Nuclear magnetic resonance studies of the nucleotide binding sites of porcine adenylate kinase.

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The alpha, beta, gamma-tridentate complex of CrATP, a paramagnetic competitive inhibitor of porcine adenylate kinase, increases the longitudinal [1/(fT1p)] and transverse [1/(fT2p)] relaxation rates of a resonance of the enzyme previously assigned by McDonald et al. [McDonald, G.G., Cohn, M., & Noda, L. (1975) J. Biol. Chem. 250, 6947-6954] to the C2 proton of histidine-36. These paramagnetic effects are diminished upon the addition of the substrate MgATP by an amount consistent with the simple displacement of CrATP. The 1/(fT2p) value sets a lower limit of 400 s-1 on the rate constant for dissociation of CrATP from the enzyme. The 1/(fT1p) value at 250 MHz and the correlation time for water protons in the same complex are used to calculate a distance of 12.9 +/- 1.0 A from Cr(III) to the C2 proton of histidine-36. A primary, negative nuclear Overhauser effect is detected on the adenine H2 resonance of enzyme-bound MgATP upon preirradiation of the C2 proton of histidine-36, indicating that these protons are approximately less than 5 A apart. These distances and negative intramolecular Overhauser effects from the ribose protons to adenine H8 of MgATP indicate an extended structure for bound MgATP with an anti conformation about the glycosidic bond. These findings require a different orientation or location of the bound metal-ATP substrate from that proposed based on X-ray studies of the binding of salicylate [Pai, E. F., Sachsenheimer, W., & Schirmer, R.H. (1977) J. Mol. Biol. 114, 37-45]. Other nuclear Overhauser effects from resonances of the protein at 1.8 and 0.9 ppm to both adenine H2 and ribose H1' of bound MgATP indicate the proximity to the substrate of at least one Arg C beta proton (at 1.8 ppm), C gamma proton (at 1.7 ppm), Lys C delta proton (at 1.7 ppm), or Leu C beta proton (at 1.6 ppm) and one or more Leu, Ile, or Val methyl groups (at 0.9 ppm). Entirely different Overhauser effects are observed from the enzyme to the adenine protons of AMP consistent with a distinct site for the other substrate.