MECHANISM OF ADENYLATE KINASE - THE ESSENTIAL LYSINE HELPS TO ORIENT THE PHOSPHATES AND THE ACTIVE-SITE RESIDUES TO PROPER CONFORMATIONS

Although how Lys21 interacts with the substrate MgATP of muscle adenyl ate kinase (AK) can now be deduced from the crystal structure of Esche richia coli AK . MgAP5A [P-1,P-5-bis(5'-adenosyl) pentaphosphate] [Mul ler, C. W., and Schulz, G. E. (1992) J. Mel. Biol. 224, 159-177], its contribution to catalysis has not yet been demonstrated by functional studies since the proton NMR of the K21M mutant was shown to be pertur bed significantly [Tian, G., Yan, H., Jiang, R.-T., Kishi, F., Nakazaw a, A., and Tsai, M.-D. (1990) Biochemistry 29, 4296-4304]. We therefor e undertook further structural and functional analyses of a conservati ve mutant K21R and a nonconservative mutant K21A. In addition to kinet ic analyses, the structures of the mutants were analyzed by one- and t wo-dimensional proton NMR spectroscopy and {H-1,N-15} heteronuclear mu ltiple-quantum coherence (HMQC) experiments. Detailed assignments were performed in reference to the total backbone assignments of the WT AK . MgAP(5)A complex [Byeon, I.-J. L., Yan, H., Edison, A. S., Mooberry , E. S., Abildgaard, F., Markley, J. L., and Tsai, M.-D. (1993) Bioche mistry 32, 12508-12521]. The analysis showed that the residues located near the active site (Gly15, Thr23, Arg97, Gln101, Arg128, Arg132, As p140, Asp141, and Tyr153) exhibit greater changes in H-1-N-15 chemical shifts. Finally, two-dimensional P-31-P-31 COSY experiments were used to examine the effects of the lysine side chain on the phosphate grou ps in the bound AP(5)A. Our data have led to the following conclusions independent of the crystal structure: (i) Because the perturbations i n the conformation of the mutants are not global and are mainly locali zed at active site residues and Tyr153, the side chain of Lys21 can be concluded to stabilize the transition state in the catalysis of AK by up to 7 kcal/mol on the basis of the 10(5)-fold decreases in the k(ca t)/K-m of mutants. (ii) The results of P-31 NMR analyses suggest that Lys21 functions by orienting the triphosphate chain of MgATP to a prop er conformation required for catalysis. (iii) The interaction between Lys21 and the phosphate chain in turn dictates the interactions betwee n the substrates and the active site residues. In the K21R . MgATP com plex, the NH chemical shifts of many of the active site residues are p erturbed. (iv) The catalytic functions of Lys21 cannot be replaced by a conservative residue arginine. In addition, since K21A and K21R beha ve similarly, the catalytic function of Lys21 should not be merely a c harge effect.