The broad, long-term objectives for the research described in this proposal are aimed at the elucidation of the complex relationships between structure and function in biological systems. The primary focus of the present application is directed at the mechanism of catalysis and structure of the bacterial phosphotriesterase. This enzyme catalyzes the detoxification or organophosphate neurotoxins through the hydrolysis of phosphorus-oxygen and phosphorus-fluorine bonds. The phosphotriesterase catalyzes the hydrolysis of optimal substrates at the diffusion- controlled limit and a divalent cation is required for catalytic activity. The role of the essential metal ion will be probed by substitution of the native Zn2+ ion with a variety of spectroscopically active cations (Cd2+, Mn2+, Ni2+, Co2+, and Cu2+). The ligand environment of the metal sites will be addressed by 113Cd-NMR spectroscopy. Interactions between the metal sites and substrates will be obtained using ESR spectroscopy with the paramagnetic metal ion derivatives. The spectroscopic studies will be complemented by replacement of potential amino acid ligands (histidine, cysteine, aspartate, and glutamate) using site-directed mutagenesis protocols. The mutant proteins will be utilized for the sequence specific assignments of the protein ligands to the metal centers and as probes for the role these metal ions play in the catalytic events. The identity and function of other amino acids at the active site of this enzyme will be determined through the design and synthesis of alkynyl phosphate esters. These suicide substrates covalently react with active site nucleophiles and result in complete inactivation of catalysis. Heavy atom oxygen-18 isotope effects with fast and slow substrates using wild type and mutant proteins will be utilized to determine the distribution of transition state structures. An X-ray crystallographic analysis will begin in an effort to determine the three-dimensional structure of the folded protein.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM033894-11
Application #
2177178
Study Section
Biochemistry Study Section (BIO)
Project Start
1984-12-01
Project End
1997-03-31
Budget Start
1995-04-01
Budget End
1996-03-31
Support Year
11
Fiscal Year
1995
Total Cost
Indirect Cost
Name
Texas A&M University
Department
Chemistry
Type
Schools of Earth Sciences/Natur
DUNS #
City
College Station
State
TX
Country
United States
Zip Code
77845
Williams, LaKenya; Fan, Fan; Blanchard, John S et al. (2008) Positional isotope exchange analysis of the Mycobacterium smegmatis cysteine ligase (MshC). Biochemistry 47:4843-50
Marti-Arbona, Ricardo; Xu, Chengfu; Steele, Sondra et al. (2006) Annotating enzymes of unknown function: N-formimino-L-glutamate deiminase is a member of the amidohydrolase superfamily. Biochemistry 45:1997-2005
Williams, LaKenya; Nguyen, Tinh; Li, Yingchun et al. (2006) Uronate isomerase: a nonhydrolytic member of the amidohydrolase superfamily with an ambivalent requirement for a divalent metal ion. Biochemistry 45:7453-62
Sakai, Ayano; Xiang, Dao Feng; Xu, Chengfu et al. (2006) Evolution of enzymatic activities in the enolase superfamily: N-succinylamino acid racemase and a new pathway for the irreversible conversion of D- to L-amino acids. Biochemistry 45:4455-62
Xu, Chengfu; Hall, Richard; Cummings, Jennifer et al. (2006) Tight binding inhibitors of N-acyl amino sugar and N-acyl amino acid deacetylases. J Am Chem Soc 128:4244-5
Li, Yingchun; Raushel, Frank M (2005) Inhibitors designed for the active site of dihydroorotase. Bioorg Chem 33:470-83
Marti-Arbona, Ricardo; Thoden, James B; Holden, Hazel M et al. (2005) Functional significance of Glu-77 and Tyr-137 within the active site of isoaspartyl dipeptidase. Bioorg Chem 33:448-58
Seibert, Clara M; Raushel, Frank M (2005) Structural and catalytic diversity within the amidohydrolase superfamily. Biochemistry 44:6383-91
Ghanem, Eman; Raushel, Frank M (2005) Detoxification of organophosphate nerve agents by bacterial phosphotriesterase. Toxicol Appl Pharmacol 207:459-70
Marti-Arbona, Ricardo; Fresquet, Vicente; Thoden, James B et al. (2005) Mechanism of the reaction catalyzed by isoaspartyl dipeptidase from Escherichia coli. Biochemistry 44:7115-24

Showing the most recent 10 out of 74 publications