Abstract

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.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
7R01GM021083-13
Application #
3270240
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1987-08-01
Project End
1988-11-30
Budget Start
1987-08-01
Budget End
1988-11-30
Support Year
13
Fiscal Year
1987
Total Cost
Indirect Cost

  Institution

Name
Albert Einstein College of Medicine
Department
Type
Schools of Medicine
DUNS #
009095365
City
Bronx
State
NY
Country
United States
Zip Code
10461

  Publications

Kline, P C; Schramm, V L (1994) Electrostatic potential surfaces of the transition state for AMP deaminase and for (R)-coformycin, a transition state inhibitor. J Biol Chem 269:22385-90
Ehrlich, J I; Schramm, V L (1994) Electrostatic potential surface analysis of the transition state for AMP nucleosidase and for formycin 5'-phosphate, a transition-state inhibitor. Biochemistry 33:8890-6
Schramm, V L; Horenstein, B A; Kline, P C (1994) Transition state analysis and inhibitor design for enzymatic reactions. J Biol Chem 269:18259-62
Sollitti, P; Merkler, D J; Estupinan, B et al. (1993) Yeast AMP deaminase. Catalytic activity in Schizosaccharomyces pombe and chromosomal location in Saccharomyces cerevisiae. J Biol Chem 268:4549-55
Merkler, D J; Schramm, V L (1993) Catalytic mechanism of yeast adenosine 5'-monophosphate deaminase. Zinc content, substrate specificity, pH studies, and solvent isotope effects. Biochemistry 32:5792-9
Merkler, D J; Kline, P C; Weiss, P et al. (1993) Transition-state analysis of AMP deaminase. Biochemistry 32:12993-3001
Horenstein, B A; Schramm, V L (1993) Correlation of the molecular electrostatic potential surface of an enzymatic transition state with novel transition-state inhibitors. Biochemistry 32:9917-25
Horenstein, B A; Schramm, V L (1993) Electronic nature of the transition state for nucleoside hydrolase. A blueprint for inhibitor design. Biochemistry 32:7089-97
Kline, P C; Schramm, V L (1992) Purine nucleoside phosphorylase. Inosine hydrolysis, tight binding of the hypoxanthine intermediate, and third-the-sites reactivity. Biochemistry 31:5964-73
Horenstein, B A; Parkin, D W; Estupinan, B et al. (1991) Transition-state analysis of nucleoside hydrolase from Crithidia fasciculata. Biochemistry 30:10788-95

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