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 HP K 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.

Elucidating Dynamics of Adenylate Kinase from Enzyme Opening to Ligand Release

“…This result aligns with the NMR studies exploring the enzyme surface involved in the initial ATP interaction with mutations at various ligand binding sites . Additionally, kinetic studies that utilized a truncated nucleoside and a phosphite dianion, together mimicking the AMP substrate, showed that ligand binding drives conformational change in catalysis and contributes to the stabilization of the reaction transition state . On the other hand, NMR and single-molecule Förster resonance energy transfer (sm-FRET) studies have reported access to both the open and closed conformations of the enzyme even in the absence of substrates (i.e., apo-state), suggesting that conformational selection is also at play. , …”

“…38 Additionally, kinetic studies that utilized a truncated nucleoside and a phosphite dianion, together mimicking the AMP substrate, showed that ligand binding drives conformational change in catalysis and contributes to the stabilization of the reaction transition state. 43 On the other hand, NMR and single-molecule Forster resonance energy transfer (sm-FRET) studies have reported access to both the open and closed conformations of the enzyme even in the absence of substrates (i.e., apo-state), suggesting that conformational selection is also at play. 22,25 This enzyme has also been extensively studied as a model system for investigating the role of close-to-open conformational motion in catalytic turnover.…”

Utilization of Cofactor Binding Energy for Enzyme Catalysis: Formate Dehydrogenase-Catalyzed Reactions of the Whole NAD Cofactor and Cofactor Pieces

“…The binding energies Δ G † summarized in Figure show the following about the activation of Cb FDH-catalyzed hydride transfer by cofactor pieces. The addition of 1.0 M of activator pieces to a solution of NR and [HCO 2 – ] = 0.20 M recovers nearly 50% of the −11.7 kcal/mol transition state stabilization by ADP at the whole NAD + cofactor. A similar balance has been observed between the transition state binding energy for the nonreacting phosphodianion or adenosyl groups that are attached to whole substrates, and the binding energy for the corresponding phosphite dianion or adenosyl activators. ,,− These differences represent partly or entirely the entropic advantage to the reaction of the single whole substrates (e.g., NAD + ) compared with the reactions of substrate pieces (e.g., NR + ADP) Significant activator binding energies Δ G † are observed for AMP, ADP, and R5P, but there is no detectable activation of hydride transfer by Ado or 1-(β- d -erythrofuranosyl)­adenine (EA, Scheme ) or by a mixture of EA or Ado and phosphite dianion.…”

“…The addition of 1.0 M of activator pieces to a solution of NR and [HCO 2 – ] = 0.20 M recovers nearly 50% of the −11.7 kcal/mol transition state stabilization by ADP at the whole NAD + cofactor. A similar balance has been observed between the transition state binding energy for the nonreacting phosphodianion or adenosyl groups that are attached to whole substrates, and the binding energy for the corresponding phosphite dianion or adenosyl activators. ,,− These differences represent partly or entirely the entropic advantage to the reaction of the single whole substrates (e.g., NAD + ) compared with the reactions of substrate pieces (e.g., NR + ADP) …”

“…This is one of many experimental studies on a variety of enzymes, which show that the binding interactions of nonreacting substrate fragments activate enzymes for catalysis . We have focused on the induced-fit mechanism because it provides a powerful structure-based guide for identifying enzymes that undergo ligand-induced activation. ,,, There have been few computational studies to evaluate critical features of this induced-fit reaction mechanism, and one disputed report that claims to provide a refutation. , Most telling is the absence of computational studies to examine whether the substrate binding energy, such as for binding of OMP to OMPDC, may be partitioned into the sum of the strong stabilizing protein–ligand interactions observed at the Michaelis complex, and the destabilization of this complex associated with the change in protein conformation from E O to E C (Scheme ). We do not know if this represents a limitation in existing computational methods, or the attitude that these results are not sufficiently important to merit attention from computational biochemists.…”