| Research Abstract |
Previously, we have designed and synthesized a new type of affinity labeling reagents specific for the lysyl residue located in the nucleotide- or sugar nucleotide-binding site in proteins, and also applied site-directed mutagenesis for amino acid residues in the glycine-rich region. Under the present title of project, we have obtained the following results.
1) By affinity labeling of potato tuber UDPG pyrophosphorylase with reagents having different structures, we have identified five lysyl residues (Lys-263, Lys-329, Lys-367, Lys-409, and Lys-410) located around the UDPG molecule at the active site of the enzyme. Of these lysyl residues, Lys-367 may participate directly in the catalytic function, whereas Lys-329 and Lys-263 play the roles, respectively, in the binding of PPi and in the conformational changes upon binding of UDPG to the enzyme.
2) We have prepared muatant enzymes of chicken skeletal muscle adenylate kinase in which Lys-66 is replaced by various other amino acids. Based on the properties of those mutant enzymes, we provide evidence for the role of Lys-66 in the recognition of the adenine ring of the substrate AMP.
3) In the chemical modification of leucine dehydrogenase of Bacillus stearothermophilus, we obtained the results suggesting the participation of Lys-80 in the catalytic function of this enzyme.
4) The catalytic site of Escherichia coli F1-ATPase was probed using areactive ATP analogue, adenosine triphosphopyridoxal. In the absence of Mg^<2+>, the alphaLys-201 and betaLys-155 residues were the major target, whereas, in the presence of the ion, predominant labeling was observed in the betaLys-155 and betaLys-211 residues. The results suggest that those three residues are located close together near the gamma-phosphate group of ATP bound to the catalytic site, and that the two beta residues and the gamma-phosphate group become closer to each other in the presence of Mg^<2+>.
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