REGULATION OF ADENINE NUCLEOTIDE POOLS

Project: Research project

Project Details

Description

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.
StatusFinished
Effective start/end date1/1/901/1/90

Funding

  • National Institute of General Medical Sciences

ASJC

  • Genetics
  • Molecular Biology

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