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.
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 k(cat) 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 epsilon-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) Biochemistry 29, 1107-1111].
Ayabe
T
T
Department of Hygiene, Miyazaki Medical College, Japan. tayabe@post.miyazaki-med.ac.jp
Takenaka
H
H
Takenaka
O
O
Sumida
M
M
Maruyama
H
H
Onitsuka
T
T
Shibata
K
K
Uesugi
S
S
Hamada
M
M
D000227:Adenine Nucleotides / Q000378:metabolism*; D000263:Adenylate Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D017931:DNA Primers; D006801:Humans; D007700:Kinetics; D008239:Lysine / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation
D000227:Adenine Nucleotides; D017931:DNA Primers; D000263:Adenylate Kinase; D008239:Lysine