Structurally and catalytically important residues in the phosphate binding loop of adenylate kinase of Escherichia coli.
Amino acids in the phosphate binding loop of adenylate kinase of Escherichia coli were mutated by site-directed mutagenesis. The mutant proteins with a Pro-9----Gly (P9G) and with a Lys-13----Gln (K13Q) exchange were overexpressed and purified. They were characterized by steady-state kinetics, fluorescence binding, and structural studies, together with the phosphate binding loop mutants P9L and G10V prepared earlier [Reinstein, J., Brune, M., & Wittinghofer, A. (1988) Biochemistry 27, 4712-4720]. The results obtained show that all these mutations change the structure of the protein as evidenced by NMR spectroscopy and temperature-stability studies. All the mutant proteins have increased dissociation constants for substrates and inhibitors, but their catalytic activity, except for K13Q, is not reduced. The results obtained with K13Q suggest that this lysine residue, which is conserved in all guanine and many adenine nucleotide proteins, might have an important role in catalysis.
Reinstein
J
J
Abteilung Biophysik, Max-Planck-Institut für medizinische Forschung, Heidelberg, West Germany.
Schlichting
I
I
Wittinghofer
A
A
D000263:Adenylate Kinase / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009154:Mutation; D010710:Phosphates / Q000378:metabolism*; D011487:Protein Conformation; D013379:Substrate Specificity; D013696:Temperature; D013816:Thermodynamics; D014961:X-Ray Diffraction
D010710:Phosphates; D000263:Adenylate Kinase