Binding Energy and Synthesis of ATP in the Absence of a Ca2 Gradient

In 1971 Makinose [22] observed that the Ca2+ -ATPase catalyzes simultaneously the hydrolysis and synthesis of ATP when the vesicles are filled with Ca2+ and a steady-state between Ca2+ influx and Ca2+efflux is reached. This was referred to as the ATP $ Pi exchange reaction and was one of the parameters used to characterize the interconversion between osmotic and chemical energy. The disruption of the vesicles integrity with either phospholipase A or diethyl ether was found to abolish the synthesis of ATP. This led to the conclusion that at steady-state the energy derived from the hydrolysis of ATP was used to maintain the Ca2+ gradient and at the same time, the energy derived from the gradient was used to synthesize ATP from ADP and Pi. At that time it was a general belief that the presence of a gradient was an absolute requirement for the ATP $ Pi exchange reaction because the osmotic energy was needed to drive the synthesis of ATP measured during the exchange reaction. This assumption was derived from studies in mitochondria where it was not possible to measure

ATP $ Pi exchange in the absence of an H+ gradient. For the Ca2+ -ATPase the synthesis of ATP is initiated by phosphorylation of the ATPase by Pi and we had already shown that this reaction is observed both in the presence and absence of a Ca2+ gradient [25, 26]. The possibility was then raised that in absence of a gradient there was no ATP synthesis because the phos-phoenzyme formed from Pi could not be converted from "low" into "high" energy. When a gradient is formed, the Ca2+ concentration inside the vesicles reaches the range of 2-10 mM. We then theorized that the conversion of the phosphoenzyme from low into high energy could be related to the binding of Ca2+ to a site of the ATPase located in a part of the protein facing the vesicles' lumen. To test this hypothesis, we prepared leaky vesicles and incubated them in media containing various Ca2+ concentrations, from a range similar to that found on the outer surface of the vesicles during transport (micromolar) up to the range found in the vesicles' lumen during Ca2+ accumulation (millimolar). The result of this experiment was very rewarding. In the presence of low Ca2+ concentrations (micromolar), the ATPase was able to catalyze only the hydrolysis of ATP, but as the Ca2+ concentration was raised to the millimolar range there was a simultaneous inhibition of ATP hydrolysis and activation of ATP synthesis. The Ca2+ concentration needed for both halfmaximal inhibition of ATP cleavage and half-maximal activation of ATP synthesis was in the range of 1-2 mM [36,37]. From these experiments we concluded that (i) during catalysis the enzyme was able to conserve some of the energy derived from ATP cleavage in such a manner as to utilize it to synthesize a new ATP molecule from ADP and Pi; (ii) the mechanism of the energy conservation involved the binding of Ca2+ to a low-affinity binding site of enzyme facing the vesicles' lumen (Ks 1-2 mM); (iii) the standard free energy derived from the binding of two Ca2+ ions to the low-affinity site is — 8.2 kcal/mol. This is sufficient for the synthesis of 1 mol of ATP for each mol of enzyme. According to these findings, the energy needed for the conversion of the phosphoenzyme from low into high energy and the subsequent synthesis of ATP was derived from the binding of Ca2+ to the low-affinity site of the enzyme. Thus, the mechanism by which the enzyme recognizes the gradient for the reversal of the catalytic cycle is related to the asymmetrical binding of Ca2+ on the two sides of the membrane. In order to phosphor-ylate the enzyme by Pi and form the low-energy phosphoenzyme, it is necessary that the Ca2+ concentration at the outer surface of the membrane be low enough so that binding to a high-affinity site of the protein facing the outer surface of the vesicle cannot occur, and in a second stage, to convert the phosphoen-zyme from low into high energy, Ca2+ must bind to a low-affinity site of the enzyme located on the inner surface of the membrane.

In the conditions used to measure ATP $ Pi exchange there is no net synthesis of ATP; the rate of ATP hydrolysis is always faster than the rate of ATP synthesis. A year after our reports on the exchange in the absence of a gradient, Knowles and Racker [38] reported that the Ca2+ -ATPase could catalyze the net synthesis of ATP in the absence of a Ca2+ gradient after a single catalytic cycle. This was achieved using leaky vesicles and a two-step procedure where initially the enzyme was phosphorylated by Pi in the absence of Ca2+ and then ADP and 3-4 mM CaCl2 were added to the medium. After the Ca2+ jump, half of the phosphoenzyme phosphate was transferred to ADP, forming ATP. Shortly after that we confirmed this experiment and in addition showed that different perturbations could drive the synthesis of ATP. These included a sudden change (jump) of pH, temperature, or water activity of the medium [39-41].

There are several similarities between the (Na++K+)ATPase and the Ca2+ -ATPase and discoveries with one enzyme frequently could be extended to the other. In 1975, Taniguchi and Post [42] reported that similar to the Ca2+ -ATPase, the (Na++K+) ATPase could be phosphorylated by Pi and could catalyze an ATP $ Pi exchange reaction in the absence of a transmembrane gradient. At this time, Robert Post visited our laboratory for a 3-week period. He is a very kind and gentle man and we learned a lot from him. Robert is an excellent athlete and one of the things I took up after his visit was jogging.

The Reaction Sequence

The study of the reversal of the Ca2+ pump led to the proposal of a basic reaction sequence [10,11,43-45] shown in Figure 1. During catalysis the enzyme cycles through two distinct conformations, E1 and E2. The enzyme form E1 binds calcium with high affinity on the outer surface of the vesicles and can be phos-phorylated by ATP but not by Pi. The enzyme form E2 binds calcium with low affinity at the inner surface of the vesicles and can be phosphorylated by Pi but not by ATP. The key feature of this cycle is the mechanism by which energy is transduced. In earlier models it was thought that the energy of hydrolysis of the different phosphate compounds would be the same regardless of whether they were in solution in the cytosol or bound to the enzyme surface, and that energy would be released and become available to the enzyme when the phosphate compound is hydrolyzed. The finding that the energy of hydrolysis of the phosphoenzyme formed from Pi varies during the reversal of the

Ca2+ -ATPase indicates that the energy becomes available for the translocation of calcium through the membrane before the cleavage of the phosphoenzyme. Thus, during the catalytic cycle there is a large change in the equilibrium constant for the hydrolysis (Keq) of the acylphosphate residue of the phosphoenzyme and the work is linked with this transition of Keq and not with the hydrolysis of the phosphate compound (Table 2). After the description of the phosphoenzyme of "high" and "low" energy in our laboratory and the "tightly bound ATP'' of the mitochondrial ATPase described in Paul Boyer's laboratory, in several laboratories it was discovered that the energy of hydrolysis of various phosphate compounds varies greatly depending on whether they are in solution or on the enzyme surface (Table 2).

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