MECHANISM OF ADENYLATE KINASE - H-1, C-13, AND N-15 NMR ASSIGNMENTS, SECONDARY STRUCTURES, AND SUBSTRATE-BINDING SITES

Backbone H-1, C-13, and N-15 NMR assignments were obtained for the com plex of chicken muscle adenylate kinase (AK) with its bisubstrate anal og, MgAP5A [magnesium P1,P5-bis(5'-adenosyl)-pentaphosphate]. The assi gnments were used to elucidate the-secondary structures and the enzyme -MgAP5A interactions. The work involves two unusual features: the mole cular weight of AK(21.6 kDa) is one of the largest, on a monomeric bas is, for which nearly complete assignment has been reported to date, an d the assignment was performed at pH 7.1 instead of the acidic pH used for most other proteins. The results are summarized as follows. First ly, unambiguous sequential assignments of backbone resonances have bee n achieved effectively by the combined use of two sequential assignmen t methods: NOE-directed assignments and the recently developed 1J-coup ling-directed assignments. The starting points of the assignments were provided by several specifically labeled enzyme samples. Over 90% of the backbone H-1, C-13, and N-15 resonances have been assigned. Second ly, spin system information was obtained from the HCCH-TOCSY and HCCH- COSY experiments as well as from 2D homonuclear NMR data. Overall, the side-chain resonances of ca. 40% of the residues, including most of t he those displaying NOEs with the adenosine moieties of MgAP5A, have b een assigned. Thirdly, secondary structural elements in the AK-MgAP5A complex were identified by extensive analyses of H-1-N-15 2D HMQC-NOES Y and 3D NOESY-HMQC spectra. Overall, the enzyme consists of ca. 60% a lpha-helices and a five-stranded parallel beta-sheet. The results are compared with the secondary structure of the free AK from porcine musc le in crystals [Dreusicke, D., Karplus, P. A., & Schulz, G. E. (1988) J. Mol. Biol. 199, 359-371]. Lastly, most of the intermolecular NOEs b etween AK and the adenosine moieties of MgAP5A have been identified: T hr39, Leu43, Gly64, Leu66, Val67, Val72, and Gln101 are in proximity t o the adenosine moiety of the adenosine 5'-monophosphate site, whereas Thr23 is in proximity to that of the adenosine 5'-triphosphate site. These data are discussed in relation to previous results from site-dir ected mutagenesis, NMR, and X-ray studies and in relation to the mecha nism of catalysis.