| Research 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 mutagensis 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 et al., 1996, Biochem.Mol.Biol.Int.38, pp.373-381). Twenty different mutants were obtained and analyzed by steady-state kinetics, and all mutants showed activity loss by K_m and/or k_<cal> effects on one or both of MgATP^<2-> and AMP^<2->. The results have led to the following conclusions. (1) Lys9 would appear to interact with both MgATP^<2-> and AMP^<2-> but to a larger extent than with AMP^<2->. (2) Lys21 is likely to play a role in substrate-binding of both MgATP^<2-> and AMP^<2-> but more strongly affects MgATP^<2->. (3) Lys27 and Lys131 would appear to paly a functional role in catalysis by interacting strongly with MgATP^<2->. (4) Lys31 would appear to interact with MgATP^<2-> and AMP^<2-> at the MgATP^<2-> site. (5) Lys63 would be more likely to interact with MgATP^<2-> than with AMP^<2->. (6) Lys194 in the flanking C-terminal domain would appear to interact not only with MgATP^<2-> but also with AMP^<2-> at the MgATP^<2-> site by stabilizing substrate-binding. The loss of the positively charged epsilon-amino group of lysine affects both affinity for the substrate and catalytic efficiency. Hence, hydrophilic lysine residues in hAK1 would appear to be essential for substrate-enzyme interaction with the coordination of the some arginine residues, reported previously (Kim et al., 1990, Biochemistry 29, pp.1107-1111).
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