ADP-ribosyl transferases are an important class of regulatory enzymes. This project proposes the design and affinity labels specific for the NAD substrate binding site of ADP-ribosyl transferases. The information sought is the identification of catalytically essential amino acid residues and elucidation of active site structure. Identification of essential amino acid residues and elucidation of active site structure. Identification of essential amino acid residues will advance the understanding of the mechanism of these unique catalysts. This information will support the development of genetically engineered vaccines against pertussis and related diseases where a microbial ADP- ribosyl transferase functions as an exotoxin component. Affinity and photoaffinity labels will be applied to the study of the mammalian NAD glycohydrolase as an example of a hydrolytic transferase and to the catalytically active . A subunits of cholera toxin, pertussis toxin, and diphtheria toxin. These toxins are medically important and are significant as representatives of the class of mono (ADP-ribosyl) transferases which has been implicated as a new cell regulatory mechanism. Neither affinity labels nor photoaffinity labels specific for the NAD substrate binding site and generally applicable to enzymes in the class are currently available. All such enzymes exhibit significant NAD glycohydrolase activity. Successful labels must therefore be noncleavable NAD analogues. The first approach to the production of affinity labels and photoaffinity labels is therefore made possible by our development of the non-cleavable analogues. The first approach to the production of affinity labels and photoaffinity labels is therefore made possible by our development of the non-cleavable analogue carba-NAD. Here a cyclopentane ring replaces the beta-D-ribotide of the nicotinamide riboside moiety of NAD. Carba-NAD has been demonstrated to be resistant to ADP-ribosyl transfer and to be an effective competitive inhibitor of NAD glycohydrolase and several mono (ADP-ribosyl) transferases. A family of related affinity labels and photoaffinity labels will be produced by modification of carba-NAD and applied to the study of the aforementioned enzymes. A second strategy for inhibitor design will utilize amino sugars as """"""""transition-state analogues"""""""" to mimic the oxocarbonium ion intermediate proposed for the mammaliam NAD glycohydrolase. A synthesis of 1,4- dideoxy-4-amino-D-ribofuranose is available and incorporation of this amino sugar into an ADP-ribose analogue will readily be accomplished. The """"""""transition-state analogue"""""""" will be tested as an inhibitor of NAD- glycohydrolase and the microbial toxins.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM032821-04A3
Application #
3281960
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1983-12-01
Project End
1992-06-30
Budget Start
1989-07-01
Budget End
1990-06-30
Support Year
4
Fiscal Year
1989
Total Cost
Indirect Cost
Name
University of Texas Health Science Center San Antonio
Department
Type
Overall Medical
DUNS #
800772162
City
San Antonio
State
TX
Country
United States
Zip Code
78229
Slama, J T; Aboul-Ela, N; Goli, D M et al. (1995) Specific inhibition of poly(ADP-ribose) glycohydrolase by adenosine diphosphate (hydroxymethyl)pyrrolidinediol. J Med Chem 38:389-93
Slama, J T; Aboul-Ela, N; Jacobson, M K (1995) Mechanism of inhibition of poly(ADP-ribose) glycohydrolase by adenosine diphosphate (hydroxymethyl)pyrrolidinediol. J Med Chem 38:4332-6
Goli, D M; Cheesman, B V; Hassan, M E et al. (1994) Synthesis of (2R,3R,4S)-2-hydroxymethylpyrrolidine-3,4-diol from (2S)-3,4-dehydroproline derivatives. Carbohydr Res 259:219-41
Slama, J T; Simmons, A M; Hernandez, T M et al. (1993) Synthesis of [35S]thiophosphoryl adenylic acid, utilizing a general procedure for [35S]thiophosphoryl chloride production. Anal Biochem 209:143-9
Slama, J T; Simmons, A M (1991) Synthesis and properties of photoaffinity labels for the pyridine dinucleotide binding site of NAD glycohydrolase. Biochemistry 30:2527-34
Slama, J T; Simmons, A M (1989) Inhibition of NAD glycohydrolase and ADP-ribosyl transferases by carbocyclic analogues of oxidized nicotinamide adenine dinucleotide. Biochemistry 28:7688-94
Slama, J T; Simmons, A M (1988) Carbanicotinamide adenine dinucleotide: synthesis and enzymological properties of a carbocyclic analogue of oxidized nicotinamide adenine dinucleotide. Biochemistry 27:183-93
Hutter, J A; Slama, J T (1987) Inhibition of thiaminase I from Bacillus thiaminolyticus. Evidence supporting a covalent 1,6-dihydropyrimidinyl-enzyme intermediate. Biochemistry 26:1969-73