Elucidation of Mg²⁺ binding activity of adenylate kinase from Mycobacterium tuberculosis H₃₇Rv using fluorescence studies.

Adenylate kinase (AK) is a small ubiquitous enzyme that catalyzes the reversible transfer of the terminal phosphate group from adenine triphosphate (ATP): magnesium ion (Mg²⁺) to adenine monophosphate (AMP) to form two molecules of adenine diphosphate (ADP). AK thus maintains the homeostasis of adenine nucleotides in eukaryotes and prokaryotes. Because the [ATP]/[ADP] ratio is an important parameter in energy regulation in cells, Mg²⁺-activated AK has an important biological role, particularly in the case of bacteria, as imbalance in the ratio of [ATP]/[ADP] has been associated with alteration in its DNA supercoiling state. In the present study, magnesium-binding assays were carried out by systematically varying the concentrations of Mg²⁺, protein, AMP, ATP, and indicator in kinetic experiments. We report evidence that during magnesium-binding assay, the fluorescence level of the indicator "Mag-Indo-1" changes with protein concentration, suggesting that magnesium ions are binding to AK. The dual activity of AK both as nucleoside monophosphate and diphosphate kinases suggests that this enzyme may have a role in RNA and DNA biosynthesis in addition to its role in intracellular nucleotide metabolism. According to the proposed model, the magnesium-activated AK exhibits an increase in its forward reaction rate compared with the inactivated form. These findings imply that Mg²⁺ could be an important regulator in the energy signaling network in cells.