Mechanism of adenylate kinase. Demonstration of a functional relationship between aspartate 93 and Mg2+ by site-directed mutagenesis and proton, phosphorus-31, and magnesium-25 NMR.
Earlier magnetic resonance studies suggested no direct interaction between Mg2+ ions and adenylate kinase (AK) in the AK.MgATP (adenosine 5'-triphosphate) complex. However, recent NMR studies concluded that the carboxylate of aspartate 119 accepts a hydrogen bond from a water ligand of the bound Mg2+ ion in the muscle AK.MgATP complex [Fry, D.C., Kuby, S.A., & Mildvan, A.S. (1985) Biochemistry 24, 4680-4694]. On the other hand, in the 2.6-A crystal structure of the yeast AK.MgAP5A [P1,P5-bis(5'-adenosyl)pentaphosphate] complex, the Mg2+ ion is in proximity to aspartate 93 [Egner, U., Tomasselli, A.G., & Schulz, G.E. (1987) J. Mol. Biol. 195, 649-658]. Substitution of Asp-93 with alanine resulted in no change in dissociation constants, 4-fold increases in Km, and a 650-fold decrease in kcat. Notable changes have been observed in the chemical shifts of the aromatic protons of histidine 36 and a few other aromatic residues. However, the results of detailed analyses of the free enzymes and the AK.MgAP5A complexes by one- and two-dimensional NMR suggested that the changes are due to localized perturbations. Thus it is concluded that Asp-93 stabilizes the transition state by ca. 3.9 kcal/mol. The next question is how. Since proton NMR results indicated that binding of Mg2+ to the AK.AP5A complex induces some changes in the proton NMR signals of WT but not those of D93A, the functional role of Asp-93 should be in binding to Mg2+.(ABSTRACT TRUNCATED AT 250 WORDS)
Yan
H G
HG
Department of Chemistry, Ohio State University, Columbus 43210.
Tsai
M D
MD
D000263:Adenylate Kinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000409:Alanine / Q000235:genetics; D000818:Animals; D001224:Aspartic Acid / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D002645:Chickens; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D010759:Phosphorus Isotopes; D011487:Protein Conformation; D014961:X-Ray Diffraction
D010759:Phosphorus Isotopes; D001224:Aspartic Acid; D000263:Adenylate Kinase; D008274:Magnesium; D000409:Alanine