The enzyme 3-oxo-delta5-steroid isomerase (KSI) is a key enzyme in the biosynthetic pathway for steroid hormones. the importance of steroid hormone deprivation protocols in current cancer therapy makes a detailed understanding of the mechanisms of the enzymes involved in this pathway of major importance. Ultimately, it is hoped that a selective inhibition of one of these enzymes will allow a pharmacological approach to hormone- dependent cancers to be achieved, surpassing current surgical methodology. This proposal for supplemental funding describes plans to investigate the 3-dimensional structure of the KSI from Pseudomonas testosteroni and its D38E mutant. These studies are required to enable us to correlate our kinetic and mechanistic results with the structure of the enzyme. Two- dimensional and three-dimensional NMR investigations will be carried out on 500 MHz and 600 MHz spectrometers and the results will be analyzed by NMR- based distance geometry methodologies. NMR studies will be extended to protein-inhibitor complexes to gain additional insights into structural aspects of catalysis. Preliminary data presented here show that this approach is feasible for the structural determination of KSI.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM038155-05
Application #
3294263
Study Section
Biochemistry Study Section (BIO)
Project Start
1987-08-01
Project End
1995-03-31
Budget Start
1992-04-01
Budget End
1993-03-31
Support Year
5
Fiscal Year
1992
Total Cost
Indirect Cost
Name
University of Maryland Balt CO Campus
Department
Type
Schools of Arts and Sciences
DUNS #
City
Baltimore
State
MD
Country
United States
Zip Code
21250
Wilde, Thomas C; Blotny, Grzegorz; Pollack, Ralph M (2008) Experimental evidence for enzyme-enhanced coupled motion/quantum mechanical hydrogen tunneling by ketosteroid isomerase. J Am Chem Soc 130:6577-85
Pollack, Ralph M (2004) Enzymatic mechanisms for catalysis of enolization: ketosteroid isomerase. Bioorg Chem 32:341-53
Petrounia, I P; Blotny, G; Pollack, R M (2000) Binding of 2-naphthols to D38E mutants of 3-oxo-Delta 5-steroid isomerase: variation of ligand ionization state with the nature of the electrophilic component. Biochemistry 39:110-6
Henot, F; Pollack, R M (2000) Catalytic activity of the D38A mutant of 3-oxo-Delta 5-steroid isomerase: recruitment of aspartate-99 as the base. Biochemistry 39:3351-9
Pollack, R M; Thornburg, L D; Wu, Z R et al. (1999) Mechanistic insights from the three-dimensional structure of 3-oxo-Delta(5)-steroid isomerase. Arch Biochem Biophys 370:9-15
Petrounia, I P; Pollack, R M (1998) Substituent effects on the binding of phenols to the D38N mutant of 3-oxo-delta5-steroid isomerase. A probe for the nature of hydrogen bonding to the intermediate. Biochemistry 37:700-5
Thornburg, L D; Henot, F; Bash, D P et al. (1998) Electrophilic assistance by Asp-99 of 3-oxo-Delta 5-steroid isomerase. Biochemistry 37:10499-506
Qi, L; Pollack, R M (1998) Catalytic contribution of phenylalanine-101 of 3-oxo-Delta 5-steroid isomerase. Biochemistry 37:6760-6
Wu, Z R; Ebrahimian, S; Zawrotny, M E et al. (1997) Solution structure of 3-oxo-delta5-steroid isomerase. Science 276:415-8
Zawrotny, M E; Hawkinson, D C; Blotny, G et al. (1996) Mechanism of proton transfer in the isomerization of 5-androstene-3, 17-dione by 3-oxo-delta 5-steroid isomerase and its D38E mutant. Biochemistry 35:6438-42

Showing the most recent 10 out of 19 publications