Abstract:
:The enzyme form of the calcium adenosinetriphosphatase of sarcoplasmic reticulum (CaATPase) that is stable in the presence of calcium, cE.Ca2, has a binding site for the catalytic Mg2+ ion with a dissociation constant of 0.94 +/- 0.15 mM at 25 degrees C, pH 7.0, and 100 mM KCl. This is approximately 10 times smaller than that reported for the free enzyme, E, (8.8 mM) under similar conditions [Punzengruber, C., Prager, R., Kolassa, N., Winkler, F., & Suko, J. (1978) Eur. J. Biochem. 92, 349-359]. This difference shows that the sites for the catalytic and the transported ions interact in the absence of ATP. The addition of ATP and EDTA to enzyme that had been incubated with Ca2+ and Mg2+ resulted in the formation of 61% phosphoenzyme. The addition of unlabeled ATP and Mg2+ to enzyme that had been incubated with 3.5 microM free Ca2+ and labeled ATP gave 39% labeled phosphoenzyme. This shows that the binding of ATP and Mg2+ to cE.Ca2 follows a random mechanism. The rate constants for dissociation of ATP and Mg2+ from cE.Ca2.ATP.Mg are different: kdiss(ATP) = 120 s-1 and kdiss(Mg2+) = 60 s-1. This shows that Mg2+ and ATP can bind and dissociate independently; they do not have to associate or dissociate from cE as a Mg.ATP complex. Calcium-free enzyme binds metal-free ATP at the active site with a dissociation constant of 44 +/- 4 microM, kdiss = 130 +/- 7 s-1, and a calculated association rate constant of 3 x 10(6) M-1 s-1. Calcium-free enzyme that was incubated with [gamma-32P]ATP gave 38% labeled phosphoenzyme when chased with unlabeled ATP, Mg2+, and Ca2+. An increase of the Mg2+ concentration did not increase the amount of E32P formed. This shows that the binding of Mg2+ and ATP to free E also follows a random mechanism. The Mg2+ ion is not buried under ATP, and ATP is not under a Mg2+ ion. Incubation of free E with Mg2+ and ATP causes a conformational change that activates the enzyme for phosphorylation and decreases the rate constant for the dissociation of ATP from kdiss = 120 s-1 to kdiss = 47 s-1.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Reinstein J,Jencks WPdoi
10.1021/bi00077a016subject
Has Abstractpub_date
1993-07-06 00:00:00pages
6632-42issue
26eissn
0006-2960issn
1520-4995journal_volume
32pub_type
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