Adenylate Kinase (Human)

Takanori Ayabe and Minoru Hamada

     

      Adenylate kinase (ATP-AMP transphosphorylase, adenylate kinase, myokinase, EC 2.7.4.3.) is a ubiquitous enzyme which is involved in the interconversion of adenine nucleotides in cells.  In vertebrates, three isozymes (AK1, AK2, and AK3) have already been characterized.  AK1 is present in the cytosol of skeletal muscle, brain, and erythrocyte, while AK2 is found in the mitochondrial intermembrane space of the liver, kidney, spleen, and heart.  These isozymes catalyze the reaction: MgATP + AMP MgADP + ADP (1), and contribute to homeostasis of the adenine nucleotide composition in the cell.  During cellular metabolism, it is considered that adenylate kinase provides AMP to the rate-limiting steps in the energy-generating pathway (e.g., glycolysis) as an allosteric activator, resulting in  rate stimulation of the pathway. AK3, which catalyzes the reaction: MgGTP + AMP MgGDP + ADP, is present in the mitochondrial matrix of the liver and heart.  GTP is generated by substrate level phosphorylation in mitochondria; therefore, AK3 is thought to be involved in transferring the phosphate moiety from the guanine nucleotide pool to the adenine nucleotide pool together with the nucleotide diphosphate kinase.

      This enzyme is a monomeric, small globular protein having molecular weights near 21,700.  Human adenylate kinase (isozyme AK1) from skeletal muscle is a single polypeptide chain of 194 amino acid residues with an acetylmethionine at the N-terminus and a lysine at the C-terminus.  The primary structure of the enzyme was determined to be Ac-Met-Glu-Glu-Lys-Leu-Lys-Lys-Thr-Lys-Ile[10]-Ile-Phe-Val-Val Gly-Gly-Pro-Gly-Ser-Gly[20]-Lys-Gly-Thr-Gln-Cys-Glu-Lys-Ile-Val-Gln[30]-Lys-Tyr-Gly-Tyr-Thr-His-Leu-Ser-Thr-Gly[40]-Asp-Leu-Leu-Arg-Ser-Glu-Val Ser-Ser-Gly[50]-Ser-Ala-Arg-Gly-Lys-Lys-Leu-Ser-Glu-Ile[60]-Met-Glu-Lys-Gly Gln-Leu-Val-Pro-Leu-Glu[70]-Thr-Val-Leu-Asp-Met-Leu-Arg-Asp-Ala-Met[80]-Val-Ala-Lys-Val-Asn-Thr-Ser-Lys-Gly-Phe[90]-Leu-Ile-Asp-Gly-Tyr-Pro-Arg-Glu-Val-Gln[100]-Gln-Gly-Glu-Glu-Phe-Glu-Arg-Arg-Ile-Gly[110]-Gln-Pro-Thr-Leu-Leu-Leu-Tyr-Val-Asp-Ala[120]-Gly-Pro-Glu-Thr-Met-Thr-Arg-Arg-Leu-Leu[130]-Lys-Arg-Gly-Glu-Thr-Ser-Gly-Arg-Val-Asp[140]-Asp-Asn-Glu-Glu Thr-Ile-Lys-Lys-Arg-Leu[150]-Glu-Thr-Tyr-Tyr-Lys-Ala-Thr-Glu-Pro-Val[160]-Ile-Ala-Phe-Tyr-Glu-Lys-Arg-Gly-Ile-Val[170]-Arg-Lys-Val-Asn-Ala-Glu-Gly Ser-Val-Asp[180]-Glu-Val-Phe-Ser-Gln-Val-Cys-Thr-His-Leu[190]-Asp-Ala-Leu-Lys (2).

      X-ray structural studies indicate that the AK protein has ten α-helices and five β-strands (3) and that the active center cleft opens to some extent.  Figure 1 shows a model of the human adenylate kinase (4).  There are two distinct nucleotide-binding sites, one for the metal-chelated MgATP2- and the other for the unchelated AMP2-.  The conserved arginine residues (Arg44, Arg97, Arg132, and Arg138) were substituted with alanine and the AMP site was assigned in the right side of the model (5).  These arginine residues were determined to be essential for catalytic activity.  The loss of the positively charged guanidino groups from the arginine residues would inhibit the catalytic efficiency, therefore, the arginine residue was suggested to be essential for catalysis because it interacted with the negative charge of the phosphates of the adenine nucleotides (5).  The loss of the positively charged ε-amino groups of the Lys9, Lys21, Lys27, Lys31, Lys63, Lys131, and Lys194 residues by random site-directed mutagenesis resulted in a decreased or unchanged affinity for the substrates and reduced catalytic efficiencies (4).  The positive charge of lysine may interact with the negatively charged phosphates of MgATP2- and AMP2-, which in turn may orient the phosphate chains of the two substrates to the proper conformation required for catalysis.  These lysine residues would take part in the phophoryl transfer reaction between MgATP2- and AMP2-, in cooperation with the other active site residues such as the arginine residues.

      The ATP-binding site of AK has features in common with the nucleotide binding domains of the other kinases.  The amino acid sequences of several ATP-binding proteins such as the F1-ATPases, myosin, rec A protein, ras P21, transducin GTPases, cAMP-dependent, and src protein kinases contain a homologous sequence which is associated with the ATP-binding site.

      In human genetic disorders, an aberrant AK isozyme has been specifically found in the serum of Duchenne dystrophic patients (6), which resembled the isolated human liver mitochondrial adenylate kinase in its kinetics, stability, and electrophoretic properties, but based on immunological detection, it appeared to structurally be a muscle type.  An AK1 deficiency associated with hemolytic anemia has been reported (7), and the replacement of Arg-128 with Trp in AK1 was considered to be the cause of the enzyme deficiency.  The same mutant prepared by site-directed mutagenesis resulted in reduced catalytic activity and decreased solubility.  Based on these findings, it seems reasonable to study the properties of various mutant AKs in order to better understand the disorders due to such mutations.  The efforts of other mutants with site-directed mutagenesis of residues on ATP-binding, AMP-one, and the catalytic region of this enzyme, are currently under investigation, and future work should be directed toward a more detailed examination of the structure-function, delineation of metabolic pathways, studies of the regulation of metabolic pathways, the search for receptors, studies of energy transduction, etc.

 

References

1.     M. Hamada and  S.A. Kuby (1978) Arch. Biochem. Biophys. 190, 772-779.

2.     I. Von Zabern, B. Wittmann-Liebold, R. Untucht-Grau, R. H. Schirmer, and E.      F. Pai (1976) Eur. J. Biochem. 68, 281-290.

3.     G. E. Shulz, M. Elzinga, F. Marx, and R. H. Schirmer (1974) Nature 250, 120-      123.

4.     T. Ayabe, H.Takenaka, O. Takenaka, M. Sumida, H. Maruyama, T. Onitsuka, K. Shibata, S. Uesugi, and M. Hamada (1997) Biochemistry 36, 4027-4033.

5.     H. J. Kim, S. Nishikawa, Y. Tokutomi, H. Takenaka, M. Hamada, S. A. Kuby, and S. Uesugi (1990) Biochemistry 29, 1107-1111.

6.     M. Hamada, H. Okuda, K. Oka, T. Watanabe, K. Ueda, M. Nojima, S. A. Kuby, M. Manship, F. H. Tyler, and F.A. Ziter (1981) Biochimica Biophysica   Acta 660, 227-237.

7.     S. Miwa, H. Fujii, K. Tani, K. Takahashi, T. Takizawa, T. Igarashi (1983) Am. J. Hematol. 14, 325.

 

Figure 1.

Side chains of Lys9, Lys21, Lys27, Lys31, Lys63, Lys131, and Lys194 are depicted in a proposed model of the binding sites for AMP and MgATP in adenylate kinase.  These lysine residues could take part in a phosphoryl transfer reaction between MgATP2- and AMP2-, in cooperation with the other active site residues, such as the arginine residues.