The objective of this proposal is to study the quantitative structure- function relationship of adenylate kinase (AK) by a combination of genetic, biochemical, bioorganic, and biophysical techniques. The system will be the AK from chicken muscle expressed in E. coli.
The Specific Aim A is to continue the """"""""iterative structure-function studies"""""""" to further identify catalytic residues and define their functions. The major areas of emphasis are: (a) interactions with the adenosine moiety of MgATP; (b) interactions with the adenosine moiety of AMP; (c) complementary substrate mutagenesis; (d) creating kinases with broader substrate specificity; (e) interactions with the phosphates; (f) metal ion specificity; and (g) correlation of ATPase activity and conformational changes.
The Specific Aim B is to manipulate the phosphorus stereospecificity of AK. The detailed interactions between the active site residues and the phosphate groups of the substrates, and their contribution to catalysis, will be probed by using various isomers of phosphorothioates. A significant perturbation in the stereospecificity, if observed, is evidence for direct interaction between the mutated residue and the phosphate site. On the other hand, knowledge of the role of the mutated residue can lead to predictions of changes in the phosphorus stereospecificity.
Specific Aim C involves total NMR assignment and structural determination of free AK, AK-AMP, AK- MgAMPPCP, AK-AMP-MgAMPPCP, and AK-MgAP5A, using enzymes uniformly labeled with 15N and/or 13C. The main purposes are to support structure-function studies in Specific Aims A and B, and to provide a detailed molecular description of conformational changes during the catalytic cycle.
The Specific Aim D is to develop the use of unnatural amino acids for structure-function studies of AK. This approach will be used to complement the structure-function studies in Specific Aims A and B, with a particular emphasis on the structural and functional roles of the phosphate-binding loop (P-loop). The P-loop residues likely to be H-bonded to the phosphate moieties of substrates via the backbone NH (Gly-16, 18, 20, and 22) will be changed to the corresponding N-methyl glycine. In addition, other possible backbone interactions with the adenosine moieties will be probed accordingly. Most importantly, the aromatic ring of Tyr-95 (which is in proximity to the adenosine moiety of AMP) will be substituted with cyclohexadiene or cyclopentene to probe the structural and functional importance of the aromaticity.
