Analysis of phosphoryl transfer mechanism and catalytic centre geometries of transport ATPase by means of spin-labelled ATP.
Spin-labelled ATP [3'-O-(1-oxyl-2,2,5,5-tetramethyl-3-carbonyl pyrrolidine)-adenosine 5'-triphosphate], abbreviated SL-ATP, is used to study firstly the occurrence of an associative phosphorane mechanism for the phosphoryl transfer from ATP to the transport-ATPase protein, and secondly the presence of two geometrically unequal catalytic centres in the two catalytic peptide chains deduced to explain the existence of two KD'(ATP) values under equilibrium conditions and two Km(ATP) values under turnover conditions. 1. In the presence of Na+, K+ and Mg2+, SL-ATP is not hydrolysed by transport-ATPase from three different sources. In the presence of Na+ and Mg2+, SL-ATP reacts initially like ATP with the enzyme, as indicated by the production of a similar ouabain-binding protein conformation. With both nucleotides, this initial reaction includes the formation of the covalent enzyme-nucleotide complex through nucleophilic attack of the aspartate carboxyanion of the catalytic centre on the terminal phosphorus atom of the triphosphate chain. This produces the ouabain-binding conformation of the enzyme. Unlike ATP, the covalent enzyme-SL-ATP complex resists further transformation. 2. In the presence of Na+, K+ and Mg2+, the influence of SL-ATP on ATP hydrolysis by transport-ATPase depends on the ATP concentration chosen. At low ATP concentration, when the enzyme works as Na+-ATPase, SL-ATP does not affect the rate of ATP cleavage. At high ATP concentration, however, when the enzyme works as (Na+ + K+)-ATPase, SL-ATP reduces the rate of ATP hydrolysis to the level of Na+-ATPase activity, apparently due to the formation of the covalent enzyme-SL-ATP complex. 3. SL-ATP in the covalent enzyme-SL-ATP complex shows an ESR spectrum which is indistinguishable regarding the overall shape, the rotational correlation time, tau, and the hyperfine coupling constant, aN, from the ESR spectrum of free SL-ATP. Consequently, the dimensions of the catalytic centre cleft of transport-ATPase provide the labelled group of SL-ATP, opposite to its 3'-O-esterification site at the ribose moiety, in a wide-cleft groove, enough free space for an essentially unhindered rotational mobility within an aqueous environment like that of the bulk medium. Judged from literature data, similarly wide grooves exist in the catalytic centre clefts of mitochondrial and myosin ATPases. 4. In the framework of present knowledge, the idea is put forward that the structural unit forming the binding site for the AMP moiety of ATP in ATPases is similar to the structural unit forming the binding site for the AMP moiety of NAD and ADP in several dehydrogenases and kinases.
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport; D001921:Brain / Q000201:enzymology; D004578:Electron Spin Resonance Spectroscopy; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008968:Molecular Conformation; D009132:Muscles / Q000201:enzymology; D011485:Protein Binding; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D013552:Swine
C026652:3'-O-(1-oxyl-2,2,5,5-tetramethyl-3-carbonyl pyrrolidine)-adenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D000254:Sodium-Potassium-Exchanging ATPase