MECHANISM OF ADENYLATE KINASE - DEMONSTRATION OF A FUNCTIONAL-RELATIONSHIP BETWEEN ASPARTATE-93 AND MG2+ BY SITE-DIRECTED MUTAGENESIS AND PROTON, P-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-angstrom 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 K(m), and a 650-fold decrease in k(cat). 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+. We then asked whether disruption of the interaction between Mg2+ and Asp-93 affects the interaction between Mg2+ and the nucleotide at the active site of AK. Analysis by P-31 NMR suggested that Mg2+ orients the conformation of the polyphosphate chain of bound AP5A in WT but not in D93A. These results raised the question of whether Mg2+ could bind to D93A.nucleotide complexes, which was then probed by Mg-25 NMR. The results suggest that Mg2+ does bind to the D93A.AP5A complex, but possibly only weakly. The weaker affinity of Mg2+ was also confirmed by kinetic analysis. However, the low activity of D93A cannot be restored by higher concentrations of Mg2+ ions. Thus Asp-93 plays critical roles in the binding and function of Mg2+ ions during the catalysis by AK. Substitution of Asp-93 with alanine also lowered the pK(a) of His-36, which supports the proximity between Asp-93 and His-36. However, disruption of the interaction between Asp-93 and His-36 did not affect the conformational stability of the enzyme.