NMR studies of active -site structures of adenylate kinase

Yan Lin, Purdue University

Abstract

Adenylate kinase (M.W. 20 ∼ 24 kDa) catalyzes the reaction: AMP+M(II)ATP [special characters omitted]ADP+M(II)ADP, where M(II) is an obligatory divalent cation, Mg(II) in vivo, and may be substituted by Mn(II) and Co(II) in vitro . The AMP-site on this enzyme specifically binds cation-free AMP and ADP, and the ATP-site binds ATP(ADP) as well as GTP(GDP) with or without the cation. The nucleotide conformations in of E. coli adenylate kinase (AKe)-bound complexes, viz. AKe•M(II)ATP and AKe•AMP•M(II)GDP, have been determined by two types of NMR measurements: (i) 2D TRNOESY, which give interproton distances allowing the determination of the conformation of the adenosine moiety, and (ii) 31P and 13C paramagnetic relaxation measurements, which yield Co(II)-31P distances as well as Mn(II)-13C distances (by using [ul- 13C] nucleotides). The active-site conformation was established by combining these results using molecular modeling methods. Attempts were made to dock the NMR-determined enzyme-bound nucleotide structures with the protein structures obtained by the x-ray crystallography. The following conclusions were reached: (1) The nucleotide conformations determined by NMR methods are significantly different from those obtained by x-ray crystallography; (2) AMPPNP, an ATP analogue, binds primarily to the Mg(II)ATP site of AKe with or without the presence of metal; (3) The binding of AMP to the Mg(II)ATP site of AKe can be inhibited by Mg(II)GDP; (4) The glycosidic orientation of Mg(II)ATP, AMP, AMPPNP, and Mg(II)AMPPNP in AKe-bound complexes corroborates the narrow range of the glycosidic: torsion angles found for other ATP-utilizing enzymes previously studied, which is suggestive that a common motif for substrate binding with respect to the adenosine moiety may exist similarly to the G-loop for the triphosphates of ATP and GTP; (5) The comparison of the docked structure of the reaction complex, AKe•M(II)ATP•AMP, with the x-ray structure of AKe•AP5A, which mimics the reaction complex, shed light on the roles of a group of arginine residues surrounding the reacting phosphates. Arg 36 and Arg 156 directly facilitate the transfer of the γ-phosphate of ATP, while other argines, such as Arg 123, Arg 88, and Arg 167 are responsible for stabilization.

Degree

Ph.D.

Advisors

Rao, Purdue University.

Subject Area

Chemistry|Biophysics

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