ESSENTIAL LYSINE RESIDUES IN THE N-TERMINAL AND THE C-TERMINAL DOMAINOF HUMAN ADENYLATE KINASE INTERACT WITH ADENINE-NUCLEOTIDES AS FOUND BY SITE-DIRECTED RANDOM MUTAGENESIS

To elucidate the minimum requirement of amino acid residues for the ac tive center in human adenylate kinase (hAK1), we carried out random si te-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. Mel. Biol. Int. 38, 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(cat) effects on MgATP(2-), AMP(2-), or both. The results hav e 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 Mg ATP(2-) and AMP(2-) but more strongly affects MgATP(2-). (3) Lys27 and Lys131 would appear to play a functional role in catalysis by interac ting 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 l ikely to interact with MgATP(2-) than with AMP(2-). (6) Lys194 in the flanking C-terminal domain would appear to interact not only with MgAT P(2-) but also with AMP(2-) at the MgATP(2-) site by stabilizing subst rate binding. The loss of the positively charged E-amino group of lysi ne affects both the affinity for the substrate and the catalytic effic iency. Hence, hydrophilic lysine residues in hAK1 would appear to be e ssential for substrate-enzyme interaction with the coordination of som e arginine residues, reported previously [Kim, H. J., et al. (1990) Bi ochemistry 29, 1107-1111].