Site-directed mutagenesis and steady-state kinetic analysis of mutant enzymes of human adenylate kinase.
Site-directed mutagenesis of human adenylate kinase (AK) was carried out on residues His36, Lys55, and the C-terminal segment (Val182, V186, and Leu193). Five mutants [(H36T, K55G, V182G, V186S, and L193Stop (deletion of residues 193-194)] were generated and analyzed by steady-state kinetics. H36T, K55G, and L193Stop mutants showed an increase of K(m) values (19.8-, 19.7-, and 11.3-fold) for AMP2- compared to that for the wild-type enzyme, and these residues appeared to interact with AMP2-. V182G showed an increased K(m) value (7.4-fold) for MgATP2-. Therefore, V182 may be essential for interaction with MgATP2-. V186S increased the K(m) value (7.0- and 7.5-fold) for MgATP2- and AMP2-. V186 may thus interact with both substrates. The C-terminal domain of AK appears to be essential for MgATP2- and AMP2- binding.
Ayabe
T
T
Department of Hygiene, Miyazaki Medical College, Japan.
Park
S K
SK
Nagahama
H
H
Maruyama
H
H
Sumida
M
M
Takenaka
H
H
Takenaka
O
O
Onitsuka
T
T
Hamada
M
M
D000263:Adenylate Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D018360:Crystallography, X-Ray; D017931:DNA Primers; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation
D017931:DNA Primers; D000263:Adenylate Kinase