Adenylate Kinase-Catalyzed Reaction of AMP in Pieces: Enzyme Activation for Phosphoryl Transfer to Phosphite Dianion

The binding of adenosine 5’-triphosphate (ATP) and adenosine 5’-monophosphate (AMP) to adenylate kinase (AdK) drives closure of lids over the substrate adenosyl groups. We test the hypothesis that this conformational change activates AdK for catalysis. The rate constants for Homo sapiens adenylate kinase 1 (HsAdK1)-catalyzed phosphoryl group transfer to AMP, k(cat)/K(m) = 7.0 x 10(6) M(−1) s(−1), and phosphite dianion, (k(HPi))(obs) ≤ 1 x 10(−4) M(−1) s(−1), show that the binding energy of the adenosyl group effects a ≥7.0 x 10(10)-fold rate acceleration of phosphoryl transfer from ATP. The third-order rate constant of k(cat)/K(HPi)K(EA) = 260 M(−2) s(−1) for 1-(β-d-erythrofuranosyl)adenine (EA)-activated phosphoryl transfer to phosphite dianion was determined, and the isohypophosphate reaction product characterized by (31)P NMR. The results demonstrate: (i) a ≥14.7 kcal/mol stabilization of the transition state for phosphoryl transfer by the adenosyl group of AMP and a ≥2.6 x 10(6)-fold rate acceleration from the EA-driven conformational change, and (ii) the recovery of ≥8.7 kcal/mol of this transition state stabilization for EA-activated phosphoryl transfer from ATP to phosphite.