Adenylate Kinase-Catalyzed Reactions of AMP in Pieces: Specificity for Catalysis at the Nucleoside Activator and Dianion Catalytic Sites

Abstract

The pressure to optimize the enzymatic rate acceleration for adenylate kinase (AK)-catalyzed phosphoryl transfer has led to the evolution of an induced-fit mechanism, where the binding energy from interactions between the protein and substrate adenosyl group is utilized to drive a protein conformational change that activates the enzyme for catalysis. The adenine group of adenosine contributes 11.8 kcal mol^-1 to the total ≥14.7 kcal mol^-1 adenosine stabilization of the transition state for AK-catalyzed phosphoryl transfer to AMP. The relative third-order rate constants for activation of adenylate kinase, by the C-5 truncated adenosine 1-(β-d-erythrofuranosyl)adenine (EA), for catalysis of phosphoryl transfer from ATP to phosphite dianion (HP, k_cat/K_HPK_Act = 260 M^-2 s^-1), fluorophosphate (47 M^-2 s^-1), and phosphate (9.6 M^-2 s^-1), show that substitution of -F for -H and of -OH for -H at HP results, respectively, in decreases in the reactivity of AK for catalysis of phosphoryl transfer due to polar and steric effects of the -F and -OH substituents. The addition of a 5'-CH₂OH to the EA activator results in a 3.0 kcal mol^-1 destabilization of the transition state for AK-activated phosphoryl transfer to HP due to a steric effect. This is smaller than the 8.3 kcal mol^-1 steric effect of the 5'-CH₂OH substituent at OMP on HP-activated OMPDC-catalyzed decarboxylation of 1-(β-d-erythrofuranosyl)orotate. The 2'-OH ribosyl substituent shows significant interactions with the transition states for AK-catalyzed phosphoryl transfer from ATP to AMP and for adenosine-activated AK-catalyzed phosphoryl transfer from ATP to HP.