Adenylate kinase
Adenylate kinase is a phosphotransferase enzyme that catalyzes the interconversion of the various adenosine phosphates. By constantly monitoring phosphate nucleotide levels inside the cell, ADK plays an important role in cellular energy homeostasis.
Substrate and products
The reaction catalyzed is:ATP + AMP ⇔ 2 ADP
The equilibrium constant varies with condition, but it is close to 1. Thus, ΔGo for this reaction is close to zero. In muscle from a variety of species of vertebrates and invertebrates, the concentration of ATP is typically 7-10 times that of ADP, and usually greater than 100 times that of AMP. The rate of oxidative phosphorylation is controlled by the availability of ADP. Thus, the mitochondrion attempts to keep ATP levels high due to the combined action of adenylate kinase and the controls on oxidative phosphorylation.
Isozymes
To date there have been nine human ADK protein isoforms identified. While some of these are ubiquitous throughout the body, some are localized into specific tissues. For example, ADK7 and ADK8 are both only found in the cytosol of cells; and ADK7 is found in skeletal muscle whereas ADK8 is not. Not only do the locations of the various isoforms within the cell vary, but the binding of substrate to the enzyme and kinetics of the phosphoryl transfer are different as well. ADK1, the most abundant cytosolic ADK isozyme, has a Km about a thousand times higher than the Km of ADK7 and 8, indicating a much weaker binding of ADK1 to AMP. Sub-cellular localization of the ADK enzymes is done by including a targeting sequence in the protein. Each isoform also has different preference for NTP's. Some will only use ATP, whereas others will accept GTP, UTP, and CTP as the phosphoryl carrier.Some of these isoforms prefer other NTP's entirely. There is a mitochondrial GTP:AMP phosphotransferase, also specific for the phosphorylation of AMP, that can only use GTP or ITP as the phosphoryl donor. ADK has also been identified in different bacterial species and in yeast. Two further enzymes are known to be related to the ADK family, i.e. yeast uridine monophosphokinase and slime mold UMP-CMP kinase. Some residues are conserved across these isoforms, indicating how essential they are for catalysis. One of the most conserved areas includes an Arg residue, whose modification inactivates the enzyme, together with an Asp that resides in the catalytic cleft of the enzyme and participates in a salt bridge.
Subfamilies
Mechanism
Phosphoryl transfer only occurs on closing of the 'open lid'. This causes an exclusion of water molecules that brings the substrates in proximity to each other, lowering the energy barrier for the nucleophilic attack by the α-phosphoryl of AMP on the γ-phosphoryl group of ATP resulting in formation of ADP by transfer of the γ-phosphoryl group to AMP. In the crystal structure of the ADK enzyme from E. coli with inhibitor Ap5A, the Arg88 residue binds the Ap5A at the α-phosphate group. It has been shown that the mutation R88G results in 99% loss of catalytic activity of this enzyme, suggesting that this residue is intimately involved in the phosphoryl transfer. Another highly conserved residue is Arg119, which lies in the adenosine binding region of the ADK, and acts to sandwich the adenine in the active site. It has been suggested that the promiscuity of these enzymes in accepting other NTP's is due to this relatively inconsequential interactions of the base in the ATP binding pocket. A network of positive, conserved residues stabilize the buildup of negative charge on phosphoryl group during the transfer. Two distal aspartate residues bind to the arginine network, causing the enzyme to fold and reduces its flexibility. A magnesium cofactor is also required, essential 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.Structure
Flexibility and plasticity allow proteins to bind to ligands, form oligomers, aggregate, and perform mechanical work. Large conformational changes in proteins play an important role in cellular signaling. Adenylate Kinase is a signal transducing protein; thus, the balance between conformations regulates protein activity. ADK has a locally unfolded state that becomes depopulated upon binding.A 2007 study by Whitford et al. shows the conformations of ADK when binding with ATP or AMP. The study shows that there are three relevant conformations or structures of ADK—CORE, Open, and Closed. In ADK, there are two small domains called the LID and NMP. ATP binds in the pocket formed by the LID and CORE domains. AMP binds in the pocket formed by the NMP and CORE domains. The Whitford study also reported findings that show that localized regions of a protein unfold during conformational transitions. This mechanism reduces the strain and enhances catalytic efficiency. Local unfolding is the result of competing strain energies in the protein.
The local stability of the substrate-binding domains ATPlid and AMPlid has been shown to be significantly lower when compared with the CORE domain in ADKE. coli. Furthermore, it has been shown that the two subdomains can fold and unfold in a "non-cooperative manner." Binding of the substrates causes preference for 'closed' conformations amongst those that are sampled by ADK. These 'closed' conformations are hypothesized to help with removal of water from the active site to avoid wasteful hydrolysis of ATP in addition to helping optimize alignment of substrates for phosphoryl-transfer. Furthermore, it has been shown that the apoenzyme will still sample the 'closed' conformations of the ATPlid and AMPlid domains in the absence of substrates. When comparing the rate of opening of the enzyme and the rate of closing that accompanies substrate binding, closing was found to be the slower process.