in 0.01 M aqueous solutions at 70° C in the pH range 0-10. Representative values of the pseudo-first order rate constants are: (a) in 1 Ν HC1, k = 13.9 x 10 min , t = 5 min; (b) in 0.1 Ν HC1, k = 1.98 x 10 min , t = 35 min; (c) at pH 8.30, k = 11.5 x 10 min , t = 100.5 hr. Hydrolysis rates were determined using a liquid-chromatographic (LC) technique that also permitted examination of the various stages of the reaction. In addition, acetonitrile solutions oftetra-n-butylammonium(M) salts of the tetra- and trianions, ATP4M and ΑΤΡΗ 3M , produced t-butyl and isopropyl phosphates at approximately equal rates when the solutions contained t-butyl and isopropyl alcohols, respectively(8). These results led to the conclusion that the tetra- and trianions of ATP undergo hydrolysis by an elimination-addition sequence via the monomeric metaphosphate anion, in accord with a previous suggestion(9), while the hydrolysis of the acid, ATPH, and the monoanion, ATPH , proceeds by addition-elimination, presumably via an oxyphosphorane intermediate. The mechanism of hydrolysis of the dianion, ATPH , remained unsettled. -2
-2
-5
-1
½
-1
½
-1
½
+
4-
+
4
3-
+
3
2-
2
We have now supplemented LC w i t h 0 l a b e l i n g as a probe o f the r e l a t i v e c o n t r i b u t i o n s o f h y d r o l y t i c a t t a c k on the three 0097-6156/81/0171-0093$05.00/0 © 1981 American Chemical Society
In Phosphorus Chemistry; Quin, Louis D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.
Downloaded by UNIV OF CALIFORNIA SAN DIEGO on February 3, 2016 | http://pubs.acs.org Publication Date: November 11, 1981 | doi: 10.1021/bk-1981-0171.ch018
94
PHOSPHORUS CHEMISTRY
phosphorus atoms o f A T P and on the two phosphorus atoms o f ADP i n nonenzymatic h y d r o l y s e s . We have a l s o compared the r e s u l t s of these h y d r o l y s e s w i t h those c a r r i e d out under c a t a l y s i s by myosin CaATPase. T h i s enzymatic r e a c t i o n has been e x t e n s i v e l y u t j j i z e d as a c o n t r o l to e s t a b l i s h the i s o t o p i c d i s t r i b u t i o n i n C Y - O ] A T P samples used i n i n v e s t i g a t i o n s of intermediate oxygen exchange during actomyosin MgATPase a c t i o n i n muscle contraction(j^O, 11). We hydrolyzed ATP and ADP i n 1 Ν and 0.1 Ν HC1 and i n b u f f e r e d s o l u t i o n s a t pH 4^gnd 8 i n which the h y d r o l y s i s medium was v a r i o u s l y e n r i c h e d i n 0 t o e i t h e r ~10% o r ~20%. To assess the i s o t o p i c enrichment of each such s o l u t i o n f o r use i n the n u c l e o t i d e h y d r o l y s i s experiments, we hydrolyzed PC1
