Asymmetric binding of the inhibitor di(adenosine-5') pentaphosphate (Ap5A) to adenylate kinase.
The effect of binding diadenosine pentaphosphate (Ap(5)A) to adenylate kinase (ATP:AMP phosphotransferase; EC 2.7.4.3) has been investigated by (31)P nuclear magnetic resonance. The symmetric molecule, Ap(5)A, is a potent inhibitor of the adenylate kinase reaction, 2 ADP right arrow over left arrow ATP + AMP. Free Ap(5)A has two groups of signals in its (31)P nuclear magnetic resonance spectrum centered at 11.1 and 22.8 parts/million (ppm) upfield from 85% H(3)PO(4) that are assigned to the end (1-P and 5-P) and middle (2-, 3-, and 4-P) phosphates, respectively. Addition of Mg(2+) shifts the centers of these resonances to 11.7 and 22.3 ppm. The spectrum of Ap(5)A bound to porcine adenylate kinase shows five groups of signals centered at 10.9, 11.9, 20.5, 22.7, and 24.0 ppm; the resonances at 11.1 ppm (1-P and 5-P) and at 22.8 ppm (2-P and 4-P) are now clearly split, indicating asymmetric binding of Ap(5)A to the enzyme. The asymmetry is strikingly enhanced in enzyme-bound MgAp(5)A, which has resonances at 10.5, 12.5, 18.6, 22.7, and 25.6 ppm. By the addition of Mn(2+) to the enzyme.MgAp(5)A complex, the observed signals in increasing order of shifts were tentatively assigned to 1-P, 5-P, 4-P, 3-P, and 2-P, where the 3-, 4-, and 5-P positions correspond to the ATP-binding site on the enzyme. The asymmetry introduced in the phosphate chain of enzyme.MgAp(5)A is indicated by the (31)P chemical shift of 7 ppm between 2- and 4-P, which is one of the largest thus far observed for phosphate substrates bound noncovalently to enzymes.
D000227:Adenine Nucleotides / Q000378:metabolism*; D000263:Adenylate Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D008274:Magnesium / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D009132:Muscles / Q000201:enzymology; D009841:Oligonucleotides / Q000378:metabolism; D010770:Phosphotransferases / Q000378:metabolism*; D013552:Swine
D000227:Adenine Nucleotides; D009841:Oligonucleotides; D010770:Phosphotransferases; D000263:Adenylate Kinase; D008274:Magnesium