In vitro mutagenesis studies at the arginine residues of adenylate kinase. A revised binding site for AMP in the X-ray-deduced model.
Although X-ray crystallographic and NMR studies have been made on the adenylate kinases, the substrate-binding sites are not unequivocally established. In an attempt to shed light on the binding sites for MgATP2- and for AMP2- in human cytosolic adenylate kinase (EC 2.7.4.3, hAK1), we have investigated the enzymic effects of replacement of the arginine residues (R44, R132, R138, and R149), which had been assumed by Pai et al. [Pai, E. F., Sachsenheimer, W., Schirmer, R. H., & Schulz, G. E. (1977) J. Mol. Biol. 114, 37-45] to interact with the phosphoryl groups of AMP2- and MgATP2-. With use of the site-directed mutagenesis method, point mutations were made in the artificial gene for hAK1 [Kim, H. J., Nishikawa, S., Tanaka, T., Uesugi, S., Takenaka, H., Hamada, M., & Kuby, S. A. (1989) Protein Eng. 2, 379-386] to replace these arginine residues with alanyl residues and yield the mutants R44A hAK1, R132A hAK1, R138A hAK1, and R149A hAK1. The resulting large increases in the Km,app values for AMP2- of the mutant enzymes, the relatively small increases in the Km,app values for MgATP2-, and the fact that the R132A, R138A, and R149A mutant enzymes proved to be very poor catalysts are consistent with the idea that the assigned substrate binding sites of Pai et al. (1977) have been reversed and that their ATP-binding site may be assigned as the AMP site.
Kim
H J
HJ
Faculty of Pharmaceutical Sciences, Osaka University, Japan.
Nishikawa
S
S
Tokutomi
Y
Y
Takenaka
H
H
Hamada
M
M
Kuby
S A
SA
Uesugi
S
S
D000249:Adenosine Monophosphate / Q000378:metabolism*; D000263:Adenylate Kinase / Q000235:genetics*; D001120:Arginine / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D003600:Cytosol / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010770:Phosphotransferases / Q000235:genetics*
D000249:Adenosine Monophosphate; D001120:Arginine; D010770:Phosphotransferases; D000263:Adenylate Kinase