Site-directed mutagenesis of Pro-17 located in the glycine-rich region of adenylate kinase.
Proline 17 in the glycine-rich region of adenylate kinase was replaced by Gly (the Gly-mutant) or Val (the Val-mutant) by site-directed mutagenesis. The mutant enzymes were purified to homogeneous states on sodium dodecyl sulfate-gel electrophoresis after solubilization of the proteins from the pellets of cell lysates of Escherichia coli. The apparent Km values of the Gly- and the Val-mutants for AMP increased approximately 7- and 24-fold, respectively, as compared with that of the wild-type enzyme. The apparent Km values for ATP also increased 7- and 42-fold in the Gly- and Val-mutants, respectively. In contrast, Vmax values of both mutant enzymes were comparable to that of the wild-type enzyme. These results suggest that Pro-17 plays an important role for the binding of substrates, but not for catalytic efficiency, although it does not directly interact with substrates. Adenosine diphosphopyridoxal, which specifically modifies Lys-21 in adenylate kinase (Tagaya, M., Yagami, T., and Fukui, T. (1987) J. Biol. Chem. 262, 8257-8261), inactivated the wild-type and mutant enzymes at almost the same rates. Interestingly, both mutant enzymes showed higher specificities for adenine nucleotides than the wild-type enzyme. Both mutant enzymes were less resistant than the wild-type enzyme against inactivation at elevated temperatures or by treatment with trypsin. It would appear that most of the properties of the mutant enzymes may be explained on the basis of a need for conformational flexibility of the loop which includes Pro-17 for substrate binding.
Tagaya
M
M
Institute of Scientific and Industrial Research, Osaka University, Japan.
Yagami
T
T
Noumi
T
T
Futai
M
M
Kishi
F
F
Nakazawa
A
A
Fukui
T
T
D000263:Adenylate Kinase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005998:Glycine; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D010770:Phosphotransferases / Q000235:genetics*; D011392:Proline; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013552:Swine
D011994:Recombinant Proteins; D011392:Proline; D010770:Phosphotransferases; D000263:Adenylate Kinase; D005998:Glycine