COMPLEXES OF ESCHERICHIA-COLI ADENYLATE KINASE AND NUCLEOTIDES - H-1-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 µM 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+. © 1990, American Chemical Society. All rights reserved.