Direct Mg2+ Binding Activates Adenylate Kinase from Escherichia coli

Abstract

We report evidence that adenylate kinase (AK) from Escherichia coli can be activated by the direct binding of a magnesium ion to the enzyme, in addition to ATP-complexed Mg 2؉ . By systematically varying the concentrations of AMP, ATP, and magnesium in kinetic experiments, we found that the apparent substrate inhibition of AK, formerly attributed to AMP, was suppressed at low magnesium concentrations and enhanced at high magnesium concentrations. This previously unreported magnesium dependence can be accounted for by a modified random bi-bi model in which Mg 2؉ can bind to AK directly prior to AMP binding. A new kinetic model is proposed to replace the conventional random bi-bi mechanism with substrate inhibition and is able to describe the kinetic data over a physiologically relevant range of magnesium concentrations. According to this model, the magnesium-activated AK exhibits a 23-؎ 3-fold increase in its forward reaction rate compared with the unactivated form. The findings imply that Mg 2؉ could be an important affecter in the energy signaling network in cells. Adenylate kinase (AK)2 is a ϳ24-kDa enzyme involved in cellular metabolism that catalyzes the reversible phosphoryl transfer reaction (1) as in Reaction 1.It is recognized to play an important role in cellular energetic signaling networks (2, 3). A deficiency in human AK function may lead to such illness as hemolytic anemia (4 -8) and coronary artery disease (9); the latter is thought to be caused by a disruption of the AMP signaling network of AK (10). The ubiquity of AK makes it an ideal candidate for investigating evolutionary divergence and natural adaptation at a molecular level (11,12). Indeed, extensive structure-function studies have been carried out for AK (reviewed in Ref. 13). Both structural and biophysical studies have suggested that large-amplitude conformational changes in AK are important for catalysis (14 -19). More recently, the functional roles of conformational dynamics have been investigated using NMR (20 -22), computer simulations (23-27), and single-molecule spectroscopy (28). Given the critical role of AK in regulating cellular energy networks and its use as a model system for understanding the functional roles of conformational changes in enzymes, it is imperative that the enzymatic mechanism of AK be thoroughly characterized and understood.The enzymatic reaction of adenylate kinase has been shown to follow a random bi-bi mechanism using isotope exchange experiments (29). Isoforms of adenylate kinases characterized from a wide range of species have a high degree of sequence, structure, and functional conservation. Although all AKs appear to follow the same random bi-bi mechanistic framework (15, 29 -33), a detailed kinetic analysis reveals interesting variations among different isoforms. For example, one of the most puzzling discrepancies is the change in turnover rates with increasing AMP concentration between rabbit muscle AK and Escherichia coli AK. Although the reactivity of rabbit muscle AK is slightly inhibited at high...

The energy landscape of adenylate kinase during catalysis

“…While some kinases are activated by a single Mg 2+ ion, additional Mg 2+ binding may either be required for full activation, result in inhibition, or be involved in structural stabilization 13 , 17 , 18 . The variety of different mechanisms attributed to the Mg 2+ cofactor has resulted in arguments about the kinetic schemes to account for the role of Mg 2+ in catalysis 19 . Other heated debates focus on the role of conformational changes in the enzymatic reaction 20 - 25 .…”

On the Roles of Substrate Binding and Hinge Unfolding in Conformational Changes of Adenylate Kinase

“…The conformational changes of AK have been shown to exhibit a strong correlation with enzyme activity (34,(38)(39)(40)(41). Kinetic experiments (16,17,35,42,43) suggested conformational change as a rate-limiting step for catalysis. Recent single-molecule Förster resonance energy transfer experiments (41) affirmed that the conformational change from a closed to an open conformation after the phosphoryl transfer is the ratelimiting step of the reverse ATP-producing reaction of AK.…”

Elucidation of Mg2+ binding activity of adenylate kinase from Mycobacterium tuberculosisH37Rv using fluorescence studies

“…The dual activity of AK, as both NMPK and NDK, suggests that this enzyme may have a role in RNA and DNA biosynthesis in addition to its role in intracellular nucleotide metabolism. Thus, mechanisms regulating the optimal supply of adenine nucleotides and balance between its production and consumption are crucial for the maintenance of kinetic and thermodynamic balance within the cell . AK is one such regulator that catalyzes the reversible phosphotransfer from ATP to AMP and thus is involved in regulating metabolic signals coupled to cellular energy utilization .…”

“…Thus, mechanisms regulating the optimal supply of adenine nucleotides and balance between its production and consumption are crucial for the maintenance of kinetic and thermodynamic balance within the cell . AK is one such regulator that catalyzes the reversible phosphotransfer from ATP to AMP and thus is involved in regulating metabolic signals coupled to cellular energy utilization . Another significant role of AK was implicated in Pseudomonas aeruginosa as a virulence factor in triggering macrophage cell death.…”

“…Conventionally, the enzymatic reaction of AK follows a random bi–bi mechanism , and different isoforms of AK belong to a wide range of species with a high degree of structure, sequence, and functional conservation. In spite of having the same random bi–bi mechanism ,, a detailed kinetic study unveils intriguing variations among different isoforms . We depict a previously unreported kinetic study of forward reaction rate in MTb AK, in which an increase in the forward reaction rate of AK was observed as the concentration of Mg 2+ increased.…”