Substrate-binding and catalytic roles of Lys194 in the C-terminus in human adenylate kinase by site-directed random mutagenesis.
Site-directed random mutagenesis of Lys194 residue in the C-terminus of human adenylate kinase (AK) was performed, and six mutants were analyzed by steady-state kinetics. K194-mutants variously affected the apparent Michaelis constants (K(m) values) for ATP and AMP, although the kcat values strikingly decreased. The Lys194 residue appears to interact not only with MgATP2- but also with the AMP2- substrates by salt bridge formation with a nucleotide and to play a functional role in stabilizing the phosphate-transfer during catalysis. Lys194 could be essential for substrate-holdings and in catalysis and not replaceable to the other amino acids.
Ayabe
T
T
Department of Hygiene, Miyazaki Medical College, Japan. tayabe@post.miyazaki-med.ac.jp
Takenaka
H
H
Takenaka
O
O
Onitsuka
T
T
Shibata
K
K
Uesugi
S
S
Hamada
M
M
D000263:Adenylate Kinase / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence / Q000502:physiology; D001483:Base Sequence; D001665:Binding Sites; D006801:Humans; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed
D000263:Adenylate Kinase; D008239:Lysine