Myokinase

Myokinase (EC 2.7.4.3, also known as Adenylate kinase or ADK) is a phosphotransferase enzyme catalyzing the interconversion of adenine nucleotides. ADK is important in cellular energy homeostasis since it can constantly monitor phosphate nucleotide levels inside the cell. The equilibrium constant is variable according to the condition, but it is adjacent to 1. Therefore, ΔG^o for this reaction is almost zero. In muscle from various species of vertebrates and invertebrates, the concentration of ATP (adenosine triphosphate) is typically 7 to10 times higher than that of ADP (adenosine diphosphate), and usually 100 times greater than that of AMP (adenosine monophosphate). The availability of ADP controls the rate of oxidative phosphorylation. Consequently, the mitochondrion attempts to keep high ATP levels owing to the combined action of ADK and the controls on oxidative phosphorylation.

Structure

Flexibility and plasticity make proteins bind with ligands to form oligomers, to aggregate, and to perform mechanical work. Large conformational changes in proteins are critical for cellular signaling. ADK is a signal transducing protein, and therefore, the balance between conformations could regulate protein activity. ADK contains a locally unfolded state that becomes depopulated upon binding.

It has been showed that there are three relevant conformations or structures of ADK-CORE, Open, and Closed. There are two small domains called LID and NMP in ADK. ATP binds in the pocket formed by the LID and CORE domains and AMP binds in the pocket formed by the NMP and CORE domains. The localized regions of a protein would unfold during conformational transitions, which reduces the strain and enhances catalytic efficiency. Local unfolding is resulted from the competing strain energies in the protein.

Figure 1. ADK enzyme skeleton in cartoon

Isozymes

Up to now, nine human ADK protein isoforms have been identified, some of which are ubiquitous throughout the body, while some are localized at specific tissues. Not only do the locations of various isoforms within the cell vary, but also the binding of substrate to the enzyme and kinetics of the phosphoryl transfer are different. Sub-cellular localization of the ADK enzymes is implemented by incorporating a targeting sequence into the protein. Each isoform also has different preference for nucleoside triphosphates (NTP’s). ADK has also been found in different bacterial species and yeasts. Some residues are conserved across these isoforms, indicating their essential for catalysis. One of the most conserved areas is the Arg residue, whose modification could inactivate the enzyme, together with an Asp that resides in the catalytic cleft of the enzyme and participates in a salt bridge.

Mechanism

Phosphoryl transfer only occurs on closing of the 'open lid', which leads to an exclusion of water molecules to bring the substrates in proximity to each other, thus lowering the energy barrier for the nucleophilic attack by the γ-phosphoryl group of ATP on the α-phosphoryl of AMP. In the crystal structure of the ADK enzyme from E. coli with inhibitor Ap5A, the Arg88 residue binds with the Ap5A at the α-phosphate group. The mutation R88G has been shown to cause 99% loss of catalytic activity of this enzyme, demonstrating the intimate involvement of that this residue in the phosphoryl transfer. Another highly conserved residue is Arg119. It resides in the adenosine binding region of the ADK, and functions to sandwich the adenine in the active site. The promiscuity of these enzymes in accepting other NTP's is caused by the relatively inconsequential interactions of the base in the ATP binding pocket. A network of positive, conserved residues (Lys13, Arg123, Arg156, and Arg167 in ADK from E. coli) is used to stabilize the buildup of negative charge on phosphoryl group during the transfer. The binding og two distal aspartate residues with the arginine network causes the enzyme to fold and reduces its flexibility. A magnesium cofactor is also required for increasing the electrophilicity of the phosphate on AMP, though this magnesium ion is only held in the active pocket by electrostatic interactions and dissociates easily.

Function

a. Metabolic monitoring

The ability for a cell to dynamically measure energetic levels makes us easy to monitor metabolic processes. ADK could regulate energy expenditure at the cellular level by continually monitoring and altering the levels of adenyl phosphates. As energy levels get changed under different metabolic stresses, ADK is then capable of generating AMP that acts as a signaling molecule in further signaling cascades. Common factors could influence adenine nucleotide levels, and therefore ADK activity are exercise, stress, changes in hormone levels and diet. ADK facilitates the decoding of cellular information by catalyzing nucleotide exchange in the intimate “sensing zone” of metabolic sensors.

b. ADK shuttle

ADK is present in mitochondrial and myofibrillar compartments in the cell, and it allows two high-energy phosphorylase (β and γ) of ATP available to be transferred between adenine nucleotide molecules. Essentially, ADK carries ATP to sites of high energy consumption and removes the generated AMP over the course of those reactions. Ultimately, these sequential phosphotransfer relays could result in the propagation of the phosphoryl groups along collections of ADK molecules. This process can be considered as a bucket brigade of ADK molecules that results in changes in local intracellular metabolic flux without apparent global changes in metabolite concentrations, which is extremely pivotal for overall homeostasis of the cell.

Reference

1. Gearhart J D, Mintz B. Creatine kinase, myokinase, and acetylcholinesterase activities in muscle-forming primary cultures of mouse teratocarcinoma cells. Cell, 1975, 6(1):61-66.