Essential Lysine Residues in the N-Terminal and the C-Terminal Domain of Human Adenylate Kinase Interact with Adenine Nucleotides As Found by Site-Directed Random Mutagenesis†,¶,#

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Abstract
To elucidate the minimum requirement of amino acid residues for the active center in human adenylate kinase (hAK1), we carried out random site-directed mutagenesis of key lysine residues (K9, K21, K27, K31, K63, K131, and K194), which were conserved in mammalian AK1 species, with the pMEX8-hAK1 plasmid [Ayabe, T., et al. (1996) Biochem. Mol. Biol. Int.38, 373−381]. Twenty different mutants were obtained and analyzed by steady-state kinetics, and all mutants showed activity loss by Km and/or kcat effects on MgATP2-, AMP2-, or both. The results have led to the following conclusions. (1) Lys9 would appear to interact with both MgATP2- and AMP2- but to a larger extent than with AMP2-. (2) Lys21 is likely to play a role in substrate binding of both MgATP2- and AMP2- but more strongly affects MgATP2-. (3) Lys27 and Lys131 would appear to play a functional role in catalysis by interacting strongly with MgATP2-. (4) Lys31 would appear to interact with MgATP2- and AMP2- at the MgATP2- site. (5) Lys63 would be more likely to interact with MgATP2- than with AMP2-. (6) Lys194 in the flanking C-terminal domain would appear to interact not only with MgATP2- but also with AMP2- at the MgATP2- site by stabilizing substrate binding. The loss of the positively charged ε-amino group of lysine affects both the affinity for the substrate and the catalytic efficiency. Hence, hydrophilic lysine residues in hAK1 would appear to be essential for substrate−enzyme interaction with the coordination of some arginine residues, reported previously [Kim, H. J., et al. (1990) Biochemistry29, 1107−1111].