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), threonine residues in the N-terminal region (T35 and T39), and hydrophobic residues in the C-terminal ƒż-helix (V182, V186, C187, L190, and L193), which were conserved in mammalian AK1 species, with the pMEX8-hAK1 plasmid (Ayabe et al., 1996, Biochem. Mol. Biol. Int. 38, pp. 373-381).@ More than forty different mutants were obtained and analyzed by steady-state kinetics, and all mutants showed activity loss by Km and/or kcat effects on one or both of MgATP2- and AMP2-.@ The results have led to the following conclusions.@ The loss of the positively charged ƒĂ-amino group of lysine affects both the 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 some arginine residues, as reported previously (Kim et al., 1990, Biochemistry 29, pp. 1107-1111).@ The Thr39 residue appears to play an important role in the binding of AMP2- and to be essential for catalysis.@ The hydroxyl group of the threonine residue in human AK appears to be important.@ The change in the hydrophobic residues in the C-terminal ƒż-helix affects the affinity for substrates (Km), that is, not for MgATP2- but for AMP2-, and the catalytic efficiency (kcat).@ The activity of all mutants decreased due to changing substrate-affinity.@ The nearer the residue is, located to the C-terminal end, the mutation affects not only MgATP2- but also AMP2- substrate binding.@ The hydrophobic alterations have disrupted the hydrophobic interactions with substrates, which might have destabilized the conformation of the active site.@ The C-terminal ƒż-helix of human adenylate kinase appears to be essential for playing a functional role in catalysis by interacting with adenine substrates.