Site-directed mutagenesis and steady-state kinetic analysis of mutantenzymes of human adenylate kinase

摘要

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 AMP(2-) compared to that for the wild-type enzyme, and these residues appeared to interact with AMP(2-). V182G showed an increased K-m value (7.4-fold) for MgATP(2-). 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 AMP(2-) binding.