REGULATION OF ADENINE NUCLEOTIDE POOLS

The pathway of AMP degradation in prokaryotic organisms involves hydrolysis of the N-glycosidic bond of AMP by AMP nucleosidase. Regulation of this enzyme in vivo appears to occur by allosteric MgATP activation, Pi inhibition and a Pi induced dissociation. The pathway for AMP degradation in eukaryotic organisms, involves AMP deaminase. AMP deaminase from yeast is also under allosteric regulation by activation with ATP (and MgATP) and by inhibition with Pi. No prokaryotes have been found to contain AMP deaminase and no eukaryotes have been found to contain AMP nucleosidase. The similarity of the proposed metabolic function and the allosteric regulation of these two enzymes suggest that AMP deaminase may have evolved from AMP nucleosidase. One goal of this project is to complete studies of the regulation and the metabolic role of AMP degradation. Magnetic resonance studies will be used to quantitate the ligands bound to Mn++ in the MnATP-AMP nucleosidase complex. Studies of the mechanism of AMP nucleosidase will be completed with the enzyme from Azotobacter vinelandii. Metabolic studies will use E. coli mutants with deficiencies in the enzymes of adenylate degradation. Special emphasis will be placed on the regulation and role of AMP nucleosidase in in vivo experiments. The metabolic consequences of adenylate regulation will be tested in mutants deficient in this pathway. Another goal of this research is to determine the primary sequence of AMP nucleosidase and AMP deaminase to gain structural information on these regulatory enzymes and to test for possible regions of sequence homology. The structure will be determined by sequencing the structural genes which are to be cloned as plasmids in E. coli K12. The structural gene for AMP nucleosidase will be amplified in E. coli to create mutants which overproduce the enzyme.