Structural Characterization of Adenine Nucleotides Bound to Escherichia coli Adenylate Kinase. 2. 31P and 13C Relaxation Measurements in the Presence of Cobalt(II) and Manganese(II)

• Published in: Biochemistry (Easton) 2000-04, Vol.39 (13), p.3647-3655
• Main Authors: Lin, Yan, Nageswara Rao, B. D
• Format: Article
• Language: English
• Subjects: Adenine Nucleotides - chemistry; Adenosine - chemistry; Adenosine Monophosphate - chemistry; Adenosine Triphosphate - chemistry; Adenylate Kinase - chemistry; Adenylyl Imidodiphosphate - chemistry; Binding Sites; Carbon Isotopes; Cations, Divalent - chemistry; Cobalt - chemistry; Escherichia coli - enzymology; Manganese - chemistry; Models, Molecular; Nuclear Magnetic Resonance, Biomolecular - methods; Nucleic Acid Conformation; Phosphorus Isotopes
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi992460e

13C spin−lattice relaxation rates have been measured for two complexes of Escherichia coli adenylate kinase (AKe), viz., AKe·[U-13C]ATP and AKe·[U-13C]AMP·GDP in the presence of the substituent activating paramagnetic cation Mn(II) for the purpose of determination of the enzyme-bound conformations of ATP and AMP. (GDP has been added to the AMP complex with the enzyme in order to hold the cation in the bound complex.) Measurements of relaxation times at three different 13C frequencies, 181.0, 125.7, and 75.4 MHz, indicate that the relaxation times in the enzyme−nucleotide complexes with the paramagnetic cation are not exchange-limited; i.e., they are larger than the effective lifetimes of cation binding to these complexes and are, therefore, dependent on the cation−13C distances. An analysis of the frequency-dependent relaxation data allowed all of the ten Mn(II)−13C distances to be determined in each of the complexes. Similar measurements of the 31P relaxation rate made on AKe·ATP and AKe·AMP·GDP complexes in the presence of Co(II) as the activating cation yielded Co(II)−31P distances for each adenine nucleotide. These distances, together with the interproton distances determined previously from TRNOESY experiments [Lin, Y., and Nageswara Rao, B. D. (2000) Biochemistry 39, 3636−3646], led to a complete characterization of both ATP and AMP conformations in AKe-bound complexes. These conformations differ significantly from the nucleotide conformations in crystals of AKe·AP5A and AKe·AMP·AMPPNP as determined by X-ray crystallography.