Insight into the Uncoupling Mechanism of Sarcoplasmic Reticulum ATPase Using the Phosphorylating Substrate UTP

Infrared spectroscopy has been used to map substrate-protein interactions: the conformational changes of the sarcoplasmic reticulum Ca 2؉ -ATPase upon nucleotide binding and ATPase phosphorylation were monitored using the substrate ATP and ATP analogues (2 -deoxy-ATP, 3 -deoxy-ATP, and inosine 5 -triphosphate), which were modified at specific functional groups of the substrate. Modifications to the 2 -OH, the 3 -OH, and the amino group of adenine reduce the extent of bindinginduced conformational change of the ATPase, with particularly strong effects observed for the latter two. This demonstrates the structural sensitivity of the nucleotide-ATPase complex to individual interactions between nucleotide and ATPase. All groups studied are important for binding and interactions of a given ligand group with the ATPase depend on interactions of other ligand groups.Phosphorylation of the ATPase was observed for ITP and 2 -deoxy-ATP, but not for 3 -deoxy-ATP. There is no direct link between the extent of conformational change upon nucleotide binding and the rate of phosphorylation showing that the full extent of the ATP-induced conformational change is not mandatory for phosphorylation. As observed for the nucleotide-ATPase complex, the conformation of the first phosphorylated ATPase intermediate E1PCa 2 also depends on the nucleotide, indicating that ATPase states have a less uniform conformation than previously anticipated.Ligand binding to proteins controls vast numbers of cellular processes and has attracted great scientific and economic interest. Protein and ligand flexibility are important determinants of the interaction and often lead to ligand binding modes that are not anticipated from structures obtained with other ligands. To these "failure(s) of the rigid receptor hypothesis" (1) is added here an impressive example: induced-fit binding of nucleotides to the Ca 2ϩ -ATPase. This finding stems from a systematic mapping of substrate-protein interactions with infrared (IR) 1 spectroscopy. New approaches like this are welcome in the field of ligand-protein recognition, since the most informative techniques, NMR and x-ray crystallography, are laborious and problematic for some systems. Methods like fluorescence and luminescence that require less expenditure also provide less molecular information. We expect that this technology gap will be bridged by IR spectroscopy.IR spectroscopy, one of the methods of vibrational spectroscopy, provides direct information on the molecular level, is cost-effective, and can be universally applied from small soluble proteins to large membrane proteins under near-physiological conditions. Work summarized in recent reviews (2-5) has shown that the vibrational spectrum changes characteristically when a ligand binds to a protein. This provides a direct observation of ligand binding: no marker compound has to be introduced to report the binding process, as with many other methods. Previous work has mostly focused on individual interactions between a ligand and a protein by monitoring the ...

Section: Discussionsupporting

confidence: 79%

Mapping Interactions between the Ca2+-ATPase and Its Substrate ATP with Infrared Spectroscopy

Liu

Barth

2003

“…An increase in internal free Ca 2ϩ produced complete uncoupling and slightly decreased the sensitivity to vanadate. Uncoupling through E 1 forms induced by lumenal Ca 2ϩ has already been characterized when the phosphorylating substrate is UTP (39).…”

Section: Ca 2ϩ -Atpase Reaction Cycle and P-nitrophenyl Phosphatementioning

confidence: 99%

“…It has been reported that SR Ca 2ϩ -ATPase is phosphorylated by and/or hydrolyzes ATP (40), UTP (39), or even the nonnucleotide substrate furylacryloylphosphate (41) in a Ca 2ϩ -free medium. In the case of pNPP, the rate of phosphorylation, which is much lower than that by ATP (25), does not allow the accumulation of phosphorylated intermediate during the enzyme turnover.…”

Section: Ca 2ϩ -Atpase Reaction Cycle and P-nitrophenyl Phosphatementioning

confidence: 99%

Testing the Versatility of the Sarcoplasmic Reticulum Ca2+-ATPase Reaction Cycle When p-Nitrophenyl Phosphate Is the Substrate

Fernández-Belda

Fortea

Soler

2001

Self Cite

A detailed characterization of p-nitrophenyl phosphate as energy-donor substrate for the sarcoplasmic reticulum Ca 2؉ -ATPase was undertaken in this study. The fact that p-nitrophenyl phosphate can be hydrolyzed in the presence or absence of Ca 2؉ by the purified enzyme is consistent with the observed phenomenon of intramolecular uncoupling. Under the most favorable conditions, which include neutral pH, intact microsomal vesicles, and low free Ca 2؉ in the lumen, the Ca 2؉ /P i coupling ratio was 0.6. A rise or decrease in pH, high free Ca 2؉ in the lumenal space, or the addition of dimethyl sulfoxide increase the intramolecular uncoupling. Alkaline pH and/or high free Ca 2؉ in the lumen potentiate the accumulation of enzyme conformations with high Ca 2؉ affinity. Acidic pH and/or dimethyl sulfoxide favor the accumulation of enzyme conformations with low Ca 2؉ affinity. Under standard assay conditions, two uncoupled routes, together with a coupled route, are operative during the hydrolysis of p-nitrophenyl phosphate in the presence of Ca 2؉ . The prevalence of any one of the uncoupled catalytic cycles is dependent on the working conditions. The proposed reaction scheme constitutes a general model for understanding the mechanism of intramolecular energy uncoupling.It is well established that the free energy released from ATP hydrolysis can be used for the vectorial translocation of cations across cellular membranes (1). In this sense, clear structural and functional similarities among P-type ATPases have been observed (2-4). These similarities include the hydrophobic transmembrane segments containing the porelike region, the extramembranous globular head where the ATP binding site is located, two major conformational states of the enzyme, the acyl phosphate intermediate and the catalytic cycle. Although these common features suggest that ion-transporting ATPases share the same energy transduction mechanism, this idea was challenged when non-nucleotide compounds were used as phosphorylating substrate (5). In fact, it was observed that the transport activity supported by non-nucleotide substrates is dependent on the cation-ATPase involved. In other words, the same non-nucleotide substrate does not always perform the same task since it is dependent on the energy transduction system. For instance, it has been reported that Ca 2ϩ is transported by sarcoplasmic reticulum (SR) 1 Ca 2ϩ -ATPase during the hydrolysis of pNPP or acetyl phosphate (6 -9). However, the hydrolysis of pNPP supports neither Ca 2ϩ transport by the erythrocyte membrane Ca 2ϩ -ATPase (10, 11) nor H ϩ transport by the H ϩ -ATPase from the yeast plasma membrane (12). Other studies have reported that reconstituted Na ϩ ,K ϩ -ATPase displays slight Na ϩ uptake during the hydrolysis of acetyl phosphate (13), whereas the H ϩ ,K ϩ -ATPase from gastric mucosa cannot actively transport H ϩ and K ϩ when hydrolyzing acetyl phosphate (5). Thus, it has been recognized that the hydrolysis of non-nucleotide substrates in the absence of transport activity is relate...

“…Data obtained in different laboratories indicate that the conversion of the phosphate compound from high to low energy is promoted by a change of water activity at the catalytic site of the enzyme, and this decrease can be explained by the solvation energy theory (33)(34)(35)(36)(37)(38)(39). forming an acyl phosphate residue of high energy (Table II), and translocation of Ca 2ϩ across the membrane occurs simultaneously with the conversion of the acyl phosphate from high into low energy (6,(13)(14)(15)(31)(32)(33). In leaky vesicles (no gradient) practically all the ATPase activity measured is coupled with the translocation of Ca 2ϩ across the membrane.…”

Section: Energy Of Hydrolysis Of Phosphate Compounds During Catalysismentioning

confidence: 99%

“…In this case, a part of the chemical energy derived from ATP cleavage is used for Ca 2ϩ transport, and a part is converted into heat. The second route is a shortcut of the transport cycle where the cleavage of ATP is completed in a step that precedes the translocation of Ca 2ϩ through the membrane, and all the energy derived from ATP hydrolysis is converted into heat (4,5,8,10,(13)(14)(15). Thus, during the course of the reaction, the amount of heat released varies depending on how much ATP is cleaved in each of these two routes.…”

mentioning

confidence: 99%

Heat of PPi Hydrolysis Varies Depending on the Enzyme Used

da‐Silva¹,

Bomfim²,

Galina³

et al. 2004

With yeast-soluble inorganic pyrophosphatase, the heat released during PP i hydrolysis was ؊6.3 kcal/mol regardless of the KCl concentration in the medium. With the membrane-bound pyrophosphatase of corn vacuoles, the heat released varies between ؊23.5 and ؊7.5 kcal/mol depending on the KCl concentration in the medium and whether or not a H ؉ gradient is formed across the vacuole membranes. The data support the proposal that enzymes are able to handle the energy derived from phosphate compound hydrolysis in such a way as to determine the parcel that is used for work and the fraction that is converted into heat (de Meis, L. (2001) J. Biol. Chem. 276, 25078 -25087).Evidence reported in the past 5 years indicates that enzymes are able to handle the energy derived from the hydrolysis of phosphate compounds in such a way as to determine the parcel that is used for work and the fraction that is converted into heat (1-12). The ability to modulate the conversion of energy into either heat or work varies depending on both the enzyme and the experimental conditions used. This was first observed with the sarco/endoplasmic reticulum Ca 2ϩ -ATPases (SERCA), 1 a family of membrane-bound ATPases that are able to translocate Ca 2ϩ ion across the membrane by using the chemical energy derived from ATP hydrolysis. With these enzymes it was found that the heat released during ATP hydrolysis may vary from 10 to 30 kcal/mol depending on the SERCA isoform used and on whether or not a Ca 2ϩ gradient is formed across the membrane (1-10). Kinetic measurements indicate that the SERCA are able to hydrolyze ATP through two catalytic routes (4 -6, 8, 13-15). In one of them hydrolysis is coupled with the translocation of Ca 2ϩ through the membrane. In this case, a part of the chemical energy derived from ATP cleavage is used for Ca 2ϩ transport, and a part is converted into heat. The second route is a shortcut of the transport cycle where the cleavage of ATP is completed in a step that precedes the translocation of Ca 2ϩ through the membrane, and all the energy derived from ATP hydrolysis is converted into heat (4,5,8,10,(13)(14)(15). Thus, during the course of the reaction, the amount of heat released varies depending on how much ATP is cleaved in each of these two routes.Recently, Bianconi (11) found that the enthalpy for the reactions of yeast hexokinase varies depending on the isozymes used, and Szöke et al. (12) in an elegant theoretical analysis proposed that enzyme could adjust the reaction path by altering the reversible interchange of different forms of energies (chemical, electrical, and mechanical). In these views, the same reaction catalyzed by different enzymes would lead to the release of different amounts of heat. In the report by Szöke et al. (12), it was proposed that the amount of heat produced during PP i hydrolysis by the soluble and membrane-bound PPases should be markedly different. The soluble PPase would convert ⌬G into heat, whereas for the membrane-bound PPases, less heat should be released because part of the ener...