The enzyme Delta5-3-ketosteriod isomerase is a key enzyme in the biosynthetic pathway for steroid hormones. The importance of 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 to be hoped that a selective inhibition of one of these enzymes will allow a pharmacological approach to hormone-dependent cancers to be achieved, surpassing the current surgical methodology. This proposal describes plans to continue our studies on the Delta5-3-ketosteroid isomerase from P. testosteroni. The bacterial enzyme can serve as a model for the corresponding mammalian enzyme which is quite difficult to investigate. Our work is primarily concerned with the mechanism of action of the isomerase and has thus far resulted in the synthesis of three types of active-site-directed irreversible inhibitors, the identification of Asp-38 at the active site, and the finding that there are at least two modes of binding of steroids to the enzyme. In addition, we have proposed a novel mechanism for the enzyme. The current proposal involves further characterization of the amino acids important in the catalytic mechanism by the use of the above inhibitors and other suitably designed affinity labels. We will also determine the energy relationships in catalysis by rapid-flow experiments and investigate in more detail the binding of steroids to the enzyme. The rapid-flow experiments, in addition to their relevance to the mechanism of the isomerase, will also shed some light on the general question of enzyme perturbation of equilibrium constants by binding, i.e. the """"""""internal thermodynamics"""""""" of enzyme reactions.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM033059-05
Application #
3282404
Study Section
Bio-Organic and Natural Products Chemistry Study Section (BNP)
Project Start
1980-09-01
Project End
1987-11-30
Budget Start
1985-12-01
Budget End
1986-11-30
Support Year
5
Fiscal Year
1986
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