Phosphorylation of the calcium-transporting adenosinetriphosphatase by lanthanum ATP: rapid phosphoryl transfer following a rate-limiting conformational change

Abstract

The calcium-transport ATPase (CaATPase) of rabbit sarcoplasmic reticulum preincubated with 0.02 mM Ca2+ (cE.Ca2) is phosphorylated upon the addition of 0.25 mM LaCl3 and 0.3 mM [gamma-32P]ATP with an observed rate constant of 6.5 s-1 (40 mM MOPS, pH 7.0, 100 mM KCl, 25 degrees C). La.ATP binds to cE.Ca2 with a rate constant of 5 X 10(6) M-1 s-1, while ATP, Ca2+, and La3+ dissociate from cE.Ca2.La.ATP at less than or equal to 1 s-1. The reaction of ADP with phosphoenzyme (EP) formed from La.ATP is biphasic. An initial rapid loss of EP is followed by a slower first-order disappearance, which proceeds to an equilibrium mixture of EP.ADP and nonphosphorylated enzyme with bound ATP. The fraction of EP that reacts in the burst (alpha) and the first-order rate constant for the slow phase (kb) increase proportionally with increasing concentrations of ADP to give maximum values of 0.34 and 65 s-1, respectively, at saturating ADP (KADPS = 0.22 mM). The burst represents rapid phosphoryl transfer and demonstrates that ATP synthesis and hydrolysis on the enzyme are fast. The phosphorylation of cE.Ca2 by La.ATP at 6.5 s-1 and the kinetics for the reaction of EP with ADP are consistent with a rate-limiting conformational change in both directions. The conformational change converts cE.Ca2.La.ATP to the form of the enzyme that is activated for phosphoryl transfer, aE.Ca2.La.ATP, at 6.5 s-1; this is much slower than the analogous conformational change at 220 s-1 with Mg2+ as the catalytic ion [Petithory & Jencks (1986) Biochemistry 25, 4493]. The rate constant for the conversion of aE.Ca2.La.ATP to cE.Ca2.La.ATP is 170 s-1. ATP does not dissociate measurably from aE.Ca2.La.ATP. Labeled EP formed from cE.Ca2 and La.ATP with leaky vesicles undergoes hydrolysis at 0.06 s-1. It is concluded that the reaction mechanism of the CaATPase is remarkably similar with Mg.ATP and La.ATP; however, the strong binding of La.ATP slows both the conformational change that is rate limiting for EP formation and the dissociation of La.ATP. An interaction between La3+ at the catalytic site and the calcium transport sites decreases the rate of calcium dissociation by greater than 60-fold. When cE-Ca2 is mixed with 0.3 mM ATP and 1.0 mM Cacl2, the phosphoenzyme is formed with an observed rate constant of 3 s-1. The phosphoenzyme formed from Ca.ATP reacts with 2.0 mM ADP and labeled ATP with a rate constant of 30 s-1; there may be a small burst (alpha less than or equal to 0.05).

Kinetic Mechanism of Nicotinic Acid Phosphoribosyltransferase: Implications for Energy Coupling

“…The k cat / K 0.5 for MgATP in phosphorylation of NAPRTase (2 × 10 4 M -1 s -1 ) provides a minimum estimate of the rate constant 5 7.0 × 10 6 M -1 s -1 experimentally determined k-5 k 6 g500 s -1 pre-steady-state formation of NAMN k-6 k 7 6.3 s -1 determined from E-32 P hydrolysis k-7 k 8 5.2 s -1 calculated from steady-state phosphorylation k-8 for ATP binding and is supported by k cat /K M (5 × 10 3 M -1 s -1 ) in the overall reaction. These rates are slow compared to rate constants for ATP binding in other enzymes, typically about 10 6 M -1 s -1 (15,16).…”

“…The k cat / K 0.5 for MgATP in phosphorylation of NAPRTase (2 × 10 4 M -1 s -1 ) provides a minimum estimate of the rate constant for ATP binding and is supported by k cat / K M (5 × 10 3 M -1 s -1 ) in the overall reaction. These rates are slow compared to rate constants for ATP binding in other enzymes, typically about 10 6 M -1 s -1 ( , ).…”

Dissociation of calcium from the phosphorylated calcium-transporting adenosine triphosphatase of sarcoplasmic reticulum: kinetic equivalence of the calcium ions bound to the phosphorylated enzyme

“…The addition of ADP plus EGTA blocks the internalization of calcium because ADP rapidly reverses the phosphorylation of the enzyme by ATP and forms a mixture of phosphorylated and nonphosphorylated enzyme at equilibrium, which is described by the equilibrium constant Áfint = 0.44 (Pickart & Jencks, 1982; Hanel & Jencks, 1990) (Scheme II). Calcium that is bound to the phosphorylated enzyme is internalized into the lumen of the vesicle with the rate constant kc, while calcium that is bound to the nonphosphorylated enzyme dissociates irreversibly into the outside medium; the presence of EGTA prevents the rebinding of calcium to the nonphosphorylated enzyme and further phosphorylation by ATP.…”

Changes in the profile structure of the sarcoplasmic reticulum membrane induced by phosphorylation of the Ca2+ ATPase enzyme in the presence of terbium: a time-resolved x-ray diffraction study

“…The effect of lanthanides on the functionality of the Ca2"ATPase (slow formation, and very long lifetime of the phosphorylated enzyme conformation) can be viewed as an extrapolation of the effects of low temperature and/or low [Mg2"], which we have shown to be mediated by changes in the structure of the SR membrane. Although it has been proposed that the inhibition of steady-state turnover of the ATPase in the presence of lanthanides is due to the replacement of Mg2" by lanthanides at the catalytic site (Hanel and Jencks, 1990;Fujimori and Jencks, 1990), this is most likely not the case under the conditions of our experiments, as discussed in an earlier section. Instead, we propose that the effect of lanthanides on the functionality of the Ca2"ATPase is also mediated by changes in the structure of the SR membrane.…”

“…\have on the membrane structure and functionality (e.g., the presence of excess lanthanide ions could cause extensive lateral phase separation of the membrane lipids and thereby z'*0 (A-i) affect the functionality of the ATPase). There are several reports in the literature (Squier et al, 1990;Girardet et al, 1989;Hanel and Jencks, 1990) that indicate that lanthanides B bind to a number of different protein sites, including highaffinity calcium-binding/transport sites on the ATPase. Evidence that, under certain conditions, lanthanides bind to calcium-binding/transport sites on the enzyme is provided by measurements of the stoichiometry and affinity of lanthanide binding, the nature of changes in the intrinsic fluorescence of tryptophan residues in the Ca2+ATPase upon displacement of Ca2+ by lanthanides, and from the results of lanthanide \ / GFSD "chase" experiments using 45Ca (Squier et al, 1990;Girardet ----mode et al, 1989).…”