MECHANISM OF ADENYLATE KINASE - STRUCTURAL AND FUNCTIONAL ROLES OF THE CONSERVED ARGININE-97 AND ARGININE-132

The structural and functional roles of two conserved active site residues, Arg-97 and Arg-132, in chicken muscle adenylate kinase (AK) were evaluated by site-directed mutagenesis in conjunction with one- and two-dimensional proton nuclear magnetic resonance (NMR), kinetics, and guanidine hydrochloride-induced denaturation. In addition, P-31 NMR analysis was used to evaluate the contribution of Arg-97 to the phosphorus stereospecificity of AK. The results and conclusions are summarized as follows: (i) Kinetic analysis of R97M reveals 6- and 28-fold increases in the dissociation constant K(i) and Michaelis constant K of AMP, respectively, and a moderate 30-fold decrease in k(cat). The K(i) and K values of MgATP are relatively unperturbed. The localized effect of AMP stabilization was independently confirmed by proton NMR titration, which showed a ca. 20-fold increase in the dissociation constant of AMP but not of MgATP. (ii) R132M affords a dramatic decrease in k(cat) by a factor of 8.0 x 10(3), With unchanged dissociation and Michaelis constants for either substrate. The lack of perturbation in the affinities toward substrates was confirmed by proton NMR titration. (iii) Although small chemical shift changes were observed for the free mutants and their complexes with substrates, further analyses by nuclear Overhauser enhanced spectroscopy with the bisubstrate analogue inhibitor, P1,P5-bis(5'-adenosyl)pentaphosphate (AP5A), indicated little perturbation in the global conformation. (iv) Contributions to conformational stability by Arg-97 and Arg- 1 3 2 are negligible on the basis of the free energy of unfolding, DELTA-G(d)H2O. (v) R97M was predicted and demonstrated to exhibit enhanced stereospecificity at the AMP site by at least 10-fold relative to WT in the conversion of adenosine 5'-monothiophosphate to adenosine 5'-(1-thiodiphosphate). This result for R97M was predicted on the basis of the orientation of Arg-97 relative to Arg-44 and AMP in the active site as observed in available crystal structures and the stereospecificity results of R44M [Jiang, R.-T., Dahnke, T., & Tsai, M.-D. (1991) J. Am. Chem. Soc. 113, 5485-54861. (vi) The above structural and functional analyses led us to conclude that Arg-97 interacts with the phosphoryl group of AMP, beginning at the binary complex (1-2 kcal/mol), continuing through the transition state (3.5 kcal/mol), and that Arg-132 stabilizes the transition state by greater than 5 kcal/mol. (vii) The functional importance of Arg-97 appears to be similar to that of Arg-44 [Yan, H., Dahnke, T., Zhou, B., Nakazawa, A., & Tsai, M.-D. (1990) Biochemistry 29, 10956-10964]. The results for R97M also clarify conflicting reports from analogous mutants in other types of AK and support conclusions based upon analysis of the mitochondrial matrix AK.AMP crystal structure [Diederichs, K., & Schulz, G. E. (1991) J. Mol. Biol. 217, 541-549].