The ATP binding site

Munkonge et al. [411] obtained distances of 20A for the quenching of the fluorescence of NCD-Ca -ATPase either by doxylstearate (5-NS), or by FITC-labeled phosphatidylethanolamine (FITC-PE) incorporated into the bilayer although the authors concluded that the NCD label was located 20A above the surface of the bilayer, the data actually support the conclusion of Scott [132] placing the NCD = 20A below the surface of the bilayer. The energy transfer distance of 

10 A for the Tb RITC-DPPE pair [412] is also consistent with Tb binding in the acyl chain region of the bilayer. 

The estimated distances between lanthanides bound at various cation binding sites on the Ca -ATPase, and FITC, TNP-ATP, Cr-ATP or eosin bound at the nucleotide binding site range between 10 and 47 A [389,390,400,401,407-409,413] (Table II). 

The longer distances (35-47 A) are usually attributed to the distance between the high-affinity Ca site and the ATP binding site [389-390]. This would be consistent with the distance of 40-50 A obtained for the NCD-TNP-ATP pair [132]. Other evidence that places the ATP binding site high above the surface of the bilayer is the distance of 34-A2 A for the FITC-RITC DPPE pair [412], the distance of 60-80 A for 

The intramolecular distances measured at room temperature with the AEDANS FITC pair were similar in the Ca2Ei and E2V states [297]. Ca and lanthanides are expected to stabilize the Ej conformation of the Ca -ATPase, since they induce a similar crystal form of Ca -ATPase [119,157] and have similar effects on the tryptophan fluorescence [151] and on the trypsin sensitivity of Ca -ATPase [119,120]. It is also likely that the vanadate-stabilized E2V state is similar to the p2 P state stabilized by Pi [418]. Therefore the absence of significant difference in the resonance energy transfer distances between the two states implies that the structural differences between the two conformations at sites recorded by currently available probes, fall within the considerable error of resonance energy transfer measurements. Even if these distances would vary by as much as 5 A the difference between the two conformations could not be established reliably. 

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Approximately 500 of the 820 amino acid residues of the myosin head are highly conserved between various species. One conserved region, located approximately at residues 170 to 214, constitutes part of the ATP-binding site. Whereas many ATP-binding proteins and enzymes employ a /3-sheet-a-helix-/3-sheet motif, this region of myosin forms a related a-f3-a structure, beginning with an Arg at (approximately) residue 192. The /3-sheet in this region of all myosins includes the amino acid sequence... 

Most of the PKIs currently in clinical trials are small molecules that compete for the ATP-binding site [3,5]. They prevent the phosphate donor ATP to bind to the protein kinase, and hence the target protein will not become phosphorylated and the perturbed signalling can be terminated. 

Scholz B, Rechter S, Drach JC, Townsend LB, Bogner E (2003) Identification of the ATP-binding site in the terminase subunit pUL56 of human cytomegalovims. Nucleic Adds Res 31 1426-1433... 

Indirect evidence for the existence of different conformations of H,K-ATPase has been gained by site-selective reagents. Eor example, Schrijen et al. [67,95] demonstrated that Mg increased exposure of an essential arginine residue near the ATP-binding site and Mg " caused an increase in the number of reactive sulfhydryl groups on the enzyme. 

Conformational changes within or near the ATP-binding site of H,K-ATPase have also been demonstrated with the reversible fluorescent probes TNP-ATP [97,98] and... 

Fig. 8. Mutagenesis of the predicted ATP binding site. ATP is shown in proximity to amino acids in four loops predicted to form the ATP binding site in the nucleotide binding domain [49,134] and a fifth loop representing the phosphorylation site at Asp351 [97], Mutations and the corresponding Ca transport activity of the mutants relative to wild-type are indicated. From Clarke et al. [103).

Chemical modification studies with fluorescein-5 -isothiocyanate support the proximity of Lys515 to the ATP binding site [98,113-117,212,339]. Fluorescein-5 -isothiocyanate stoichiometrically reacts with the Ca -ATPase in intact or solubilized sarcoplasmic reticulum at a mildly alkaline pH, causing inhibition of ATPase activity, ATP-dependent Ca transport, and the phosphorylation of the Ca " -ATPase by ATP the Ca uptake energized by acetylphosphate, carbamylphos-phate or j -nitrophenyl phosphate is only partially inhibited [113,114,212,339]. The reaction of -ATPase with FITC is competitively inhibited by ATP, AMPPNP, TNP-ATP, and less effectively by ADP or ITP the concentrations of the various nucleotides required for protection are consistent with their affinities for the ATP binding site of the Ca -ATPase [114,212,340]. 

Similarly, the rate of inhibition of phosphoenzyme formation by diethylpyrocarbonate (DEPC) was much slower than the loss of ATPase activity [368], Even when the reaction approached completion with more than 90% inhibition of ATP hydrolysis, about 70% of the Ca -ATPase could still be phosphorylated by ATP (2.3nmoles of E P/mg protein). The remaining 30% of E P formation and the corresponding ATPase activity was not reactivated by hydroxylamine treatment, suggesting some side reaction with other amino acids, presumably lysine. When the reaction of the DEPC-modified ATPase with P-ATP was quenched by histidine buffer (pH 7.8) the P-phosphoenzyme was found to be exceptionally stable under the same conditions where the phosphoenzyme formed by the native ATPase underwent rapid hydrolysis [368]. The nearly normal phosphorylation of the DEPC-trea-ted enzyme by P-ATP implies that the ATP binding site is not affected by the modification, and the inhibition of ATPase activity is due to inhibition of the hydrolysis of the phosphoenzyme intermediate [368]. This is in contrast to an earlier report by Tenu et al. [367], that attributed the inhibition of ATPase activity by... 

In a fourth experiment, a paramagnetic probe can be used to determine the proximity from the ATP-binding site of non-competitive inhibitors. An MnATP probe is generated by the addition of Mn ions to ATP [51]. The... 

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