Dissertation

Conformational changes of the calcium-transporting adenosine triphosphatase: A kinetic investigation

Hanel, Arthur Marshall

1992

Overview

Phosphorylation of the calcium transporting adenosinetriphosphatase of rabbit sarcoplasmic reticulum by La$\\sp{3+}\\cdot$ ATP and the dissociation of calcium from the phosphorylated enzyme formed from Mg$\\cdot$ATP were investigated with rapid-quench reaction techniques. Conditions for the experiments were 25$\\sp\\circ$C, 100 mM KCl and 40 mM MOPS, pH 7.0. The enzyme with bound calcium ($\\sp{\\rm c}$E$\\cdot$Ca$\\sb2$) is phosphorylated by 200 $\\mu$M ($\\gamma$-$\\sp{32}$P) La$\\cdot$ATP with an observed rate constant of k = 6.5 s$\\sp{-1}$. The reaction of the labeled phosphoenzyme (EP) with ADP in the presence of labeled ATP is biphasic; a burst of phosphoenzyme disappearance is followed by a slower first-order disappearance, which proceeds gives an equilibrium mixture of EP, EP$\\cdot$ADP and nonphosphorylated forms of the enzyme with bound ATP. The fraction of EP that reacts in the burst and the first-order rate constant for the slow phase increase proportionally with increasing concentrations of ADP. The kinetics of EP formation and disappearance are consistent with a rate-limiting conformational change in both directions. The conformational change converts $\\sp{\\rm c}$E$\\cdot$Ca$\\sb2\\cdot$La$\\cdot$ATP to the form of the enzyme that is activated for phosphoryl transfer, $\\sp{\\rm c}$E$\\cdot$Ca$\\sb2\\cdot$La$\\cdot$ATP, at 6.5 s$\\sp{-1}$, the analogous reaction with Mg$\\cdot$ ATP is faster with a rate constant of k = 220 s$\\sp{-1}$. The internalization of radiolabeled calcium into SR vesicles containing ambient ($\\sim$10 $\\mu$M) or 20 mM unlabeled calcium by the phosphoenzyme formed from Mg$\\cdot$ATP was measured using a quench of 7 mM ADP and 10 mM EGTA. Two Ca$\\sp{2+}$ ions are internalized into empty and loaded vesicles during a single turnover of the enzyme in a first-order reaction with a rate constant of k = $\\sim$30 s$\\sp{-1}$. The individual Ca$\\sp{2+}$ ions are internalized with the same rate constant; there is no indication of a biphasic reaction or inhibition by 20 mM lumenal calcium. The data are consistent with a rate-limiting conformational change that precedes rapid dissociation of the Ca$\\sp{2+}$ ions from the phosphoenzyme. The absence of inhibition by 20 mM lumenal calcium suggests that the ions may dissociate rapidly from independent sites on the phosphoenzyme. Alternatively, the Ca$\\sp{2+}$ ions could rapidly exchange their positions on the phosphoenzyme and dissociate sequentially from a single site.

• Publisher: ProQuest Dissertations & Theses
• Subject: Biochemistry
• ISBN: 9798207733180