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 adenylate kinase (AK) can now be deduced from the crystal structure of Escherichia coli AK.MgAP5A [P1,P5-bis(5'-adenosyl) pentaphosphate] [Müller, C. W., & Schulz, G. E. (1992) J. Mol. 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 perturbed significantly [Tian, G., Yan., H., Jiang, R.-T., Kishi, F., Nakazawa, A., & Tsai, M.-D. (1990) Biochemistry 29, 4296-4304]. We therefore undertook further structural and functional analyses of a conservative mutant K21R and a nonconservative mutant K21A. In addition to kinetic analyses, the structures of the mutants were analyzed by one- and two-dimensional proton NMR spectroscopy and (1H, 15N) heteronuclear multiple-quantum coherence (HMQC) experiments. Detailed assignments were performed in reference to the total backbone assignments of the WT AK.MgAP5A complex [Byeon, I.-J. L., Yan, H., Edison, A. S., Mooberry, E. S., Abildgaard, F., Markley, J. L., & Tsai, M.-D. (1993) Biochemistry 32, 12508-12521]. The analysis showed that the residues located near the active site (Gly15, Thr23, Arg97, Gln101, Arg128, Arg132, Asp140, Asp141, and Tyr153) exhibit greater changes in 1H-15N chemical shifts. Finally, two-dimensional 31P-31P COSY experiments were used to examine the effects of the lysine side chain on the phosphate groups in the bound AP5A. Our data have led to the following conclusions independent of the crystal structure: (i) Because the perturbations in the conformation of the mutants are not global and are mainly localized 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 kcat/Km of mutants. (ii) The results of 31P NMR analyses suggest that Lys21 functions by orienting the triphosphate chain of MgATP to a proper conformation required for catalysis. (iii) The interaction between Lys21 and the phosphate chain in turn dictates the interactions between the substrates and the active site residues. In the K21R.MgATP complex, the NH chemical shifts of many of the active site residues are perturbed. (iv) The catalytic functions of Lys21 cannot be replaced by a conservative residue arginine. In addition, since K21A and K21R behave similarly, the catalytic function of Lys21 should not be merely a charge effect.
Byeon
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Department of Chemistry, Ohio State University, Columbus 43210.
Shi
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Tsai
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D000255:Adenosine Triphosphate / Q000378:metabolism; D000263:Adenylate Kinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002645:Chickens; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D015226:Dinucleoside Phosphates / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008239:Lysine / Q000737:chemistry / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009154:Mutation; D010710:Phosphates / Q000737:chemistry; D011487:Protein Conformation
D015226:Dinucleoside Phosphates; D010710:Phosphates; C012275:P(1),P(5)-di(adenosine-5'-)pentaphosphate; D000255:Adenosine Triphosphate; D004247:DNA; D000263:Adenylate Kinase; D008239:Lysine