The pathway of AMP degradation in prokaryotic organisms involves AMP nucleosidases, which are N-glycohydrolases. AMP nucleosidases exhibit allosteric activation by MgATP and inhibition by Pi. The pathway for AMP degradation in eukaryotes involves AMP deaminase. Regulation of AMP deaminase is similar to AMP nucleosidase by allosteric activation with ALP and inhibition by Pi. No prokaryotes have been found to contain AMP nucleosidase. The purpose of this proposal is to establish structure, catalytic mechanism, allosteric regulatory mechanism and genetic regulation for enzymes in this family of AMP degrading enzymes. The catalytic and allosteric mechanisms of AMP nucleosidase and AMP deaminase will be investigated by heavy-atom kinetic isotope effects in the presence and absence of allosteric activators. Transition state structures can be established for both of these enzymes. The role of specific amino acids in stabilizing the transition state will be investigated by determination of transition state structure in site- directed enzyme mutants. The amino acid sequences of E. coli AMP nucleosidase and yeast AMP deaminase have been established from the deoxynucleotide sequence of the genomic DNA. X-ray crystal structure will be determined for AMP nucleosidase and will be initiated for yeast AMP deaminase. Crystal structure and chemical modification will e used to establish the amino acids involved in catalysis and in allosteric regulation. The role of enzyme-bound zinc in AMP deaminase will be investigated by EPR studies in which Zn(II) is replaced by Mn(II). Expression of AMP nucleosidase in E. coli is regulated by cAMP and inorganic phosphate levels. The promoter region contains overlapping consensus sequences for phosphate regulatory protein and cAMP receptor protein. Experiments are designed to isolate the phosphate regulatory protein and to quantitate its interaction with phosphate and promoter region of the AMP nucleosidase gene. Competition studies with cyclic receptor protein will establish the nature of the overlapping regulatory regions.
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