Complexes of Escherichia coli adenylate kinase and nucleotides: 1H NMR studies of the nucleotide sites in solution.
One- and two-dimensional nuclear magnetic resonance (NMR) studies, in particular substrate--protein nuclear Overhauser effect (NOESY) measurements, as well as nucleotide and P1,P5-bis-(5'-adenosyl) pentaphosphate (AP5A) titrations and studies of the temperature-dependent unfolding of the tertiary structure of Escherichia coli adenylate kinase (AKEC) were performed. These experiments and comparison with the same type of experiments performed with the porcine enzyme [Rösch, P., Klaus, W., Auer, M., & Goody, R. S. (1989) Biochemistry 28, 4318-4325] led us to the following conclusions: (1) At pH 8 and concentrations of approximately 2.5-3 mM, AKEC is partially unfolded at 318 K. (2) ATP.Mg2+ binds to the ATP site with a dissociation constant of approximately 40 microM under the assumption that ATP binds to one nucleotide site only. (3) AP5A.Mg2+ binds to both nucleotide sites and thus simulates the active complex. (4) The ATP.Mg2+ adenine in the AKEC.AP5A.Mg2+ complex is located close to His134 and Phe19. (5) The AKEC "G-loop" with bound ATP.Mg2+ is structurally highly homologous to the loop region in the oncogene product p21 with bound GTP.Mg2+.
Vetter
I R
IR
Department of Biophysics, Max Planck Institute for Medical Research, Heidelberg, FRG.
Reinstein
J
J
Rösch
P
P
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000263:Adenylate Kinase / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D006358:Hot Temperature; D006859:Hydrogen; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D011487:Protein Conformation; D011489:Protein Denaturation; D012996:Solutions; D013552:Swine; D014961:X-Ray Diffraction
D012996:Solutions; D006859:Hydrogen; D000255:Adenosine Triphosphate; D000263:Adenylate Kinase