| Budget Amount *help |
¥6,600,000 (Direct Cost: ¥6,600,000)
Fiscal Year 1989: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1988: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1987: ¥5,000,000 (Direct Cost: ¥5,000,000)
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| Research Abstract |
Although extensive studies including X-ray crystallographic and NMR studies have been made on adenylate kinases, the substrate binding sites are still not unequivocally established.
In an attempt to identify the binding sites for MgATP^<2-> and for AMP^<2-> in human ncytosolic nadenylate kinase (EC 2.7.4.3, hAK1), we have investigated the functions of arginine residues (R44, R132, R138 and R149) which are assumed to interact with the nphosphoryl groups of AMP^<2-> and MgATP^<2->. Using the site-directed mutagenesis method, point mutations were made in the artificial gene for hAK1 that we previously nsynthesized (Kim et al.,(1989) Protein Eng. 2, 379-3861 by replacing these arginines with nalanines to create the mutants, R44A hAK1, R132A hAK1, R138A hAK1 and R149A hAK1. The increase in K_m values of the mutant enzymes for AMP^<2-> was in the following order; R44A (44-fold), R138A (21-fold), R149A (18-fold) , R132A (13-fold) . That for MgATP^<2-> was in the order; R132A (18-fold), R149A (8-fold), R44A (4-fold), R138A (1.3-fold).
Moreover, mutants R132A, R138A and R149A were very inefficient in catalysis. These results strongly suggest that Arg44 is important in maintaining the proper geometry of the AMP binding pocket and Argl38 is a catalytically indispensable residue which makes contacts with the nphosphoryl group of AMP. This finding leads us to the conclusion that the binding site for AMP is the one previously assigned as ATP binding site by Pai et al.[(1977) J. Mol. Biol. 114.37-45).
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